Article

The crystal and molecular structure of the third domain of silver pheasant ovomucoid (OMSVP3)

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

OMSVP3 and OMTKY3 (third domains of silver pheasant and turkey ovomucoid inhibitor) are Kazal-type serine proteinase inhibitors. They have been isomorphously crystallized in the monoclinic space group C2 with cell dimensions of a = 4.429 nm, b = 2.115 nm, c = 4.405 nm, beta = 107 degrees. The asymmetric unit contains one molecule corresponding to an extremely low volume per unit molecular mass of 0.0017 nm3/Da. Data collection was only possible for the OMSVP3 crystals. Orientation and position of the OMSVP3 molecules in the monoclinic unit cells were determined using Patterson search methods and the known structure of the third domain of Japanese quail ovomucoid (OMJPQ3) [Papamokos, E., Weber, E., Bode, W., Huber, R., Empie, M. W., Kato, I. and Laskowski, M., Jr (1982) J. Mol. Biol. 158, 515-537]. The OMSVP3 structure has been refined by restrained crystallographic refinement yielding a final R value of 0.199 for data to 0.15 nm resolution. Conformation and hydrogen-bonding pattern of OMSVP3 and OMJPQ3 are very similar. Large deviations occur at the NH2 terminus owing to different crystal packing, and at the C terminus of the central helix, representing an intrinsic property and resulting from amino acid substitutions far away from this site. The deviation of OMSVP3 from OMTKY3 complexed with the Streptomyces griseus protease B is very small [Fujinaga, M., Read, R. J., Sielecki, A., Ardelt, W., Laskowski, M., Jr and James, M. N. G. (1982) Proc. Natl Acad. Sci. USA, 79, 4868-4872].

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Unfortunately, there are few uncomplexed inhibitor structures solved with B-values present. Of those that have been solved, the reactive site loops are typically reported to have higher than average temperature factors , Bode et al., 1985, McPhalen & James, 1987, Hubbard et al., 1991. This is illustrated in figure 2.9(b), (d) and (f). ...
... This is also the case for OMJPQ3. The reactive site loop of another ovomucoid third domain OMSVP3 is also seen to be flexible (Bode et al., 1985) along with mutants of OMJPQ3 and OMSVP3 (Musil et al., 1991). ...
... This confirms the observation that inhibitors possess a certain degree of flexibility that allows a small amount of hinge bending, to allow the binding loop to fit into the cognate enzyme's active site. Such available mobility may broaden specificity by permitting the adaption of the reactive site loop to different active sites as suggested by many authors (Greenblatt et al., 1989, Read & James,1986, Fujinaga et al., 1987, Bode et al., 1985. This helps to explain the ability of inhibitors such as eglin to inhibit such a wide range of proteinases. ...
Thesis
The molecular recognition exhibited between particular protein molecules has been analyzed, utilising the Brookhaven databank of atomic resolution protein structures. Specifically, an extensive study of the recognition between serine proteinases, and their protein inhibitors and substrates has been made. The analyses involve comparisons of the molecular structure of these protein inhibitors, specifically at their recognition regions, and further comparisons with protein substrates of the proteinases. These comparisons reveal a common recognition motif possessed by the binding loops of serine proteinase inhibitors, which is absent in the substrates (limited proteolytic sites). This conserved recognition motif is maintained despite the sequence and global structural dissimilarity between inhibitor families. In contrast, the recognition regions of the substrates are structurally diverse, and could not be bound to their target enzymes in their crystal conformations. This implies mobility is vital for their recognition, to allow structural rearrangement. Modelling experiments are carried out to ascertain the degree to which the limited proteolytic sites may adopt inhibitor-like conformations, thus mimicking the expected recognition conformation. This was done by first testing the ability of the substrates to adopt such a conformation geometrically, and secondly to simulate the types of motions that might occur. Finally, an algorithm is presented that predicts the likely location of limited proteolytic sites within proteins. The predictive power of the method and its individual component parameters are critically assessed.
... The Kazal family is well-characterized, especially at the structural level [4][5][6]. A typical Kazal domain comprises 40-60 amino acids that include six cysteine residues capable of forming three intra-domain disulfide bridges with the pattern 1-5, 2-4 and 3-6, resulting in a characteristic three-dimensional structure comprising one a-helix surrounded by an adjacent three-stranded anti-parallel b-sheet. ...
Article
Unlabelled: Greglin is an 83-residue serine protease inhibitor purified from the ovaries of the locust Schistocerca gregaria. Greglin is a strong inhibitor of subtilisin and human neutrophil elastase, acting at sub-nanomolar and nanomolar concentrations, respectively; it also inhibits neutrophil cathepsin G, α-chymotrypsin and porcine pancreatic elastase, but to a lesser extent. In the present study, we show that greglin resists denaturation at high temperature (95 °C) and after exposure to acetonitrile and acidic or basic pH. Greglin is composed of two domains consisting of residues 1-20 and 21-83. Mass spectrometry indicates that the N-terminal domain (1-20) is post-translationally modified by phosphorylations at three sites and probably contains a glycosylation site. The crystal structure of the region of greglin comprising residues 21-78 in complex with subtilisin was determined at 1.75 Å resolution. Greglin represents a novel member of the non-classical Kazal inhibitors, as it has a unique additional C-terminal region (70-83) connected to the core of the molecule via a supplementary disulfide bond. The stability of greglin was compared with that of an ovomucoid inhibitor. The thermostability and inhibitory specificity of greglin are discussed in light of its structure. In particular, we propose that the C-terminal region is responsible for non-favourable interactions with the autolysis loop (140-loop) of serine proteases of the chymotrypsin family, and thus governs specificity. Database: The atomic coordinates and structure factors for the greglin-subtilisin complex have been deposited with the RCSB Protein Data Bank under accession number 4GI3. Structured digital abstract: Greglin and Subtilisin Carlsberg bind by X-ray crystallography (View interaction).
... 42 The PDB code of the complex is 1cgi. 43 2. CHO, ovomucoid (2ovo) 44 bound to ␣-chymotrypsin (5cha). 45 The PDB code of the complex is 1cho. ...
Article
Empirical residue–residue pair potentials are used to screen possible complexes for protein–protein dockings. A correct docking is defined as a complex with not more than 2.5 Å root-mean-square distance from the known experimental structure. The complexes were generated by “ftdock” (Gabb et al. J Mol Biol 1997;272:106–120) that ranks using shape complementarity. The complexes studied were 5 enzyme-inhibitors and 2 antibody-antigens, starting from the unbound crystallographic coordinates, with a further 2 antibody-antigens where the antibody was from the bound crystallographic complex. The pair potential functions tested were derived both from observed intramolecular pairings in a database of nonhomologous protein domains, and from observed intermolecular pairings across the interfaces in sets of nonhomologous heterodimers and homodimers. Out of various alternate strategies, we found the optimal method used a mole-fraction calculated random model from the intramolecular pairings. For all the systems, a correct docking was placed within the top 12% of the pair potential score ranked complexes. A combined strategy was developed that incorporated “multidock,” a side-chain refinement algorithm (Jackson et al. J Mol Biol 1998;276:265–285). This placed a correct docking within the top 5 complexes for enzyme-inhibitor systems, and within the top 40 complexes for antibody–antigen systems. Proteins 1999;35:364–373. © 1999 Wiley-Liss, Inc.
... In fig. lc [9]; OMSVP3, ovomucoid [10]; PSTI, pancreatic secretory trypsin inhibitor [11]; UBQ, ubiquitin [12]; WGA, wheat germ agglutinin [13]; RNST, ribonuclease St [14]; TLN, thermolysin [15]; RBP, retinol-binding protein [16]; LYSC, chicken lysozyme [17]; HSUB, H-subunit of the photosynthetic reaction center [ 18]; CI-2, chymotrypsin inhibitor 2 [ 19]; LYST4, phage T4 lysozyme [20]; PLA, plastocyanin [21]; RNT~, ribonuclease T1 [22]; PRE, prealbumin [23]; CHYM, chymotrypsin [24]; RNS, ribonuclease S [25]. Proteins indicated by asterisks are those for which conformations of residues are established taking into account C~-atom stereo plots only. ...
Article
Protein standard structures formed by two consecutive β-strands connected by short loops are considered in this paper. A stereochemical analysis of each standard structure has been performed to determine the necessary conditions which must be fulfilled in the amino acid sequence encoding the standard structure. It is shown that amino acid sequences coding for the same standard structure in ditterent proteins have practically the same order of hydrophobic, hydrophilic and glycine residues. The results of the stereochemical analysis are confirmed by a large number of examples from known protein structures.
... This assumption is not valid for the P 2 Thr-17-P 1 Ј Glu-19 residue pair (Fig. 2). The side chains of these two residues are hydrogenbonded to each other in the free inhibitor (37). The hydrogen bond shortens on complex formation with SGPB (29) and CHYM (30) but not with HLE (31). ...
