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NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive‐site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)‐III of the squash family and comparison with those of counterparts of CMTI‐V of the potato I family
Abstract
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.
... The average S 2 value, <S 2 >, for free MCoTI-I was 0.83 ± 0.03. This value similar to that found for the six Cys residues involved in the cystine-knot (<S 2 > = 0.84 ± 0.02) and is considerably larger than those found for other linear squash trypsin inhibitors (<S 2 > = 0.71 for trypsin inhibitor from Cucurbita maxima (CMTI-III, 78% homology with MCoTI-I) [22] thus indicating the importance of the backbone cyclization to rigidify the overall structure. Loops 2 through 5 in free MCoTI-I showed <S 2 > values ≥ 0.8. ...
... Residue Leu 6 in loop 1 also required chemical exchange terms to be considered indicating the existence of intramolecular conformational exchange on the μs-ms time scale. The mobility observed in loop 1 at both ns-ps and ms time scales have been also described in other trypsin inhibitors [22,24,25] and it has been suggested to play an important role in receptor-ligand binding. [11] To explore if that was the case in the MCoTI cyclotides, we next studied the effect of ligandbinding on the backbone dynamics of MCoTI-I (Figs. 1 and 2). ...
... The average S 2 value, <S 2 >, for free MCoTI-I was 0.83 ± 0.03. This value similar to that found for the six Cys residues involved in the cystine-knot (<S 2 > = 0.84 ± 0.02) and is considerably larger than those found for other linear squash trypsin inhibitors (<S 2 > = 0.71 for trypsin inhibitor from Cucurbita maxima (CMTI-III, 78% homology with MCoTI-I) [22] thus indicating the importance of the backbone cyclization to rigidify the overall structure. Loops 2 through 5 in free MCoTI-I showed <S 2 > values ≥ 0.8. ...
... Residue Leu 6 in loop 1 also required chemical exchange terms to be considered indicating the existence of intramolecular conformational exchange on the μs-ms time scale. The mobility observed in loop 1 at both ns-ps and ms time scales have been also described in other trypsin inhibitors [22,24,25] and it has been suggested to play an important role in receptor-ligand binding. [11] To explore if that was the case in the MCoTI cyclotides, we next studied the effect of ligandbinding on the backbone dynamics of MCoTI-I (Figs. 1 and 2). ...
Showing some backbone: Most of the backbone NH groups of cyclotide MCoTI-I are constrained in the free state, which results in a well-folded compact structure, as indicated by {15N,1H} NMR spectroscopy (see picture). According to the backbone order parameter S2, the backbone mobility in trypsin-bound MCoTI-I is significantly increased.
... This view is based on the observation that X-ray structures of several free and complexed inhibitors show little structural differences in their protease binding loop, as measured by the deviation of backbone dihedral angles [3]. However, heteronuclear NMR studies have revealed that the interaction site of canonical protease inhibitors in complex with the enzyme is flexible on the ps-ns timescale [5][6][7][8][9]. These observations are in line with recent experimental and theoretical results emphasizing the role of internal dynamics in protein function in general [10][11][12] and are in apparent contradiction with the rigid lock-and-key model widely accepted for canonical serine protease inhibitors. ...
... Therefore, we propose that conformational changes inevitably occur upon enzyme binding, in contrast to the lock-and-key mechanism. Solution-state NMR and computational studies on a variety of unrelated canonical inhibitors corroborate this finding [5][6][7][8][9]25]. ...
The efficiency of canonical serine protease inhibitors is conventionally attributed to the rigidity of their protease binding loop with no conformational change upon enzyme binding, yielding an example of the lock-and-key model for biomolecular interactions. However, solution-state structural studies revealed considerable flexibility in their protease binding loop. We resolve this apparent contradiction by showing that enzyme binding of small, 35-residue inhibitors is actually a dynamic conformer selection process on the nanosecond-timescale. Thus, fast timescale dynamics enables the association rate to be solely diffusion-controlled just like in the rigid-body model.
... An increasing amount of structural data indicates that a ¯exible proteinase binding loop is not an uncommon feature of proteinase inhibitors. The reported solution structure of the Cucurbita maxima P1 trypsin inhibitor V (CMTI-V) [31] and R-ela®n [32], as well as experimental and theoretical studies of backbone dynamics of a recombinant form of CMTI-V [33,34], CMTI-III [34] and chymotrypsin inhibitor 2 [35,36] provide the evidence for various serine proteinase inhibitors . Thus, our results indicating the somewhat increased ¯exibility of the proteinase-binding loop compared to the rest of the molecules agree well with the ®ndings obtained from recently determined NMR structures of other proteinase inhibitors. ...
... An increasing amount of structural data indicates that a ¯exible proteinase binding loop is not an uncommon feature of proteinase inhibitors. The reported solution structure of the Cucurbita maxima P1 trypsin inhibitor V (CMTI-V) [31] and R-ela®n [32], as well as experimental and theoretical studies of backbone dynamics of a recombinant form of CMTI-V [33,34], CMTI-III [34] and chymotrypsin inhibitor 2 [35,36] provide the evidence for various serine proteinase inhibitors . Thus, our results indicating the somewhat increased ¯exibility of the proteinase-binding loop compared to the rest of the molecules agree well with the ®ndings obtained from recently determined NMR structures of other proteinase inhibitors. ...
The solution structure of three small serine proteinase inhibitors, two natural and one engineered protein, SGCI (Schistocerca gregaria chymotrypsin inhibitor), SGCI[L30R, K31M] and SGTI (Schistocerca gregaria trypsin inhibitor), were determined by homonuclear NMR-spectroscopy. The molecules exhibit different specificities towards target proteinases, where SGCI is a good chymotrypsin inhibitor, its mutant is a potent trypsin inhibitor, and SGTI inhibits both proteinases weakly. Interestingly, SGTI is a much better inhibitor of insect proteinases than of the mammalian ones used in common assays. All three molecules have a similar fold composed from three antiparallel beta-pleated sheets with three disulfide bridges. The proteinase binding loop has a somewhat distinct geometry in all three peptides. Moreover, the stabilization of the structure is different in SGCI and SGTI. Proton-deuterium exchange experiments are indicative of a highly rigid core in SGTI but not in SGCI. We suggest that the observed structural properties play a significant role in the specificity of these inhibitors.
... The binding loop includes the amino acid residues between positions 40 and 49. The scissile bond is formed by Lys44-Asp45 (Table 4) [159,161]. ...
Coagulation factor XII (FXII), a S1A serine protease, was discovered more than fifty years ago. However, its in vivo functions and its three-dimensional structure started to be disclosed in the last decade. FXII was found at the crosstalk of several physiological pathways including the intrinsic coagulation pathway, the kallikrein-kinin system, and the immune response. The FXII inhibition emerges as a therapeutic strategy for the safe prevention of artificial surface-induced thrombosis and in patients suffering from hereditary angioedema. The anti-FXII antibody garadacimab discovered by phage-display library technology is actually under phase II clinical evaluation for the prophylactic treatment of hereditary angioedema. The implication of FXII in neuro-inflammatory and neurodegenerative disorders is also an emerging research field. The FXII or FXIIa inhibitors currently under development include peptides, proteins, antibodies, RNA-based technologies, and, to a lesser extent, small-molecular weight inhibitors. Most of them are proteins, mainly isolated from hematophagous arthropods and plants. The discovery and development of these FXII inhibitors and their potential indications are discussed in the review.
A study was conducted to demonstrate the use of NMR as a as a tool to determine the structures, folding behavior, dynamics, and interactions of plant proteins. Investigations revealed that one-dimensional (1D) spectra were useful for rapid characterization of the folding or structure of smaller proteins. Correct folding was associated with sharp spectral lines and good dispersion of the amide signals or the observation of some upfield-shifted methyl groups. Upfield or down field dispersion of the α-H shifts provided a measure of helical or β-sheet secondary structure. A quick measure of the folding of proteins was also obtained from the pattern of chemical shifts in two-dimensional (2D) HSQC spectra for larger proteins. This was conveniently done with 15N-labeled samples, but the sensitivity of modern instruments equipped with cryo-probes allowed 2D HSQC spectra to be recorded for natural abundance samples.
Protease inhibitors (PIs) are present in a wide variety of living organisms including microorganisms, plants, and animals. These molecules act as regulators of endogenous proteolytic activity, as participants in many developmental processes and as host's defense components. Because of their important role in living organisms, PIs have been extensively studied in order to allow a better understanding of their structural and functional properties. Intensive works have reported that proteinaceous PIs are potential compounds in several distinct economical and clinical fields. This review aims to describe proteinaceous PIs structure and design, focusing on their multiple potential applications in agriculture; immune system; parasitic, bacterial, fungal, and viral infection; hemostasis; and cancer.
Plants are consumed by bacteria, fungi and animals and utilization of plant proteins requires their hydrolysis, a process which is catalyzed by proteases. Plants defend themselves against other organisms by elaborating physical and chemical defenses, the latter including protein and non-protein inhibitors of proteases. Plant protease inhibitor proteins can be either constitutive or inducible as a result of wounding or pathogen invasion. Proteases are classified into the aspartic proteases, cysteine proteases, metalloproteases and serine proteases on the basis of the involvement of aspartate, cysteine, metal ions and serine, respectively, in the catalytic mechanism. Extracellular proteases are involved in digestion, blood clotting, inflammatory responses to invasion, and extracellular matrix digestion required for angiogenesis and tissue remodelling. Intracellular proteolysis must be exquisitely regu to avoid autolysis and intracellular proteases are involved in proprotein processing, lysosome- and proteasome-mediated protein destruction, cell division and apoptosis. A large variety of plant protease inhibitor proteins and peptides have been resolved including aspartic protease inhibitor proteins, cysteine protease inhibitory phytocystatins, metallocarboxypeptidase inhibitor proteins and serine protease inhibitor proteins such as the Bowman-Birk, cereal bifunctional, Kunitz, potato type I, potato type II, mustard family, squash family, serpin and other protease inhibitors. Plant protease inhibitor proteins have potential transgenic applications for crop plant defense. A variety of non-protein protease inhibitors have also been resolved from plants. Plant protease inhibitors have potential for pharmaceutical development especially in relation to Alzheimer's disease, angiogenesis, cancer, inflammatory disease and viral and protozoal infection.
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.
Cyclization via head-to-tail linkage of the termini of a peptide chain occurs in only a small percentage of proteins, but engenders the resultant cyclic proteins with exceptional stability. The mechanisms involved are poorly understood and this review attempts to summarize what is known of the events that lead to cyclization. Cyclic proteins are found in both prokaryotic and eukaryotic species. The prokaryotic circular proteins include the bacteriocins and pilins. The eukaryotic circular proteins in mammals include the theta defensins, found in rhesus macaques, and the retrocyclins. Two types of cyclic proteins have been found in plants, the sunflower trypsin inhibitor and the larger, more prolific, group known as cyclotides. The cyclotides from Oldenlandia affinis, the plant in which these cyclotides were first discovered, are processed by an asparaginyl endopeptidase which is a cysteine protease. Cysteine proteases are commonly associated with transpeptidation reactions, which, for suitable substrates can lead to cyclization events. These proteases cleave an amide bond and form an acyl enzyme intermediate before nucleophilic attack by the amine group of the N-terminal residue to form a peptide bond, resulting in a cyclic peptide. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 573–583, 2010.