Article
Full-text available
An additivity-based sequence to reactivity algorithm for the interaction of members of the Kazal family of protein inhibitors with six selected serine proteinases is described. Ten consensus variable contact positions in the inhibitor were identified, and the 19 possible variants at each of these positions were expressed. The free energies of interaction of these variants and the wild type were measured. For an additive system, this data set allows for the calculation of all possible sequences, subject to some restrictions. The algorithm was extensively tested. It is exceptionally fast so that all possible sequences can be predicted. The strongest, the most specific possible, and the least specific inhibitors were designed , and an evolutionary problem was solved.
... Pairs of proteins were superposed on their Cα atoms by the least squares fitting algorithm of McLachlan, 1979. This algorithm takes two equivalent sets of atoms, a and b, and calculates the transformation matrix that minimises the RMSD between them (see "Root Ovomucoid Unbound 2ovo (Bode et al., 1985) 1.5 ...
... If the dissociation is slow, the predominant product is likely to be the modified inhibitor (Ardelt & Laskowski, 1983, 1991). However, at low pH the rate of dissociation to virgin inhibitor is much more rapid than to modified (Ardelt & Laskowski, 1985 ), therefore kinetically controlled dissociation at very low pH yields virgin inhibitor. Therefore, 3 min after the complex was formed, the pH value was lowered by adding with stirring an amount of 1 M HCl required to lower the pH value to 1 .O. ...
Article
In the association of serine proteinases with their cognate substrates and inhibitors an important interaction is the fitting of the P1 side chain of the substrate or inhibitor into a preformed cavity of the enzyme called the S1 pocket. In turkey ovomucoid third domain, which is a canonical protein proteinase inhibitor, the P1 residue is Leu¹⁸. Here we report the values of equilibrium constants, Ka, for turkey ovomucoid third domain and 13 additional Leu18X variants with six serine proteinases: bovine α chymotrypsin A, porcine pancreatic elastase, subtilisin Carlsberg, Streptomyces griseus proteinases A and B, and human leukocyte elastase. Eight of the Xs are coded amino acids: Ala, Ser, Val, Met, Gln, Glu, Lys, and Phe, and five are noncoded: Abu, Ape, Ahx, Ahp, and Hse. They were chosen to simplify the interamino acid comparisons. In the homologous series of straight-chain side chains Ala, Abu, Ape, Ahx, Ahp, free energy of binding decreases monotonically with the side-chain length for chymotrypsin with large binding pocket, but even for this enzyme shows curvature. For the two S. griseus enzymes a minimum appears to be reached at Ahp. A minimum is clearly evident for the two elastases, where increasing the side-chain length from Ahx to Ahp greatly weakens binding, but much more so for the apparently more rigid pancreatic enzyme than for the more flexible leukocyte enzyme. β-Branching (Ape/Val) is very deleterious for five of the six enzymes; it is only slightly deleterious for the more flexible human leukocyte elastase. The effect of γ-branching (Ahx/Leu), of introduction of heteroatoms (Abu/Ser), (Ape/Hse), and (Ahx/Met), and of introduction of charge (Gln/Glu) and (Ahp/Lys) are tabulated and discussed. An important component of the free energy of interaction is the distortion of the binding pocket by bulky or branched side chains.
... 42 The PDB code of the complex is 1cgi. 43 2. CHO, ovomucoid (2ovo) 44 bound to ␣-chymotrypsin (5cha). 45 The PDB code of the complex is 1cho. ...
Article
Empirical residue–residue pair potentials are used to screen possible complexes for protein–protein dockings. A correct docking is defined as a complex with not more than 2.5 Å root-mean-square distance from the known experimental structure. The complexes were generated by “ftdock” (Gabb et al. J Mol Biol 1997;272:106–120) that ranks using shape complementarity. The complexes studied were 5 enzyme-inhibitors and 2 antibody-antigens, starting from the unbound crystallographic coordinates, with a further 2 antibody-antigens where the antibody was from the bound crystallographic complex. The pair potential functions tested were derived both from observed intramolecular pairings in a database of nonhomologous protein domains, and from observed intermolecular pairings across the interfaces in sets of nonhomologous heterodimers and homodimers. Out of various alternate strategies, we found the optimal method used a mole-fraction calculated random model from the intramolecular pairings. For all the systems, a correct docking was placed within the top 12% of the pair potential score ranked complexes. A combined strategy was developed that incorporated “multidock,” a side-chain refinement algorithm (Jackson et al. J Mol Biol 1998;276:265–285). This placed a correct docking within the top 5 complexes for enzyme-inhibitor systems, and within the top 40 complexes for antibody–antigen systems. Proteins 1999;35:364–373. © 1999 Wiley-Liss, Inc.
... References: IGDl (Skarzynski et al., 1987), IGOX (Lindqvist, 1989), IIlB (Finzel et al., 1984), IPCY (Guss & Freeman, 1983), ZAPR (Suguna et al., 1987), ZCAB (Kannan et al., 1984), 2CPP (Poulos et al., 1987). 20VO (Bode et al., 1985), 2PRK (Betzel et al., 1988), 3DFR (Bolin et al., 1982). 6LDH (Abad-Zapatero et al., 1987). ...
Article
Local determinants of 310-helix stabilization have been ascertained from the analysis of the crystal structure data base. We have clustered all 5-length substructures from 51 nonhomologous proteins into classes based on the conformational similarity of their backbone dihedral angles. Several clusters, derived from 310-helices and multiple-turn conformations, had strong amino acid sequence patterns not evident among α-helices. Aspartate occurred over twice as frequently in the N-cap position of 310-helices as in the N-cap position of α-helices. Unlike α-helices, 310-helices had few C-termini ending in a left-handed α conformation; most 310 C-caps adopted an extended conformation. Differences in the distribution of hydrophobic residues among 310- and α-helices were also apparent, producing amphipathic 310-helices. Local interactions that stabilize 310-helices can be inferred both from the strong amino acid preferences found for these short helices, as well as from the existence of substructures in which tertiary interactions replace consensus local interactions. Because the folding and unfolding of α-helices have been postulated to proceed through reverse-turn and 310-helix intermediates, sequence differences between 310- and α-helices can also lend insight into factors influencing α-helix initiation and propagation.
... Laskowski and coworkers have reported Khyd values for 42 avian ovomucoid third domains (M, -6 kDa; , which are elastase inhibitors of the Kazal family (Laskowski ranges from -2.1 to +0.9 kcal/mol at 25 "c). Changes in AGzyd values have been rationalized in terms of changes in residue-residue interactions, utilizing the X-ray crystal structures determined for intact and nicked inhibitors (Weber et al., 1981;Papamokos et al., 1982;Bode et al., 1985;Musil et al., 1991), and the complex formed between turkey ovomucoid third domain and Streptomyces griseus protease B (Fujinaga et al., 1982). ...
Article
Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49593-626), whereby an enzyme-catalyzed equilibrium between intact (I) and reactive-site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI-III; Mr 3 kDa) of the squash family and rCMTI-V (Mr ∼ 7 kDa) of the potato I family. These two inhibitors have different binding loop-scaffold interactions and different Khyd values—2.4 (CMTI-III) and 9 (CMTI-V)—at 25°C. The reactive-site peptide bond (P1-P'1) is that between Arg5 and Ile6 in CMTI-III, and that between Lys44 and Asp45 in CMTI-V. The order parameters (S2) of backbone NHs of uniformly 15N-labeled rCMTI-III and rCMTI-III* were determined from measurements of 15N spin-lattice and spin-spin relaxation rates, and {1H}-15N steady-state heteronuclear Overhauser effects, using the model-free formalism, and compared with the data reported previously for rCMTI-V and rCMTI-V*. The backbones of rCMTI-III (〈S2〉 = 0.71) and rCMTI-III* (〈S2) = 0.63) are more flexible than those of rCMTI-V (〈S2〉 = 0.83) and rCMTI-V* (〈S2) = 0.85). The binding loop residues, P4-P1, in the two proteins show the following average order parameters: 0.57 (rCMTI-III) and 0.44 (rCMTI-III*); 0.70 (rCMTI-V) and 0.40 (rCMTI-V*). The P1'-P4' residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI-III) and 0.47 (rCMTI-III*); and 0.73 (rCMTI-V) and 0.83 (rCMTI-V*). The newly formed C-terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI-III* due to the Cys3-Cys20 crosslink. In contrast, the newly formed N-terminal (Pn' residues) becomes more flexible only in rCMTI-III*, most likely due to lack of an interaction between the P1' residue and the scaffold in rCMTI-III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn' residues seem to facilitate the resynthesis of the P1-P1' bond, leading to a lower Khyd value.
Article
Until recently, the "substrate-like" "canonical" inhibition by the "small" serine proteinase inhibitors, and the product-like inhibition by the carboxypeptidase inhibitor, provided the only models for protein inhibitor-proteinase interactions. The recently published structures of cystatin/stefin-papain complexes and of hirudin-thrombin complexes reveal novel modes of interactions of only partial substrate-like character. Despite considerable progress in understanding the native-cleaved transition of the serpins, the mechanisms of their interaction with their cognate serine proteinases is still a matter of conjecture.