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4-C41 and C8-C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were individually removed and replaced by alanine residues. Trypsin inhibition assays show that wild-type T1 has a K(i) of <5 nM, C4A/C41A-T1 has a weaker K(i) of approximately 350 nM, and the potency of the C8A/C37A variant is further decreased to a K(i) of approximately 1.8 microM. To assess the influence of the disulfide bonds on the structure of T1, we determined the structure and dynamics of both disulfide variants by NMR spectroscopy. The structure of C4A/C41A-T1 and the amplitude of intrinsic flexibility in the reactive-site loop resemble that of the wild-type protein closely, despite the lack of the C4-C41 disulfide bond, whereas the timescale of motions is markedly decreased. The rescue of the structure despite loss of a disulfide bond is due to a previously unrecognized network of interactions, which stabilizes the structure of the reactive-site loop in the region of the missing disulfide bond, while allowing intrinsic motions on a fast (picosecond-nanosecond) timescale. In contrast, no comparable interactions are present around the C8-C37 disulfide bond. Consequently, the reactive-site loop becomes disordered and highly flexible in the structure of C8A/C37A-T1, making it unable to bind to trypsin. Thus, the reactive-site loop of T1 is stabilized differently by the C8-C37 and C4-C41 disulfide bonds. The C8-C37 disulfide bond is essential for the inhibitory activity of T1, whereas the C4-C41 disulfide bond is not as critical for maintaining the three-dimensional structure and function of the molecule but is responsible for maintaining flexibility of the reactive-site loop on a microsecond-nanosecond timescale.
Three peptides modelling a highly potent, 35-residue chymotrypsin inhibitor (Schistocerca gregaria chymotrypsin inhibitor) were designed and synthesized by convergent peptide synthesis. For each model peptide, the inhibitory constant (Ki) on chymotrypsin and the solution structure were determined. In addition, molecular dynamics calculations were performed for all of them. Two models containing approximately half of the parent inhibitor (17 of 35 residues) were designed and subsequently found to have no substantial inhibitory activity (Ki values in the mM range). The third model composed of 24 amino acid residues proved to be an effective (Ki approximately 10(-7)) inhibitor of bovine chymotrypsin. Both the solution structure properties determined by NMR spectroscopy and the dynamic behaviour of the latter model system are comparable to the native inhibitor. In contrast, the structure and dynamics of the first two related model peptides show characteristic differences. We suggest that the conformation and flexibility of the modelled protease inhibitor are crucial for its biological efficiency. Moreover, the structural and dynamic features of the binding loop (28-33) and those of the rest of the molecule appear to be interdependent. Most importantly, these structural characteristics can be rationally modified, at least partially, by peptide design.
SGCI (Schistocerca gregaria chymotrypsin inhibitor) and SGTI (Sch. gregaria trypsin inhibitor) are small, 35-residue serine protease inhibitors with intriguing taxon specificity: SGTI is specific for arthropod proteases while SGCI is an excellent inhibitor on both mammalian and arthropodal enzymes. Here we report the cloning, expression, and (15)N backbone dynamics investigations of these peptides. Successful expression could be achieved by a "dimeric" construct similar to the natural precursor of the inhibitors. An engineered methionine residue between the two modules served as a unique cyanogen bromide cleavage site to cleave the precursor and physically separate SGCI and SGTI. The overall correlation time of the precursor (5.29 ns) as well as the resulted SGCI (3.14 ns) and SGTI (2.96 ns) are as expected for proteins of this size. General order parameters (S(2)) for the inhibitors are lower than those characteristic of well-folded proteins. Values in the binding loop region are even lower. Interestingly, the distribution of residues for which a chemical exchange (R(ex)) term should be considered is strikingly different in SGCI and SGTI. Together with H-D exchange studies, this indicates that the internal dynamics of the two closely related molecules differ. We suggest that the dynamic properties of these inhibitors is one of the factors that determine their specificity.
The structure of the Bowman-Birk inhibitor from Vigna unguiculata seeds (BTCI) in complex with beta-trypsin was solved and refined at 1.55 A to a crystallographic R(factor) of 0.154 and R(free) of 0.169, and represents the highest resolution for a Bowman-Birk inhibitor structure to date. The BTCI-trypsin interface is stabilized by hydrophobic contacts and hydrogen bonds, involving two waters and a polyethylene glycol molecule. The conformational rigidity of the reactive loop is characteristic of the specificity against trypsin, while hydrophobicity and conformational mobility of the antichymotryptic subdomain confer the self-association tendency, indicated by atomic force microscopy, of BTCI in complex and free form. When BTCI is in binary complexes, no significant differences in inhibition constants for producing a ternary complex with trypsin and chymotrypsin were detected. These results indicate that binary complexes present no conformational change in their reactive site for both enzymes confirming that these sites are structurally independent. The free chymotrypsin observed in the atomic force microscopy assays, when the ternary complex is obtained from BTCI-trypsin binary complex and chymotrypsin, could be related more to the self-association tendency between chymotrypsin molecules and the flexibility of the reactive site for this enzyme than to binding-related conformational changes.
This paper describes the application of recently developed nuclear magnetic resonance (NMR) pulse sequences to obtain information about the internal dynamics of isotopically enriched hydrophobic side chains in proteins. The two-dimensional spectra provided by the pulse sequences enable one to make accurate measurements of nuclear Overhauser effects (NOE) and longitudinal (T1) and transverse (T2) relaxation times of enriched methyl carbons in proteins. Herein, these techniques are used to investigate the internal dynamics of the 11 leucine side chains of staphylococcal nuclease (SNase), a small enzyme having Mr = 16.8K, in the absence and presence of ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+. We report the synthesis of [5,5'-13C2]leucine, the preparation of SNase containing the labeled leucine, the sequential assignment of the leucine methyl carbons and protons in the liganded and unliganded proteins, and the measurement of the 13C T1, T2, and NOE values for the SNase leucine methyl carbons. Analysis of the relaxation parameters using the formalism of Lipari and Szabo shows that the internal motions of the leucine methyl carbons are characterized by effective correlation times tau f (5-80 ps) and tau s (less than 2 ns). The fast motion is identified with the rapid rotation of the methyl group about the C gamma-C delta bond axis, while the slow motion is associated with reorientation of the C gamma-C delta bond axis itself. The mean squared order parameters associated with the latter motion, Ss2, lie in the range 0.34-0.92. The values of Ss2 correlate reasonably well with the temperature factors of the leucine methyl carbons obtained from the crystal structures, but some are smaller than anticipated on the basis of the fact that nearly all leucine methyl carbons are buried and have temperature factors no larger than that of the leucine backbone atoms. Five leucine residues in liganded SNase and eight in unliganded SNase have values of Ss2 less than 0.71. These order parameters correspond to large amplitude motions (angular excursions of 27-67 degrees) of the C gamma-C delta bond axis. These results indicate that, in solution, the internal motions of the leucine side chains of SNase are significantly larger than suggested by the X-ray structures or by qualitative analysis of NOESY spectra. Comparison of Ss2 values obtained from liganded and unliganded SNase reveals a strong correlation between delta Ss2 and distance between the leucine methyl carbon and the ligands.(ABSTRACT TRUNCATED AT 400 WORDS)
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.
The study of protein internal motions from analysis of 13C and 15N NMR relaxation data and auto- and cross-correlation spectral densities is being pursued in many labs. Model-free approaches and derived motional order parameters are normally used to interpret NMR relaxation data and internal mobility in proteins and peptides. Correlated motions can substantially modify the behavior of NMR auto- and cross-correlation spectral density functions and the values of derived motional order parameters. Here, a simple model is proposed to describe small amplitude (less than about 60° or 1 rad), internally restricted correlated rotations (IRCR) in peptides and proteins in order to analyze order parameters. Bond rotations are represented by vectors whose motions are correlated by a correlation coefficient, cij, which is the cosine of the angle between these vectors. Order parameters for NH, CαH and CβH bond motions have been calculated from molecular dynamics simulations performed on short peptides with well-defined α-helix and β-sheet structures in order to derive values for cij. General equations relating dipolar auto- and cross-correlation order parameters for CαH, CβH, and NH bonds to cij have been derived. The sign of cij depends on the specific motional correlation within a particular molecular conformation. For glycine φ, ψ rotations, the sign of cij can be derived from analysis of dipolar auto- and cross-correlation order parameters. Long-range motional correlations are observed with hydrogen bonds modulating internal mobility. In general, backbone NH order parameters, S2NH, are more sensitive to structure than are CαH order parameters, S2CH. S2CH is increased and S2NH is decreased when correlation coefficients cψ‘φ and cφψ are decreased and increased, respectively.
This paper describes the use of novel two-dimensional nuclear magnetic resonance (NMR) pulse sequences to provide insight into protein dynamics. The sequences developed permit the measurement of the relaxation properties of individual nuclei in macromolecules, thereby providing a powerful experimental approach to the study of local protein mobility. For isotopically labeled macromolecules, the sequences enable measurement of heteronuclear nuclear Overhauser effects (NOE) and spin-lattice (Tâ) and spin-spin (Tâ) ¹âµN or ¹³C relaxation times with a sensitivity similar to those of many homonuclear ¹H experiments. Because Tâ values and heteronuclear NOEs are sensitive to high-frequency motions (10â¸-10¹²sâ»Â¹) while Tâ values are also a function of much slower processes, it is possible to explore dynamic events occurring over a large time scale. The authors have applied these techniques to investigate the backbone dynamics of the protein staphylococcal nuclease (S. Nase) complexed with thymidine 3â²,5â²-bisphosphate (pdTp) and Ca{sup 2+} and labeled uniformly with ¹âµN. Tâ, Tâ, and NOE values were obtained for over 100 assigned backbone amide nitrogens in the protein. Values of the order parameter (S), characterizing the extent of rapid ¹H-¹âµN bond motions, have been determined. These results suggest that there is no correlation between these rapid small amplitude motions and secondary structure for S. Nase. In contrast, ¹âµN line widths suggest a possible correlation between secondary structure and motions on the millisecond time scale. In particular, the loop region between residues 42 and 56 appears to be considerably more flexible on this slow time scale than the rest of the protein.
Structural biology has made important contributions to the understanding of biological processes. In recent years an increasing amount of structural information has also been derived from NMR spectroscopic studies, often with special emphasis on dynamic aspects. The introduction of three‐ and four‐dimensional techniques has greatly simplified protein structure determination by NMR Spectroscopy, which has in fact become routine. In the past it was more of an art to interpret the complicated NOESY spectra of proteins, but the application of three‐dimensional techniques now makes the interpretation of protein spectra straightforward.