Article
The substrate-like 'canonical' inhibition by the 'small' serine proteinase inhibitors and the product-like inhibition by the carboxypeptidase inhibitor have provided the only atomic models of protein inhibitor--proteinase interactions for about 15 years. The recently published structures of cystatin/stefin--papain complexes and of hirudin--thrombin complexes reveal novel non-substrate-like interactions. In addition, the structure of pro-carboxypeptidase shows a model of inactivation which bears resemblance to proteinase/protein inhibitor systems. Considerable progress in understanding the transition between native and cleaved states of the serpins has also been made by several recent structural studies.
Article
A novel super-secondary structure common for many non-homological proteins is considered. This folding pattern, consisting of adjacent along the chain alpha-helix and beta-hairpin, has an aligned packing. It is found that one of the two possible 'mirror-symmetrical' topologies is observed in proteins. The alpha-helix + beta-hairpin structures have a similar pattern of hydrophobic residues in their amino acid sequences. The remaining part of a molecule or a domain is almost always located on the same side of the considered folding pattern. These results can be used in the prediction of three-dimensional protein structure and protein design.
Article
A data collection which merges protein structural and sequence information is described. Structural superpositions amongst proteins with similar main-chain fold were performed or collected from the literature. Sequences taken from the protein primary structure databases were associated with the multiple structural alignments providing they were at least 50% homologous in residue identity to one of the structural sequences and at least 50% of the structural sequence residues were alignable. Such restrictions allow reasonable confidence that the primary sequences share the conformation of the tertiary structural templates, except in the less conserved loop regions. Multiple structural superpositions were collected for 38 familial groups containing a total of 209 tertiary structures; 45 structures had no superposable mates and were used individually. Other information is also provided as main-chain and side-chain conformational angles, secondary structural assignments and the like. Wedding the primary and tertiary structural data resulted in an 8-fold increase of data bank sequence entries over those associated with the known three-dimensional architectures alone.
Article
A modified version of the human pancreatic trypsin inhibitor (PSTI), generated in a protein-design project, has been crystallized in spacegroup P4(3) with lattice constants a = 40.15 A, c = 33.91 A. The structure has been solved by molecular replacement. Refinement of the structure by simulated annealing and conventional restrained least-squares yielded for 8.0 to 2.3 A data a final R-value of 19.1%. Differences to the known structures of porcine PSTI complexed with trypsinogen and modified human PSTI complexed with chymotrypsinogen occur at the flexible N-terminal part of the molecule. These differences are influenced by crystal packing, as are low temperature factors for the binding loop. The geometry of the binding loop is similar to the complexed structures.
Article
Segment match modeling uses a data base of highly refined known protein X-ray structures to build an unknown target structure from its amino acid sequence and the atomic coordinates of a few of its atoms (generally only the C alpha atoms). The target structure is first broken into a set of short segments. The data base is then searched for matching segments, which are fitted onto the framework of the target structure. Three criteria are used for choosing a matching data base segment: amino acid sequence similarity, conformational similarity (atomic co-ordinates), and compatibility with the target structure (van der Waals' interactions). The new method works surprisingly well: for eight test proteins ranging in size from 46 to 323 residues, the all-atom root-mean-square deviation of the modeled structures is between 0.93 A and 1.73 A (the average is 1.26 A). Deviations of this magnitude are comparable with those found for protein co-ordinates before and after refinement against X-ray data or for co-ordinates of the same protein in different crystal packings. These results are insensitive to errors in the C alpha positions or to missing C alpha atoms: accurate models can be built with C alpha errors of up to 1 A or by using only half the C alpha atoms. The fit to the X-ray structures is improved significantly by building several independent models based on different random choices and then averaging co-ordinates; this novel concept has general implications for other modeling tasks. The segment match modeling method is fully automatic, yields a complete set of atomic co-ordinates without any human intervention and is efficient (14 s/residue on the Silicon Graphics 4D/25 Personal Iris workstation.
Article
Protein Data Bank (PDB), jointly founded in 1971 by Brookhaven National Laboratory, U.S.A. and the Cambridge Crystallographic Data Centre, U.K, is the single global archive of experimentally determined biological macromolecular structures. PDB deposition is mandatory for publication in most scientific journals, which means “no PDB deposition, no structural publication”. The current PDB archive contains more than 180,000 entries and includes many structures from Asian institutions. The first protein structure from Asia was that of Cytochrome c determined by Prof. Masao Kakudo’s group at the Institute for Protein Research, Osaka University in 1971 at a resolution of 4 Å, and a subsequent atomic structure at 2.3 Å resolution was deposited to PDB in 1976 as the 21st entry of the entire PDB archive. Since then, 317 protein structures whose primary citation was the Journal of Biochemistry (J. Biochem.) have been deposited to PDB. Based on this long history between PDB and J. Biochem., I carried out a statistical analysis of all structural reports in J. Biochem. using the relational database system at PDBj (https://pdbj.org) and reviewed the yearly distribution, resolution, quality of structure, type of target protein, number of citations and comparison against other major journals.
Article
The conformations of known tryptic limited proteolytic sites have been analysed and compared to the structures of the binding regions of serine proteinase inhibitors, as they are found when complexed to a serine proteinase. Conformational parameters studied include main-chain torsion angles, root-mean-square fits, accessibility, mobility and protrusion indices. As observed before, the inhibitors share a common main-chain conformation at the binding loop from P3-P'3 (Schechter & Berger notation), which is maintained throughout all the serine proteinase inhibitor families for which X-ray data is available, despite lack of similarity in the rest of the protein. This canonical structure is not found amongst the limited proteolytic sites (or nicksites), which differ markedly from the inhibitor binding loop conformation, and also amongst themselves. The experimentally determined nicksites are in general both accessible and protruding; as are the inhibitor binding loops, as well as being typically flexible regions of structure, as denoted by elevated temperature factors from crystallographic determinations. For cleavage by serine proteinases these loops must radically alter their local conformations and a large motion of the loop relative to the structure, in some cases, would be required to orientate these sites for cleavage.
Article
Tetragonal and triclinic crystals of two ovomucoid inhibitor third domains from silver pheasant and Japanese quail, modified at their reactive site bonds Met18Glu19 (OMSVP3∗) and Lys18Asp19 (OMJPQ3∗), respectively, were obtained. Their molecular and crystal structures were solved using X-ray data to 2.5 Å and 1.55 Å by means of Patterson search methods using truncated models of the intact (virgin) inhibitors as search models. Both structures were crystallographically refined to R-values of 0.185 and 0.192, respectively, applying an energy restraint reciprocal space refinement procedure.
Article
Variants of the human pancreatic secretory trypsin inhibitor (PSTI) have been created during a protein design project to generate a high-affinity inhibitor with respect to some serine proteases other than trypsin. Two modified versions of human PSTI with high affinity for chymotrypsin were crystallized as a complex with chymotrypsinogen. Both crystallize isomorphously in space group P4(1)2(1)2 with lattice constants a = 84.4 A, c = 86.7 A and diffract to 2.3 A resolution. The structure was solved by molecular replacement. The final R-value after refinement with 8.0 to 2.3 A resolution data was 19.5% for both complexes after inclusion of about 50 bound water molecules. The overall three-dimensional structure of PSTI is similar to the structure of porcine PSTI in the trypsinogen complex (1TGS). Small differences in the relative orientation of the binding loop and the core of the inhibitors indicate flexible adaptation to the proteases. The chymotrypsinogen part of the complex is similar to chymotrypsin. After refolding induced by binding of the inhibitor the root-mean-square difference of the active site residues A186 to A195 and A217 to A222 compared to chymotrypsin was 0.26 A.
Article
Analysis of 13 high-resolution protein X-ray crystal structures shows that 1204 (24%) of all the 4974 hydrogen bonds are of the bifurcated three-center type with the donor X-H opposing two acceptors A1, A2. They occur systematically in alpha-helices where 90% of the hydrogen bonds are of this type; the major component is (n + 4)N-H ... O = C(n) as expected for a 3.6(13) alpha-helix, and the minor component is (n + 4)N-H ... O = C(n + 1), as observed in 3(10) helices; distortions at the C-termini of alpha-helices are stabilized by three-center bonds. In beta-sheets 40% of the hydrogen bonds are three-centered. The frequent occurrence of three-center hydrogen bonds suggests that they should not be neglected in protein structural studies.
Article
We have measured equilibrium constants, Khyd, at pN 6 for the hydrolysis of the reactive site peptide bond (bond between residues 18 and 19) in 42 sequenced variants (39 natural, 3 semisynthetic) of avian ovomucoid third domains. The values range from 0.4 to approximately 35. In 35 cases the effect of a single amino acid replacement on Khyd could be calculated, 13 are without effect and 22 range from a factor of 1.25 to 5.5. Several, but not all, of the effects can be rationalized in terms of residue-residue interactions that are affected by the reactive site hydrolysis. As the measurements are very precise it appears that additional measurements on designed rather than natural variants should allow for the precise measurement of side-chain--side-chain interaction energies.