In this review we discuss the most important multidimensional NMR techniques along with suitable applications. The emphasis is put less on the discussion of individual pulse sequences than on their application to the structure determination of proteins.
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The zinc finger DNA-binding domain Xfin-31 is a 25-residue peptide that binds a single zinc atom and forms a compact globular structure in solution. To characterize the intramolecular dynamics of Xfin-31, the C-13 spin-lattice and spin-spin relaxation rate constants and the {H-1}-C-13 nuclear Overhauser effect (NOE) enhancements have been measured for the backbone and side chain methine carbons by two-dimensional proton-detected heteronuclear NMR spectroscopy at a C-13 Larmor frequency of 125 MHz and natural C-13 abundance. The relaxation rate constants and the NOE enhancements have been analyzed by using a model-free formalism that depends on the overall rotational correlation time of the molecule, tau-m, the order parameter S, and effective internal correlation time, tau-e, for each methine carbon. The optimized global value of tau-m is 1.88 +/- 0.02 ns. The backbone C-alpha carbons are grouped into four categories based on the values of the order parameters: the N-terminal residue Tyr1 with S2 = 0.73 +/- 0.04; the C-terminal residues Lys24 and Asn25 with S2 < 0.5; residues Phe10-Lysl3 with an average S2 = 0.77 +/- 0.03; and the remainder of the backbone carbon nuclei with an average S2 = 0.89 +/- 0.05. For the side chain C-beta of Val11 and Val22 and the C-gamma of Leu5, the values of S2 are 0.62 +/- 0.03, 0.66 +/- 0.04, and 0.47 +/- 0.03, respectively. Estimates of tau-e could be obtained for 13 of the backbone and 3 of the side chain methine carbons. Excluding the terminal residues, the average value of tau-e for the backbone carbon nuclei was 34 +/- 16 ps. With the exception of the terminal residues, the motions of the backbone carbon nuclei of Xfin-31 are highly restricted. Residues 10-13, which form a turn between the beta-sheet and the helix present in the three-dimensional structure of Xfin-31, have a slightly higher mobility than the rest of the interior backbone, and the two residues at the C-terminus have considerable conformational flexibility. The side chains of the hydrophobic Val and Leu residues are more mobile than the backbone but are still significantly restricted, which indicates that Xfin-31 is compact despite its small size. Systematically large spin-spin relaxation rates for residues in the zinc binding site imply that conformational exchange may occur in this region on a time scale longer than the overall rotational correlation time.
The solution structure of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI-V), whose N-terminal is unacetylated and carries an extra glycine residue, was determined by means of two-dimensional (2D) homo and 3D hetero NMR experiments in combination with a distance geometry and simulated annealing algorithm. A total of 927 interproton distances and 123 torsion angle constraints were utilized to generate 18 structures. The root mean squared deviation (RMSD) of the mean structure is 0.53 Å for main-chain atoms and 0.95 Å for all the non-hydrogen atoms of residues 3−40 and 49−67. The average structure of rCMTI-V is found to be almost the same as that of the native protein [Cai, M., Gong, Y., Kao, J.-L., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201−5211]. The backbone dynamics of uniformly 15N-labeled rCMTI-V were characterized by 2D 1H−15N NMR methods. 15N spin−lattice and spin−spin relaxation rate constants (R1 and R2, respectively) and {1H}−15N steady-state heteronuclear Overhauser effect enhancements were measured for the peptide NH units and, using the model-free formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546−4559, 4559−4570], the following parameters were determined: overall tumbling correlation time for the protein molecule (τm), generalized order parameters for the individual N−H vectors (S2), effective correlation times for their internal motions (τe), and terms to account for motions on a slower time scale (second) due to chemical exchange and/or conformational averaging (Rex). Most of the backbone NH groups of rCMTI-V are found to be highly constrained (S2 = 0.83) with the exception of those in the binding loop (residues 41−48, S2 = 0.71) and the N-terminal region (S2 = 0.73). Main-chain atoms in these regions show large RMSD values in the average NMR structure. Residues involved in turns also appear to have more mobility (S2 = 0.80). Dynamical properties of rCMTI-V were compared with those of two other inhibitors of the potato I familyeglin c [Peng, J. W., & Wagner, G. (1992) Biochemistry 31, 8571−8586] and barley chymotrypsin inhibitor 2 [CI-2; Shaw, G. L., Davis, B., Keeler, J., & Fersht, A. R. (1995) Biochemistry 34, 2225−2233]. The Cys3−Cys48 linkage found only in rCMTI-V appears to somewhat reduce the N-terminal flexibility; likewise, the C-terminal of rCMTI-V, being part of a β-sheet, appears to be more rigid.
In the preceding paper it has been shown that the unique dynamic information on fast internal motions in an NMR relaxation experiment on macromolecules in solution is specified by a generalized order parameter, 8, and an effective correlation time, 7,. This paper deals with the extraction and interpretation of this information. The procedure used to obtain S2 and T, from experimental data by using a least-squares method and, in certain favorable circumstances, by using an analytical formula is described. A variety of experiments are then analyzed to yield information on the time scale and spatial restriction of internal motions of isoleucines in myoglobin, methionines in dihydrofolate reductase and myoglobin, a number of aliphatic residues in basic pancreatic trypsin inhibitor, and ethyl isocyanide bound to myoglobin, hemoglobin, and aliphatic side chains in three random-coil polymers. The numerical values of S2 and 7, can be readily interpreted within the framework of a variety of models. In this way, one can obtain the same physical picture of internal motions as that obtained by using complicated spectral densities to fit the data. The numerical value of the order parameter, unlike the effective correlation time T,, plays a crucial role in determining what models can be used to describe the experiment; models in which the order parameter cannot be reproduced are eliminated. Conversely, any model that can yield the correct value of S works.
A spin echo method adapted to the measurement of long nuclear relaxation times (T 2 ) in liquids is described. The pulse sequence is identical to the one proposed by Carr and Purcell, but the rf of the successive pulses is coherent, and a phase shift of 90° is introduced in the first pulse. Very long T 2 values can be measured without appreciable effect of diffusion.
An investigation has been made into several aspects of the acquisition and processing of NMR data used to obtain heteronuclear relaxation parameters in proteins. Pulse sequences are described that reduce the time required to acquire relaxation data by a factor of two without sacrificing precision or accuracy of the results. In addition, a number of factors that influence the accuracy and precision of measured relaxation parameters, including uncertainties in peak intensities, overlap between resonances, and amide-proton exchange with solvent, are examined theoretically and experimentally. The results provide guidelines for maximizing the reliability of investigations of spin relaxation in proteins.
A new approach to the interpretation of nuclear magnetic resonance relaxation experiments on macromolecules in solution is presented. This paper deals with the theoretical foundations and establishes the range of validity of this approach, and the accompanying paper demonstrates how a wide variety of experimental relaxation data can be successfully analyzed by using this approach. For both isotropic and anisotropic overall motion, it is shown that the unique imformation on fast internal motions contained in relaxation experiments can be completely specified by two model-independent quantities; (1) a generalized order parameter, S, which is a measure of the spatial restriction of the motion, and (2) an effective correlation time, T/sub e/, which is a measure of the rate of motion. A simple expression for the spectral density involving these two parameters is derived and is shown to be exact when the internal (but not overall) motions are in the extreme narrowing limit. The model-free approach (so called because S² and T/sub e/ have model-independent significance) consists of using the above spectral density to least-squares fit relaxation data by treating S² and T/sub e/ as adjustable parameters. The range of validity of this approach is illustrated by analyzing error-free relaxation data generated by using sophisticated dynamical models. Empirical rules are presented that allow one to estimate the of S² and T/sub e/ extracted by using the model-free approach by considering their numerical values, the resonance frequencies, and the parameters for the overall motion. For fast internal motions, it is unnecessary to use approaches based on complicated spectral densities derived within the framework of a model because all models that can give the correct value of S² work equally well.
Thesis (M.S.)--Kansas State University, 1992. Includes bibliographical references (leaves 85-91).
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.
Sequence-specific hydrogen-1 NMR assignments were made to all of the 29 amino acid residues of reactive-site-hydrolyzed Cucurbita maxima trypsin inhibitor I (CMTI-I*) by the application of two-dimensional NMR (2D NMR) techniques, and its secondary structural elements (two tight turns, a 3(10)-helix, and a triple-stranded beta-sheet) were identified on the basis of short-range NOESY cross peaks and deuterium-exchange kinetics. These secondary structural elements are present in the intact inhibitor [Holak, T. A., Gondol, D., Otlewski, J., & Wilusz, T. (1989) J. Mol. Biol. 210, 635-648] and are unaffected by the hydrolysis of the reactive-site peptide bond between Arg5 and Ile6, in accordance with the earlier conclusion reached for CMTI-III* [Krishnamoorthi, R., Gong, Y.-X., Lin, C. S., & VanderVelde, D. (1992) Biochemistry 31, 898-904]. Chemical shifts of backbone hydrogen atoms, peptide NH's, and C alpha H's, of CMTI-I* were compared with those of the intact inhibitor, CMTI-I, and of the reactive-site-hydrolyzed, natural, E9K variant, CMTI-III*. Cleavage of the Arg5-Ile6 peptide bond resulted in changes of chemical shifts of most of the backbone atoms of CMTI-I, in agreement with the earlier results obtained for CMTI-III. Comparison of chemical shifts of backbone hydrogen atoms of CMTI-I* and CMTI-III* revealed no changes, except for residues Glu9 and His25. However, the intact forms of the same two proteins, CMTI-I and CMTI-III, showed small but significant perturbations of chemical shifts of residues that made up the secondary structural elements of the inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)
15N NMR assignments were made to the backbone amide nitrogen atoms at natural isotopic abundance of intact and reactive-site (Arg5-Ile6) hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III and CMTI-III*, respectively) by means of 2D proton-detected heteronuclear single bond chemical shift correlation (HSBC) spectroscopy, utilizing the previously made sequence-specific 1H NMR assignments (Krishnamoorthi et al. (1992) Biochemistry 31, 898-904). Comparison of the 15N chemical shifts of the two forms of the inhibitor molecule revealed significant changes not only for residues located near the reactive-site region, but also for those distantly located. Residues Cys3, Arg5, Leu7, Met8, Cys10, Cys16, Glu19, His25, Tyr27, Cys28 and Gly29 showed significant chemical shift changes ranging from 0.3 to 6.1 ppm, thus indicating structural perturbations that were transmitted throughout the molecule. These findings confirm the earlier conclusions based on 1H NMR investigations.