Article
Predicting the structures of protein-protein complexes is a difficult problem owing to the topographical and thermodynamic complexity of these structures. Past efforts in this area have focussed on fitting the interacting proteins together using rigid body searches, usually with the conformations of the proteins as they occur in crystal structure complexes. Here we present work which uses a rigid body docking method to generate the structures of three known protein complexes, using both the bound and unbound conformations of the interacting molecules. In all cases we can regenerate the geometry of the crystal complexes to high accuracy. We also are able to find geometries that do not resemble the crystal structure but nevertheless are surprisingly reasonable both mechanistically and by some simple physical criteria. In contrast to previous work in this area, we find that simple methods for evaluating the complementarity at the protein-protein interface cannot distinguish between the configurations that resemble the crystal structure complex and those that do not. Methods that could not distinguish between such similar and dissimilar configurations include surface area burial, solvation free energy, packing and mechanism-based filtering. Evaluations of the total interaction energy and the electrostatic interaction energy of the complexes were somewhat better. Of the techniques that we tried, energy minimization distinguished most clearly between the "true" and "false" positives, though even here the energy differences were surprisingly small. We found the lowest total interaction energy from amongst all of the putative complexes generated by docking was always within 5 A root-mean-square of the crystallographic structure. There were, however, several putative complexes that were very dissimilar to the crystallographic structure but had energies that were close to that of the low energy structure. The magnitude of the error in energy calculations has not been established in macromolecular systems, and thus the reliability of the small differences in energy remains to be determined. The ability of this docking method to regenerate the crystallographic configurations of the interacting proteins using their unbound conformations suggests that it will be a useful tool in predicting the structures of unsolved complexes.
Article
The trypsin inhibitor DE-3 from Erythrina caffra (ETI) belongs to the Kunitz-type soybean trypsin inhibitor (STI) family and consists of 172 amino acid residues with two disulphide bridges. The amino acid sequence of ETI shows high homology to other trypsin inhibitors from the same family but ETI has the unique ability to bind and inhibit tissue plasminogen activator. The crystal structure of ETI has been determined using the method of isomorphous replacement and refined using a combination of simulated annealing and conventional restrained least-squares crystallographic refinement. The refined model includes 60 water molecules and 166 amino acid residues, with a root-mean-square deviation in bond lengths from ideal values of 0.016 A. The crystallographic R-factor is 20.8% for 7770 independent reflections between 10.0 and 2.5 A. The three-dimensional structure of ETI consists of 12 antiparallel beta-strands joined by long loops. Six of the strands form a short antiparallel beta-barrel that is closed at one end by a "lid" consisting of the other six strands coupled in pairs. The molecule shows approximate 3-fold symmetry about the axis of the barrel, with the repeating unit consisting of four sequential beta-strands and the connecting loops. Although there is no sequence homology, this same fold is present in the structure of interleukin-1 alpha and interleukin-1 beta. When the structure of ETI and interleukin-1 beta are superposed, the close agreement between the alpha-carbon positions for the beta-strands is striking. The scissile bond (Arg63-Ser64) is located on an external loop that protrudes from the surface of the molecule and whose architecture is not constrained by secondary structure elements, disulphide bridges or strong electrostatic interactions. The hydrogen bonds made by the side-chain amide group of Asn12 play a key role in maintaining the three-dimensional structure of the loop. This residue is in a position corresponding to that of a conserved asparagine in the Kazal inhibitor family. Although the overall structure of ETI is similar to the partial structure of STI, the scissile bond loop is displaced by about 4 A. This displacement probably arises from the fact that the structure of STI has been determined in a complex with trypsin but could possibly be a consequence of the close molecular contact between Arg63 and an adjacent molecule in the crystal lattice.
Article
We describe a largely automatic procedure for building protein structures from sequence alignments with homologues of known structure. This procedure uses simple rules by which multiple sequence alignments can be translated into distance and chirality constraints, which are then used as input for distance geometry calculations. By this means one obtains an ensemble of conformations for the unknown structure that are compatible with the rules employed, and the differences among these conformations provide an indication of the reliability of the structure prediction. The overall approach is demonstrated here by applying it to several Kazal-type trypsin inhibitors, for which experimentally determined structures are available. On the basis of our experience with these test problems, we have further predicted the conformation of the human pancreatic secretory trypsin inhibitor, for which no experimentally determined structure is presently available.
Article
The crystal structures of the complexes formed between subtilisin Novo and three inhibitors, eglin c, Arg45-eglin c and Lys53-eglin c have been determined using molecular replacement and difference Fourier techniques and refined at 2.4 A, 2.1 A, and 2.4 A resolution, respectively. The mutants Arg45-eglin c and Lys53-eglin c were constructed by site-directed mutagenesis in order to investigate the inhibitory specificity and stability of eglin c. Arg45-eglin became a potent trypsin inhibitor, in contrast to native eglin, which is an elastase inhibitor. This specificity change was rationalized by comparing the structures of Arg45-eglin and basic pancreatic trypsin inhibitor and their interactions with trypsin. The residue Arg53, which participates in a complex network of hydrogen bonds formed between the core and the binding loop of eglin c, was replaced with the shorter basic amino acid lysine in the mutant Lys53-eglin. Two hydrogen bonds with Thr44, located in the binding loop, can no longer be formed but are partially restored by a water molecule bound in the vicinity of Lys53. Eglin c in complexes with both subtilisin Novo and subtilisin Carlsberg was crystallized in two different space groups. Comparison of the complexes showed a rigid body rotation for the eglin c core of 11.5 degrees with respect to the enzyme, probably caused by different intermolecular contacts in both crystal forms.
Article
Ovomucoids were isolated from 25 avian species other than the 101 studied in Laskowskiet al. (1987,Biochemistry 26, 202–221). These were subjected to limited proteolysis with an appropriate enzyme, and connecting peptide extended ovomucoid third domains were isolated and sequenced to the end in a protein sequencer. Of the 25 new sequences, 13 duplicate ones were already known, and 12 are unique. Probably the most striking findings are a Pro14 → Ser14 replacement in weka, an Ala14→Thr15 replacement in Bulwer's pheasant, the discovery of two additional amino acid residues Ile18 and Gly18 at the P1 reactive site position in Kalij pheasant and tawny frogmouth, respectively, and the first finding of a negative (Glu34) rather than positive (Lys34 or Arg34) amino acid residue at the NH2 terminus of the α helix in caracara ovomucoid third domain. These results complete the determination of all the sequences of ovomucoid third domains in the four species genusGallus, in the five species genusSyrmaticus, and in the two species generaAix andPavo.
Article
Conformational studies have been carried out on the X-cis-Pro tripeptide system (a system of three linked peptide units, in the trans-cis-trans configuration) using energy minimization techniques. For X, residues Gly, L-Ala, D-Ala and L-Pro have been used. The energy minima have been classified into different groups based upon the conformational similarity. There are 15, 20, 18 and 6 minima that are possible for the four cases respectively and these fall into 11 different groups. A study of these minima shows that, (i) some minima contain hydrogen bonds--either 4-->1 or 1-->2 type, (ii) the low energy minima qualify themselves as bend conformations, (iii) cis' and trans' conformations are possible for the prolyl residue as also the C gamma-endo and C gamma-exo puckerings, and (iv) for Pro-cis-Pro, cis' at the first prolyl residue is ruled out, due to the high energy. The available crystal structure data on proteins and peptides, containing cis-Pro segment have been examined with a view to find the minima that occur in solid state. The data from protein show that they fall under two groups. The conformation at X in X-cis-Pro is near extended when it is a non-glycyl residue. In both peptides and proteins there exists a preference for trans' conformation at prolyl residue over cis' when X is a non-glycyl residue. The minima obtained can be useful in modelling studies.
Article
There has been considerable effort to predict the structure of proteins from their amino acid sequences. A major problem in all prediction efforts has been that, short of a direct comparison with crystallographic co-ordinates, it is often difficult to evaluate the merit of a model, or “proposed” protein structure. Here, we present a method for evaluating proposed protein structures that does not require a structural model of complete atomic detail. Our method evaluates residue-residue packing density using a simplified model of the polypeptide chain where amino acids are represented as one, two (histidine, tyrosine and phenylalanine), or three (tryptophan) spheres. This method also gives a measure of the appropriateness of residue-residue contacts, thus giving a measure of the amino acid distribution throughout the protein. Amino acid packing and amino acid distribution, as evaluated by this technique, are consistent with the accuracy of model-built structures. We have been able to select the best structures from a set of combinatorially generated models using this method, and we anticipate that it will be useful as a general tool for model-building.
Article
P14C/N39C is the disulfide variant of the ovomucoid third domain from silver pheasant (OMSVP3) introducing an engineered Cys¹⁴-Cys³⁹ bond near the reactive site on the basis of the sequence homology between OMSVP3 and ascidian trypsin inhibitor. This variant exhibits a narrower inhibitory specificity. We have examined the effects of introducing a Cys¹⁴-Cys³⁹ bond into the flexible N-terminal loop of OMSVP3 on the thermodynamics of the reactive site peptide bond hydrolysis, as well as the thermal stability of reactive site intact inhibitors. P14C/N39C can be selectively cleaved by Streptomyces griseus protease B at the reactive site of OMSVP3 to form a reactive site modified inhibitor. The conversion rate of intact to modified P14C/N39C is much faster than that for wild type under any pH condition. The pH-independent hydrolysis constant (K(hyd) °) is estimated to be approximately 5.5 for P14C/N39C, which is higher than the value of 1.6 for natural OMSVP3. The reactive site modified form of P14C/N39C is thermodynamically more stable than the intact one. Thermal denaturation experiments using intact inhibitors show that the temperature at the midpoint of unfolding at pH 2.0 is 59 °C for P14C/N39C and 58 °C for wild type. There have been no examples, except P14C/N39C, where introducing an engineered disulfide causes a significant increase in K(hyd) °, but has no effect on the thermal stability. The site-specific disulfide introduction into the flexible N-terminal loop of natural Kazal-type inhibitors would be useful to further characterize the thermodynamics of the reactive site peptide bond hydrolysis.
Article
Proteinase inhibitors are classified into synthetic and naturally occurring substances. Among the latter, the protein-like inhibitors have received the greatest amount of attention. These inhibitors are found in numerous animal tissues and fluids, in many plant tissues, particularly in legume seeds and other storage organs, and in microorganisms. They have attracted the attention of nutritionists because of their presence in valuable plant foods and their subsequent possible involvement in nutritive properties. Inhibitor proteins have been studied as model systems for elucidation of the mechanism of inhibition of proteinases, and for studies of protein–protein association. The inhibitors are also being used as valuable tools in medical research because of unique pharmacological properties that suggest clinical application. This chapter focuses on the biological, chemical, and physical properties of the inhibitors. It also discusses their roles and significance. Proteinase inhibitors are often discovered by noting their ability to readily inhibit available proteinases, such as bovine trypsin, chymotrypsin or elastase, rather than genuine target proteinases of known or unknown specificities.
Article
A common approach to computing protein pKas uses a continuum dielectric model in which the protein is a low dielectric medium with embedded atomic point charges, the solvent is a high dielectric medium with a Boltzmann distribution of ionic charges, and the pKa is related to the electrostatic free energy which is obtained by solving the Poisson–Boltzmann equation. Starting from the model pKa for a titrating residue, the method obtains the intrinsic pKa and then computes the protonation probability for a given pH including site–site interactions. This approach assumes that acid dissociation does not affect protein conformation aside from adding or deleting charges at titratable sites. In this work, we demonstrate our treecode-accelerated boundary integral (TABI) solver for the relevant electrostatic calculations. The pKa computing procedure is enclosed in a convenient Python wrapper which is publicly available at the corresponding author’s website. Predicted results are compared with experimental pKas for several proteins. Among ongoing efforts to improve protein pKa calculations, the advantage of TABI is that it reduces the numerical errors in the electrostatic calculations so that attention can be focused on modeling assumptions.
Chapter
Most of the current knowledge concerning the water structure in and around biological macromolecules has been derived at near-atomic resolution from high-resolution X-ray and neutron diffraction studies of crystals (for review, see Finney, 1979; Edsall and McKenzie, 1983; Baker and Hubbard; 1984; Savage and Wlodawer, 1986; Saenger, 1987; Westhof, 1988; Westhof and Beveridge, 1990; Rupley and Caveri, 1991). Protein crystals, like nucleic acid crystals, are made up of a lattice of macromolecules which delimit channels of variable dimensions filled with solvent molecules. They constitute, thus, a suitable system to study the interactions between protein and water molecules at the atomic level, under conditions which are, to some extent, close to those found in solution.
Chapter
Proteinases play an important role in the normal functioning of biological systems. They are involved in crucial processes such as food digestion, blood coagulation and fibrinolysis, blood pressure regulation, and fertilization. Certain proteinases release peptide hormones and neuromodulators from inactive precursors or degrade message-transmitting peptides, thus initiating or terminating a variety of biological responses. It is clear that besides a pronounced cleavage specificity, a tight regulation of proteolytic activity by endogenous proteinase inhibitors is necessary. The number of proteinaceous inhibitors that have been isolated and characterized so far is large and growing steadily. The majority are directed toward members of the serine proteinases, one of the four classes of endopeptidases. Serine proteinase inhibitors can be grouped into at least sixteen different inhibitor families on the basis of sequence similarity, topological relationships between the disulfide bridges, and the location of the binding site for the cognate proteinase. Detailed knowledge of the structure and reactivity of the inhibitors is indispensable for a thorough understanding of the controlling functions they exercise in a variety of fundamental physiological proteolytic processes. This chapter discusses these inhibitors and some approaches that might lead to satisfactory therapeutic compounds.
Article
Molecular docking explores the binding modes of two interacting molecules. The technique is increasingly popular for studying protein-ligand interactions and for drug design. A fundamental problem problem with molecular docking is that orientation space is very large and grows combinatorially with the number of degrees of freedom of the interacting molecules. Here, we describe and evaluate algorithms that improve the efficiency and accuracy of a shape-based docking method. We use molecular organization and sampling techniques to remove the exponential time dependence on molecular size in docking calculations. The new techniques allow us to study systems that were prohibitively large for the original method. The new algorithms are tested in 10 different protein-ligand systems, including 7 systems where the ligand is itself a protein. In all cases, the new algorithms successfully reproduce the experimentally determined configurations of the ligand in the protein.
Article
A novel super-secondary structure common for many non-homological proteins is considered. This folding pattern, consisting of adjacent along the chain α-helix and β-hairpin, has an aligned packing. It is found that one of the two possible ‘mirror-symmetrical’ topologies is observed in proteins. The α-helix + β-hairpin structures have a similar pattern of hydrophobic residues in their amino acid sequences. The remaining part of a molecule or a domain is almost always located on the same side of the considered folding pattern. These results can be used in the prediction of three-dimensional protein structure and protein design.
Article
Full-text available
A new method, dubbed "HAXIS" is introduced to describe local and global shape properties of a protein helix via its axis. HAXIS is based on coarse-graining and spline-fitting of the helix backbone. At each Cα anchor point of the backbone, a Frenet frame is calculated, which directly provides the local vector presentation of the helix. After cubic spline-fitting of the axis line, its curvature and torsion are calculated. This makes a rapid comparison of different helix forms and the determination of helix similarity possible. Distortions of the helix caused by individual residues are projected onto the helix axis and presented either by the rise parameter per residue or by the local curvature of the axis. From a non-redundant set of 2,017 proteins, 15,068 helices were investigated in this way. Helix start and helix end as well as bending and kinking of the helix are accurately described. The global properties of the helix are assessed by a polynomial fit of the helix axis and the determination of its overall curving and twisting. Long helices are more regular shaped and linear whereas short helices are often strongly bent and twisted. The distribution of different helix forms as a function of helix length is analyzed.
Article
We propose a fast clustering and reranking method, CyClus, for protein-protein docking decoys. This method enables comprehensive clustering of whole decoys generated by rigid-body docking using cylindrical approximation of the protein-protein interface and hierarchical clustering procedures. We demonstrate the clustering and reranking of 54,000 decoy structures generated by ZDOCK for each complex within a few minutes. After parameter tuning for the test set in ZDOCK benchmark 2.0 with the ZDOCK and ZRANK scoring functions, blind tests for the incremental data in ZDOCK benchmark 3.0 and 4.0 were conducted. CyClus successfully generated smaller subsets of decoys containing near-native decoys. For example, the number of decoys required to create subsets containing near-native decoys with 80% probability was reduced to 22% to 50% of the number required in the original ZDOCK. Although specific ZDOCK and ZRANK results were demonstrated, the CyClus algorithm was designed to be more general and can be applied to a wide range of decoys and scoring functions by adjusting just two parameters, p and T. CyClus results were also compared to those from ClusPro. Proteins 2013. © 2013 Wiley Periodicals, Inc.
Article
It has become standard practice to compare new amino-acid and nucleotide sequences with existing ones in the rapidly growing sequence databases. This has led to the recurring identification of certain sequence patterns, usually corresponding to less than 300 amino-acids in length. Many of these identifiable sequence regions have been shown to fold up to form a ‘domain’ structure; they are often called protein ‘modules’ (see definitions below). Proteins that contain such modules are widely distributed in biology, but they are particularly common in extracellular proteins.
Article
A computationally tractable strategy has been developed to refine protein-protein interfaces that models the effects of side-chain conformational change, solvation and limited rigid-body movement of the subunits. The proteins are described at the atomic level by a multiple copy representation of side-chains modelled according to a rotamer library on a fixed peptide backbone. The surrounding solvent environment is described by "soft" sphere Langevin dipoles for water that interact with the protein via electrostatic, van der Waals and field-dependent hydrophobic terms. Energy refinement is based on a two-step process in which (1) a probability-based conformational matrix of the protein side-chains is refined iteratively by a mean field method. A side-chain interacts with the protein backbone and the probability-weighted average of the surrounding protein side-chains and solvent molecules. The resultant protein conformations then undergo (2) rigid-body energy minimization to relax the protein interface. Steps (1) and (2) are repeated until convergence of the interaction energy. The influence of refinement on side-chain conformation starting from unbound conformations found improvement in the RMSD of side-chains in the interface of protease-inhibitor complexes, and shows that the method leads to an improvement in interface geometry. In terms of discriminating between docked structures, the refinement was applied to two classes of protein-protein complex: five protease-protein inhibitor and four antibody-antigen complexes. A large number of putative docked complexes have already been generated for the test systems using our rigid-body docking program, FTDOCK. They include geometries that closely resemble the crystal complex, and therefore act as a test for the refinement procedure. In the protease-inhibitors, geometries that resemble the crystal complex are ranked in the top four solutions for four out of five systems when solvation is included in the energy function, against a background of between 26 and 364 complexes in the data set. The results for the antibody-antigen complexes are not as encouraging, with only two of the four systems showing discrimination. It would appear that these results reflect the somewhat different binding mechanism dominant in the two types of protein-protein complex. Binding in the protease-inhibitors appears to be "lock and key" in nature. The fixed backbone and mobile side-chain representation provide a good model for binding. Movements in the backbone geometry of antigens on binding represent an "induced-fit" and provides more of a challenge for the model. Given the limitations of the conformational sampling, the ability of the energy function to discriminate between native and non-native states is encouraging. Development of the approach to include greater conformational sampling could lead to a more general solution to the protein docking problem.