The solution structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III*) was investigated by two-dimensional proton nuclear magnetic resonance (2D NMR) spectroscopy. CMTI-III*, prepared by reacting CMTI-III with trypsin which cleaved the Arg5-Ile6 peptide bond, had the two fragments held together by a disulfide linkage. Sequence-specific 1H NMR resonance assignments were made for all the 29 amino acid residues of the protein. The secondary structure of CMTI-III*, as deduced from NOESY cross peaks and identification of slowly exchanging hydrogens, contains two turns (residues 8-12 and 24-27), a 3(10)-helix (residues 13-16), and a triple-stranded beta-sheet (residues 8-10, 29-27, and 21-25). This secondary structure is similar to that of CMTI-I [Holak, T. A., Gondol, D., Otlewski, J., & Wilusz, T. (1989) J. Mol. Biol. 210, 635-648], which has a Glu instead of a Lys at position 9. Sequential proton assignments were also made for the virgin inhibitor, CMTI-III, at pH 4.71, 30 degrees C. Comparison of backbone hydrogen chemical shifts of CMTI-III and CMTI-III* revealed significant changes for residues located far away from the reactive-site region as well as for those located near it, indicating tertiary structural changes that are transmitted through most of the 29 residues of the inhibitor protein. Many of these residues are functionally important in that they make contact with atoms of the enzyme in the trypsin-inhibitor complex, as revealed by X-ray crystallography [Bode, W., Greyling, H. J., Huber, R., Otlewski, J., & Wilusz, T. (1989) FEBS Lett. 242, 285-292].(ABSTRACT TRUNCATED AT 250 WORDS)
Tetragonal and triclinic crystals of two ovomucoid inhibitor third domains from silver pheasant and Japanese quail, modified at their reactive site bonds Met18Glu19 (OMSVP3∗) and Lys18Asp19 (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.
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.
More than 30 years ago it seemed that the forces that cause inactive, newly formed proteins to fold into their intricate, active state could be explained by the laws of chemistry and physics. But scientists are still unable to predict how a sequence of amino acids will coil. Solutions to the folding problem-with their implications for biotechnology-are getting nearer.
The squash inhibitors of serine proteinases have been discovered as proteins, which inhibit the catalytic activity of bovine trypsin. In this report we show, that three human enzymes of trypsin-like specificity - i.e. plasmin, plasma kallikrein and thrombin - are also inhibited by squash inhibitors. Moreover, rather strong inhibition was demonstrated for human cathepsin G. Lower association constants were found for Streptomyces griseus proteinase B (SGPB) and subtilisin BPN'. No association was detected for bovine chymotrypsin, even at millimolar concentrations of the inhibitors. Porcine pancreatic elastase showed extremely weak inhibition by squash inhibitors. Most of the enzymes examined did not exhibit a clear discrimination between P1 Arg and P1 Lys inhibitors. However, human plasma kallikrein and human thrombin formed much stronger complexes with CMTI I (P1-Arg) than with CPTI II (P1-Lys).
A protein inhibitor (CMTI-V; Mr 7106) of trypsin and activated Hageman factor (Factor XIIa), a serine protease involved in blood coagulation, has been isolated for the first time from pumpkin (Cucurbita maxima) seeds by means of trypsin-affinity chromatography and reverse phase high performance liquid chromatography (HPLC). The dissociation constants of the inhibitor complexes with trypsin and Factor XIIa have been determined to be 1.6 x 10(-8) and 4.1 x 10(-8) M, respectively. The primary structure of CMTI-V is reported. The protein has 68 amino acid residues and one disulfide bridge and shows a high level of sequence homology to the Potato I inhibitor family. Furthermore, its amino terminus consists of an N-acetylates Ser. The reactive site has been established to be the peptide bond between Lys44-Asp45. The modified inhibitor which has the reactive site peptide bond hydrolyzed inhibits trypsin but not the Hageman factor.
Many inhibitors of trypsin and human beta-factor XIIa have been isolated from squash and related seeds and sequenced (Wieczorek et al., Biochem. Biophys. Res. Comm. (1985) 126, 646-652). The association equilibrium constants (Ka) of several of these inhibitors have now been determined with human beta-factor XIIa using a modification of the method of Green and Work (Park et al., Fed. Proc. Fed. Am. Soc. Exp. Biol. (1984) 43, 1962). The Ka's range from 7.8 x 10(4) M-1 to 3.3 x 10(8) M-1. Two isoinhibitors from Cucurbita maxima seeds, CMTI-I and CMTI-III, differ in only a single glutamate to lysine change in the P'4 position. This results in a factor of 62 increase in the Ka of the lysine inhibitor, CMTI-III (Ka = 3.3 x 10(8) M-1). To our knowledge, this is the largest effect ever seen for a residue substitution at the P'4 position of a serine proteinase inhibitor. The result is even more surprising because beta-factor XIIa's natural substrate, Factor XI, contains Gly in the P'4 position.
A comparison of the solution nuclear magnetic resonance (n.m.r.) structures of squash trypsin inhibitor from seeds of the squash Cucurbita maxima with the X-ray structure of a trypsin complex of the inhibitor shows that the n.m.r. and X-ray structures are similar in terms of the global folding and secondary structure. The average atomic root-mean-square difference between the 36 n.m.r. structures on the one hand and the X-ray structure is 0.96 A for the backbone atoms and 1.95 A for all heavy atoms. The n.m.r. and X-ray structures exhibit extremely similar conformations of the primary proteinase binding loop. Despite the overall similarity, there are small differences between the mean computed structure and the X-ray structure. The n.m.r. structures have slightly different positions of the segments from residues 16 to 18, and 24 and 25. The n.m.r. results show that the inclusion of stereospecific assignments and precise distance constraints results in a significant improvement in the definition of the n.m.r. structure, making possible a detailed analysis of the local conformations in the protein.