Article
A combination of two complementary approaches that are widely used to predict protein structure-homology and energy-based methods-is proposed. The algorithm is discussed in detail and is demonstrated to be a useful tool for optimization. The properties of the combination are exemplified on a simple model system: structural optimization of 2D heteropolymers on a square lattice. The algorithm is considerably more efficient than simulated annealing in the system studied. The relevance of the results of the protein folding problem is discussed.
Article
In this communication we describe an approach in which guanidine hydrochloride-induced dissociation of a protein inhibitor-serine protease complex is used to explore the molecular basis of protein denaturation. The rationale behind this approach is that the inhibitor-protease complex is stabilized by the same types of non-covalent interactions that stabilize the native state of a protein. The dissociation of inhibitor-protease complex can be performed at concentrations of guanidine hydrochloride at which the inhibitor and the protease retain their native conformations. Here, we present our results on the effect of 0.1M to 0.4M guanidine hydrochloride concentrations on the association equilibrium constants (reciprocal of dissociation constant) of P1G, P1A, P1V, P1N, and P1S variants of turkey ovomucoid third domain with bovine α-chymotrypsin. We use these results to calculate the free energy change in the dissociation of inhibitor-protease complexes (the m value) per mol of guanidine hydrochloride concentration. Our results agree with the general consensus that the denaturing effect of guanidine hydrochloride is due to its favorable interaction with the polar parts of proteins and that the non-polar side chains have no or little favorable interaction with guanidine hydrochloride.
Article
The central ingredient of any structure modeling tool is a molecular model or force field that accounts for proper geometry and energy calculation. For protein and peptide modeling based on entire amino acids as building blocks, we describe a peptide force field in which each amino acid is represented by a single point in space, taken at the position of the α-carbon atom. Apart from the positional coordinates, these units carry the two torsional angles φ and Ψ as additional degrees of freedom to account for the orientations of the peptide links. While some of the energy terms are analogous to expressions in atomic force fields, the presence of the angular variables leads to fundamental differences with new features and additional terms with no atomic counterparts. The force field reproduces secondary structure elements with very good accuracy. Globular parts of tertiary packing stay near the experimental structures with a rms deviation in C-α positions of 0.1–0.3 nm and about 25° in φ and Ψ depending on the size of the structure. A tendency for larger discrepancies is observed in exposed loops or terminal segments the conformations of which may be strongly influenced by neighboring domains. Finally, a scope of possible applications is presented. They range from modeling activities, such as model building by homology, to coarse scanning of conformation space in conformation analysis and structure determination. An extension to a dynamics model would offer the possibility to eliminate the less interesting high-frequency modes that in all-atom force-field dynamics absorb most of the computational effort.
Article
The crystal structure of the molecular complex formed by bovine -chymotrypsin and the recombinant serine proteinase inhibitor eglin c from Hirudo medicinalis has been solved using monoclinic crystals of the complex, reported previously. Four circle diffractometer data at 3.0 Å resolution were employed to determine the structure by molecular replacement techniques. Bovine -chymotrypsin alone was used as the search model; it allowed us to correctly orient and translate the enzyme in the unit cell and to obtain sufficient electron density for positioning the eglin c molecule. After independent rigid body refinement of the two complex components, the molecular model yielded a crystallographic R factor of 0.39. Five iterative cycles of restrained crystallographic refinement and model building were conducted, gradually increasing resolution. The current R factor at 2.6 Å resolution (diffractometer data) is 0.18. The model includes 56 solvent molecules. Eglin c binds to bovine -chymotrypsin in a manner consistent with other known serine proteinase/inhibitor complex structures. The reactive site loop shows the expected conformation for productive binding and is in tight contact with bovine -chymotrypsin between subsites P3 and P′2; Leu 45I acts as the P1 residue, located in the primary specificity S1 site of the enzyme. Hydrogen bonds equivalent to those observed in complexes of trypsin(ogen) with the pancreatic basic- and secretory- inhibitors are found around the scissile peptide bond.
Article
Network-editing experiments are variants of the basic NOESY experiment that allow more accurate direct measurement of interproton distances in macromolecules by defeating specific spin-diffusion pathways. Two network-editing approaches, block-decoupled NOESY and complementary-block-decoupled-NOESY, were applied as three-dimensional, heteronuclear-edited experiments to distance measurement in a small protein, turkey ovomucoid third domain (OMTKY3). Two-hundred and twelve of the original 655 distance constraints observed in this molecule (Krezel AM et al., 1994, J Mol Biol 242:203–214) were improved by their replacement by distances derived from network-edited spectra, and distance geometry/simulated annealing solution structure calculations were performed from both the unimproved and improved distance sets. The resulting two families of structures were found to differ significantly, the most important differences being the hinge angle of a β-turn and an expansion of the sampled conformation space in the region of the reactive-site loop. The structures calculated from network-editing data are interpreted as a more accurate model of the solution conformation of OMTKY3.
Thesis
The aims of the work presented in this thesis were two-fold. Firstly, an existing protein-protein docking algorithm was re-implemented on a type of computer more available than that used originally, and its behaviour was analysed in detail. This analysis led to changes in the scoring function, a treatment of electrostatic complementarity, and side-chain truncation. The algorithm had problems with its representation of surface, but more generally it pointed to difficulties in dealing with conformational change on association. Thus such changes were the second problem studied. They were measured in thirty-nine pairs of structures of complexed and unbound proteins, averaged over interface and non-interface regions and for individual residues. The significance of the changes was evaluated by comparison with the differences seen in twelve pairs of independently solved structures of identical proteins. Just over half had some substantial overall movement. Movements involved main-chains as well as side-chains, and large changes in the interface were closely involved with complex formation, while those of exposed non-interface residues were caused by flexibility and disorder. Interface movements in enzymes were similar in extent to those of inhibitors. All eight of the complexes that had structures of both components in an unbound form available showed some significant interface movement. An algorithm that was tested on five of these complexes was seen to be successful even when some of the largest changes occurred. The situation may be different in systems other than the enzyme-inhibitors which dominate this study. Thus the general model of protein-protein recognition was found to be induced fit. However, because there is only limited conformational change in many systems, recognition can be treated as lock and key to a first approximation.
Article
A molecular dynamics simulation using the OPLS nonbonded potential functions has been carried out for the third domain of silver pheasant ovomucoid in aqueous solution. Insights have been obtained on the quality of the force field, the convergence of such calculations, differences in the protein's structure in the crystal and in aqueous solution, protein hydration, and the dynamics of water molecules near a protein. The simulation covered 100 ps at 25°C, which allowed complete equilibration prior to averaging and analysis of the results. Continuous monitoring of the potential energy, root-mean-square deviations from the crystal structure, and other properties indicated that convergence to a stable structure was achieved after 30-40 ps. The RMS deviation of the instantaneous structure from the crystal structure after 100 ps is 1.43 Å for the backbone atoms of residues 8-56 and 1.61 Å for all residues. There is substantial reorganization of hydrogen bonds that do not involve secondary structure in comparing the crystal and solution structures, though in the simulation Ala-44 is displaced from the α-helix and Lys-29, Thr-30, and Gly-32 are moved out of hydrogen-bonding distance in the triple-stranded antiparallel β-sheet. Analyses of the protein-water hydrogen bonding were also carried out and are compared with results from previous simulations and NMR experiments.
Article
The structure of helices within proteins is often distorted from the ideal linear topology. Curvature of the helix axis can be measured by determining the radius of a circle fit to the axis. Described here is a method of defining a curved path that places backbone atoms (usually Cα) equidistantly from the path. The variance in the distance of backbone atoms from the helix axis is minimised to produce the parametric equations that describe the intersection of a sphere and a plane. The geometric properties of the helix (including helix radius, radius of curvature, and pitch) can be readily obtained from these equations. The approach is applicable to any form of helix, can use any atom in the peptide to determine the axis, can be applied to any polypeptide including mixed α/β peptides, and does not rely on a regular spacing of peptide monomers in the polypeptide chain.
Article
A search procedure is described for making stereospecific assignments at prochiral centers in proteins on the basis of nuclear Overhauser enhancement and coupling constant data derived from nmr experiments. A data base comprising torsion angles, associated 1H-1H coupling constants and interproton distances is searched by a computer algorithm for sets of values that match the experimental data within specified error limits. Two different data bases are used. The first is a crystallographic data base derived from 34 well-refined crystal structures; the second is a systematic data base derived from conformations of a short peptide fragment with idealized geometry by systematically varying the phi, psi, and chi 1 torsion angles. Both approaches are tested for beta-methylene groups with model data obtained from 20 crystal structures. The results for the two methods are similar though not identical, so that a combination of the two methods appears to be useful. With an appropriate choice of error estimates, around 80% of the beta-methylene groups could be assigned in the test calculations. In addition, results with experimental nmr data indicate that a similar percentage of stereospecific assignments can be made in practical situations.
Article
Full-text available
A method of structure-factor least-squares refinement of constrained groups linked by distance restraints has been developed for the refinement of macromolecular structures. Each constrained group can have any number of variable dihedral rotation parameters within the group in addition to the rigid-body translational, rotational and thermal parameters. The matrix of normal equations may be either full or sparse and provision is made for solution by matrix inversion or the conjugate-gradient iterative method. This procedure has been successfully used for 3 Å data and should be applicable even for lower-resolution data and especially for cases with a poorer data-per-atom ratio. The structure of yeast phenylalanine tRNA has been refined with this procedure from a starting crystallographic R value of 42% to a final R value of 25% with isotropic 'group" thermal parameters and 22% with isotropic atomic thermal parameters for 8207 independent reflections at 2.7 Å resolution. The proper stereochemistry of bond distances, angles and van der Waals contacts for the restrained atoms was maintained within reasonable limits throughout the refinement. Although originally developed for nucleic acids, this procedure is directly applicable to the refinement of protein structures. In addition, a combination of applying distance restraints between groups and least-squares fitting of these groups to target coordinates has been used purely as an idealization process for imposing proper stereochemistry on an approximate model.
Article
Full-text available
We have determined the crystal structure of the molecular complex between Streptomyces griseus protease B (SGPB), a bacterial serine protease, and the third domain of the ovomucoid inhibitor from turkey. Restrained-parameter least-squares refinement of the structure with the 1.8-A intensity data set has resulted in an R factor of 0.125. The carbonyl carbon atom of the reactive bond between Leu-18 and Glu-19 in the inhibitor lies at a distance of 2.71 A from the O gamma atom of the nucleophilic Ser-195 in SGPB; this distance is 0.5 A shorter than a normal van der Waals contact. Unlike the reactive bond in the pancreatic trypsin inhibitor complexed with bovine trypsin, the Leu--Glu bond of the ovomucoid inhibitor is not distorted from planarity towards a pyramidal configuration.
Article
Full-text available
Japanese quail ovomucoid exists in two polymorphic forms. One has serine, the other glycine at position 162. The tryptic peptide corresponding to positions 160 to 164 was purified from ovomucoids isolated from egg whites of eggs laid by 11 different hens and subjected to amino acid analysis. The quantitative distribution of serine and glycine in this pentapeptide is consistent with the interpretation that the ovomucoid gene exists in two codominant allelic forms at one locus. Even though the gene product is apparently expressed only in the female, these results indicate that the ovomucoid structural gene is transmitted as a simple Mendelian character which is neither sex-linked nor shows dominance. Intact third domains (positions 131 to 186) isolated from the two allelic forms of ovomucoid interact with bovine beta-trypsin in a similar but not identical manner; the complex with the glycine form dissociates more rapidly. Evidence is presented which suggests that glycine is the ancestral residue at position 162; yet, the serine form is the more frequent phenotype.
Chapter
Beyond the purely descriptive phases, long-term biochemical investigations may have algorithmic or mechanistic objectives. The ultimate hope of the algorithmic approach is the elucidation of the organismic characteristics from the nucleotide sequence of the genome (Fig. 1).
Article
The structure of the complex between the serine protease Streptomyces griseus protease B (SGPB) and the third domain of the Kazal-type ovomucoid inhibitor from turkey has been solved at 1.8-A resolution and refined to a conventional R factor of 0.125. As others have reported previously for analogous complexes of proteases and protein inhibitors, the inhibitor binds in a fashion similar to that of a substrate; it is not cleaved, but there is a close approach (2.7 A) of the active site nucleophile Ser-195 O gamma to the carbonyl carbon of the reactive peptide bond of the inhibitor. Contrary to the structural reports regarding the other enzyme-inhibitor complexes, we conclude that there is no evidence for a significant distortion of this peptide bond from planarity. The mechanism of inhibition can be understood in terms of the equilibrium thermodynamic parameters Ka, the enzyme-inhibitor association constant, and Khyd, the equilibrium constant for inhibitor hydrolysis. These thermodynamic parameters can be rationalized in terms of the observed structure.
Article
A model building and refinement system is described for use with a Vector General 3400 display. The system allows the user to build models using guide atoms and angles to arrive at the final conformation. It has been used to assist in difference Fourier map interpretation at medium and high resolution, and to build a protein molecule into a multiple isomorphous replacement phased electron density map.
Article
The experimental determination of the transmission surface, which represents an approximation to the transmission of X-rays in crystals, is described. Experimental transmission surfaces are compared with values calculated from the crystal shape. Also structural parameters of some crystal structures obtained from data with and without experimental absorption correction are compared.
Article
A new stereoelectronic theory for the cleavage of the tetrahedral intermediate in the hydrolysis of esters and amides is presented. In this new theory, the precise conformation of the intermediate hemi-orthoester or hemi-orthoamide controls the nature of the hydrolysis products. It is postulated that the breakdown of a conformer of a tetrahedral intermediate depends upon the orientation of the lone pair orbitals of the hetero-atoms. Specific cleavage of a carbon-oxygen or a carbon-nitrogen bond in any conformer is allowed only if the other two hetero-atoms (oxygen or nitrogen) each have an orbital oriented antiperiplanar to the leaving O-alkyl or N-alkyl group. Experimentally, the oxidation of acetals by ozone and the acid hydrolysis of a series of cyclic orthoesters demonstrates clearly that there is indeed a stereoelectronic control in the cleavage of hemiorthoesters. Similarly, a study of the basic hydrolysis of a variety of N,N-dialkylated imidate salts shows that the same stereoelectronic control is operating in the cleavage of hemiorthoamides.
Article
The Protein Data Bank is a computer-based archival file for macromolecular structures. The Bank stores in a uniform format atomic co-ordinates and partial bond connectivities, as derived from crystallographic studies. Text included in each data entry gives pertinent information for the structure at hand (e.g. species from which the molecule has been obtained, resolution of diffraction data, literature citations and specifications of secondary structure). In addition to atomic co-ordinates and connectivities, the Protein Data Bank stores structure factors and phases, although these latter data are not placed in any uniform format. Input of data to the Bank and general maintenance functions are carried out at Brookhaven National Laboratory. All data stored in the Bank are available on magnetic tape for public distribution, from Brookhaven (to laboratories in the Americans), Tokyo (Japan), and Cambridge (Europe and worldwide). A master file is maintained at Brookhaven and duplicate copies are stored in Cambridge and Tokyo. In the future, it is hoped to expand the scope of the Protein Data Bank to make available co-ordinates for standard structural types (e.g. α-helix, RNA double-stranded helix) and representative computer programs of utility in the study and interpretation of macromolecular structures.
Article
The Protein Data Bank is a computer-based archival file for macromolecular structures. The Bank stores in a uniform format atomic co-ordinates and partial bond connectivities, as derived from crystallographic studies. Text included in each data entry gives pertinent information for the structure at hand (e.g. species from which the molecule has been obtained, resolution of diffraction data, literature citations and specifications of secondary structure). In addition to atomic co-ordinates and connectivities, the Protein Data Bank stores structure factors and phases, although these latter data are not placed in any uniform format. Input of data to the Bank and general maintenance functions are carried out at Brookhaven National Laboratory. All data stored in the Bank are available on magnetic tape for public distribution, from Brookhaven (to laboratories in the Americas), Tokyo (Japan), and Cambridge (Europe and worldwide). A master file is maintained at Brookhaven and duplicate copies are stored in Cambridge and Tokyo. In the future, it is hoped to expand the scope of the Protein Data Bank to make available co-ordinates for standard structural types (e.g. alpha-helix, RNA double-stranded helix) and representative computer programs of utility in the study and interpretation of macromolecular structures.
Article
The reverse turn, involving four consecutive amino acids, as a tertiary conformation in globular proteins is defined in terms of dihedral angles, the C(1) (alpha)...C(4) (alpha) distance and the O(1)...H-N(4) hydrogen bond distance. In seven proteins we find 125 examples of turns, comprising 33% of the amino acids in these proteins, as compared with 34% of the residues forming helices and only 17% forming beta-sheets. The amino-acid compositions of turns, helices, and beta-sheets are analyzed in some detail. We find Asn and Gly mainly in turns, Pro in turns (and at the beginning of helices), and Glu in helices. In these turns a statistical survey indicates that 19% of Asp residues are in the first position, 33% of Pro residues are in the second position, 24% of Asn residues are in the third position, and 26% of Trp residues are in the fourth position.
Article
A program is described for drawing the van der Waal's surface of a protein molecule. An extension of the program permits the accessibility of atoms, or groups of atoms, to solvent or solute molecules of specified size to be quantitatively assessed. As defined in this study, the accessibility is proportional to surface area. The accessibility of all atoms in the twenty common amino acids in model tripeptides of the type Ala-X-Ala are given for defined conformation. The accessibilities are also given for all atoms in ribonuclease-S, lysozyme and myogoblin. Internal cavities are defined and discussed. Various summaries of these data are provided. Forty to fifty per cent of the surface area of each protein is occupied by non-polar atoms. The actual numerical results are sensitive to the values chosen for the van der Waal's radii of the various groups. Since there is uncertainty over the correct values for these radii, the derived numbers should only be used as a qualitative guide at this stage.The average change in accessibility for the atoms of all three proteins in going from a hypothetical extended chain to the folded conformation of the native protein is about a factor of 3. This number applies to both polar (nitrogen and oxygen) and non-polar (carbon and sulfur) atoms considered separately. The larger non-polar amino acids tend to be more “buried” in the native form of all three proteins. However, for all classes and for residues within a given class the accessibility changes on folding tend to be highly variable.
Article
The structure of the complex between the serine protease Streptomyces griseus protease B (SGPB) and the third domain of the Kazal-type ovomucoid inhibitor from turkey has been solved at 1.8-A resolution and refined to a conventional R factor of 0.125. As others have reported previously for analogous complexes of proteases and protein inhibitors, the inhibitor binds in a fashion similar to that of a substrate; it is not cleaved, but there is a close approach (2.7 A) of the active site nucleophile Ser-195 O gamma to the carbonyl carbon of the reactive peptide bond of the inhibitor. Contrary to the structural reports regarding the other enzyme-inhibitor complexes, we conclude that there is no evidence for a significant distortion of this peptide bond from planarity. The mechanism of inhibition can be understood in terms of the equilibrium thermodynamic parameters Ka, the enzyme-inhibitor association constant, and Khyd, the equilibrium constant for inhibitor hydrolysis. These thermodynamic parameters can be rationalized in terms of the observed structure.
Article
Japanese quail ovomucoid third domain (OMJPQ3), a Kazal-type inhibitor, was crystallographically refined with energy constraints. The final R-value is 0.20 at 1.9 Å resolution. The four molecules in the asymmetric unit are very similar, with deviations of main-chain atoms between 0.2 and 0.3 Å. An analysis of the side-chain hydrogen-bonding pattern and amino acid variability in the Kazal family shows a high correlation between hydrogen-bonding and conservation.The conformation of the reactive site loop (P2-P2′) of OMJPQ3 is similar to those of basic pancreatic trypsin inhibitor, Streptomyces subtilisin inhibitor, and soybean trypsin inhibitor. This suggests a common binding mode and justifies model-building studies of complexes.Complexes of OMJPQ3 with trypsin, chymotrypsin and elastase were modelled on the basis of the trypsin-basic pancreatic trypsin inhibitor complex structure and inspected by use of a computer graphics system. Stereochemically satisfying models were constructed in each case and detailed interactions are proposed. The complex with elastase is of particular interest, showing that leucine and methionine are good P1 residues. A good correlation is observed between functional properties of ovomucoid variants and the position of the exchanged residues with respect to the modelled inhibitor-protease contact.
Article
Sequence determinations in our laboratory have yielded the primary structures of ovomucoid third domains from 35 avian species. From that list, 12 sequences could be arranged into a contiguous set such that each sequence differs from a second by a single amino acid replacement. For this set of domains and for five additional domains of special interest, we report here the association equilibrium constants for their binding with bovine alpha-chymotrypsin, elastase I, and subtilisin Carlsberg. The results are interpreted with the aid of the three-dimensional structure of highly homologous Japanese quail ovomucoid third domain and of computer-generated models of the complexes of the inhibitor with the respective enzymes. The results show that (i) changes in inhibitor residues other than the primary recognition residue (P1) even sequentially far from the reactive site, may exert large effects on association equilibrium constant values provided these residues make contact with the enzyme, (ii) changes in residues other than P1 often exert large differential effects toward the different enzymes, i.e., the same change can make the inhibitor stronger for one enzyme and weaker for another, (iii) the sign and to some extent the magnitude of the changes can be rationalized from the known structures of the inhibitor and the enzyme, (iv) changes in surface residues which do not contact the enzyme in complex are virtually without effect, and (v) glycosylated and nonglycosylated inhibitors have the same constants. For confirmation of the validity of the equilibrium constant comparisons in a few cases, the rate constants kon and kd were determined and the resultant calculated equilibrium constant values compared to the directly determined numbers. An additional test of validity is provided by experiments where a glycosylated domain of one species is allowed to compete with an unglycosylated domain of another for the same enzyme.
Article
The three-dimensional structure of the proteic complex formed by bovine trypsinogen and the porcine pancreatic secretory trypsin inhibitor (Kazal type) has been solved by means of Patterson search techniques, using a predicted model of the trypsin-ovomucoid complex (Papamokos et al., 1982). The structure of the complex, including 162 solvent molecules, has been refined at 1.8 Å resolution (26,341 unique reflections) to a conventional crystallographic R factor of 0.195. The inhibitor molecule binds to trypsinogen via hydrogen bonds and/or apolar interactions at sites P9, P7, P6, P5, P3, P1, P1′, P2′ and P3′ of the contact area. The structure of the inhibitor itself resembles closely that of the third domain of Japanese quail ovomucoid inhibitor, recently reported by Weber et al. (1981). The trypsinogen part of the complex resembles trypsin, as is the case in the trypsinogen-basic pancreatic trypsin inhibitor complex, but two segments of the activation domain adopt a different conformation. Most notably in the N-terminal region the Ile16-Gly19 loop, which is disordered in free trypsinogen and in the trypsinogen-basic pancreatic trypsin inhibitor complex (Huber & Bode, 1978), assumes a regular structure and the polypeptide chain can be traced as far as residue Asp14. This new and fixed structure allows the formation of a buried salt link between the side-chains of Lys156 and Asp194. Conformations differing from those of trypsin are also found for residues 20 to 28 and residues 141 to 155. Some structural perturbation is observed in other parts of the molecule, including the calcium loop.
Article
The third domain of Japanese quail ovomucoid, a Kazal type inhibitor, has been crystallized and its crystal structure determined at 2.5 Å resolution using multiple isomorphous replacement techniques. The asymmetric unit contains four molecules. In the crystal the molecules are arranged in two slightly different octamers with approximate D4 symmetry. The molecules are held together mainly by interactions of the N-terminal residues, which form a novel secondary structural element, a β-channel.The molecule is globular with approximate dimensions 35 Å × 27 Å × 19 Å. The secondary structural elements are a double-stranded anti-parallel β-sheet of residues Pro22 to Gly32 and an α-helix from Asn33 to Ser44. The reactive site Lys18-Asp19 is located in an exposed loop. It is close to Asn33 at the N terminus of the helical segment. The polypeptide chain folding of ovomucoid bears some resemblance to other inhibitors in the existence of an anti-parallel double strand following the reactive site loop.
  • M Bolognesi
  • G Gatti
  • E Menegatti
  • M Guarneri
  • M Marquart
  • E Papamokos
  • R Huber
Bolognesi, M., Gatti, G., Menegatti, E., Guarneri, M., Marquart, M., Papamokos, E. & Huber, R. (1982) J. Mol. Biol. 162,
  • P Deslongcharnps
Deslongcharnps, P. (1975) Tetrahedron 31,2463 -2490.
  • I Schechter
  • A Berger
Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Cornrnun.
  • M W Empie
  • M Laskowski
  • Jr
Empie, M. W. & Laskowski, M., Jr (1982) Biochemistry 21,
  • A Jack
  • M Levitt
Jack, A. & Levitt, M. (1978) Acta Crystallogr. A34.931-935.
  • F C Bernstein
  • T F Koetzle
  • G J B Williams
  • E F Meyer
  • M D Brice
  • J R Rogers
  • O Kennard
  • T Shimanouchi
  • M Tasumi
Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F., Brice, M. D., Rogers, J. R., Kennard, O., Shimanouchi, T. & Tasumi, M. (1977) J. Mol. Biol. 112, 535-5542, 27,157-162. 839-868. 2274-2284.
  • M Fujinaga
  • R J Read
  • A Sielecki
  • W Ardelt
  • M Laskowski
  • M N G James
Fujinaga, M., Read, R. J., Sielecki, A., Ardelt, W., Laskowski, M., Jr & James, M. N. G. (1982) Proc. Natl Acad. Sci. USA 79,4868-4872.
  • T A Jones
Jones, T. A. (1978) J. Appl. Crystallogr. 11,268-272.
  • J L Crawford
  • W N Lipscomb
  • C G Schellrnan
Crawford, J. L., Lipscomb, W. N. & Schellrnan, C. G. (1973) Proc. Natl Acad. Sci. USA 70, 538 -542.
  • M Laskowski
  • Jr
  • I Kato
  • W C Kohr
  • C J March
  • W C Bogard
Laskowski, M., Jr, Kato, I., Kohr, W. C., March, C. J. & Bogard, W. C. (1980) Protides Biol. Fluids Proc. Colloq. 28, 123-128.