The complete three-dimensional structure of the trypsin inhibitor from seeds of the squash Cucurbita maxima in aqueous solution was determined on the basis of 324 interproton distance constraints, 80 non-nuclear Overhauser effect distances, and 22 hydrogen-bonding constraints, supplemented by 27 phi backbone angle constraints derived from nuclear magnetic resonance measurements. The nuclear magnetic resonance input data were converted to the distance constraints in a semiquantitative manner after a sequence specific assignment of 1H spectra was obtained using two-dimensional nuclear magnetic resonance techniques. Stereospecific assignments were obtained for 17 of the 48 prochiral centers of the squash trypsin inhibitor using the floating chirality assignment introduced at the dynamical simulated annealing stage of the calculations. A total of 34 structures calculated by a hybrid distance geometry-dynamical simulated annealing method exhibit well-defined positions for both backbone and side-chain atoms. The average atomic root-mean-square difference between the individual structures and the minimized mean structure is 0.35(+/- 0.08) A for the backbone atoms and 0.89(+/- 0.17) A for all heavy atoms. The precision of the structure determination is discussed and correlated to the experimental input data.
Novel peptide inhibitors of human leukocyte elastase (HLE) and cathepsin G (CG) were prepared by solid-phase peptide synthesis of P1 amino acid sequence variants of Curcurbita maxima trypsin inhibitor III (CMTI-III), a 29-residue peptide found in squash seed. A systematic study of P1 variants indicated that P1, Arg, Lys, Leu, Ala, Phe, and Met inhibit trypsin; P1, Val, Ile, Gly, Leu, Ala, Phe, and Met inhibit HLE; P1 Leu, Ala, Phe, and Met inhibit CG and chymotrypsin. Variants with P1, Val, Ile, or Gly were selective inhibitors of HLE, while inhibition of trypsin required P1 amino acids with an unbranched {beta} carbon. Studies of Val-5-CMTI-III (P1 Val) inhibition of HLE demonstrated a 1:1 binding stoichiometry with a (K{sub i}){sub app} of 8.7 nM. Inhibition of HLE by Gly-5-CMTI-III indicated a significant role for reactive-site structural moieties other than the P1 side chain. Val-5-CMTI-III inhibited both HLE and human polymorphonuclear leukocyte (PMN) proteolysis of surface-bound {sup 125}I-labeled fibronectin. Val-5-CMTI-III was more effective at preventing turnover of a peptide p-nitroanilide substrate than halting dissolution of {sup 125}I-labeled fibronectin. It was about as effective as human serum {alpha}{sub 1}-proteinase inhibitor in preventing PMN degradation of the connective tissue substrate. In addition to providing interesting candidates for controlling inflammatory cell proteolytic injury, the CMTI-based inhibitors are ideal for studying molecular recognition because of their small size, their ease of preparation, and the availability of sensitive and quantitative assays for intermolecular interactions.
The stoichiometric complex formed between bovine beta-trypsin and the Cucurbita maxima trypsin inhibitor I (CMTI-I) was crystallized and its X-ray crystal structure determined using Patterson search techniques. Its structure has been crystallographically refined to a final R value of 0.152 (6.0-2.0 A). CMTI-I is of ellipsoidal shape; it lacks helices or beta-sheets, but consists of turns and connecting short polypeptide stretches. The disulfide pairing is CYS-3I-20I, Cys-10I-22I and Cys-16I-28I. According to the polypeptide fold and disulfide connectivity its structure resembles that of the carboxypeptidase A inhibitor from potatoes. Thirteen of the 29 inhibitor residues are in direct contact with trypsin; most of them are in the primary binding segment Val-2I (P4)-Glu-9I (P4') which contains the reactive site bond Arg-5I-Ile-6I and is in a conformation observed also for other serine proteinase inhibitors.
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].
Six amino acid sequences for trypsin inhibitors isolated from squash, summer squash, zucchini, and cucumber seeds were determined. All these inhibitors along with the two previously sequenced squash inhibitors (1) form the squash inhibitor family. The striking characteristic of the family is that its member inhibitors are very small (29-32 residues, 3 disulfide bridges). The association equilibrium constants with bovine beta trypsin for 6 squash family inhibitors were determined and range from 5.9 X 10(10) to 9.5 X 10(11) M-1.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
A strong inhibitor of human Hageman factor fragment (HFf, beta-factor XIIa) and bovine trypsin was isolated from pumpkin (Cucurbita maxima) seed extracts by acetone fractionation, by chromatography on columns of diethyl-aminoethylcellulose and carboxylmethyl-Sephadex C-25, and by Sephadex G-50 gel filtration. Pumpkin seed Hageman factor inhibitor (PHFI) is unusual in its lack of inhibition of several other serine proteinases tested--human plasma, human urinary, and porcine pancreatic kallikreins, human alpha-thrombin, and bovine alpha-chymotrypsin. Human plasmin and bovine factor Xa are only weakly inhibited. PHFI also inhibits the HFf-dependent activation of plasma prekallikrein and clotting of plasma. Other properties of PHFI are a pI of 8.3, 29 amino acid residues, amino-terminal arginine, carboxyl-terminal glycine, 3 cystine residues, undetectable sulfhydryl groups and carbohydrate, and arginine at the reactive site. The minimum molecular weight of PHFI is 3268 by amino acid analysis. PHFI may be the smallest protein inhibitor of trypsin known.
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.
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.
Reactive-site (Lys44-Asp45 peptide bond) hydrolyzed Cucurbita maxima trypsin inhibitor-V (CMTI-V*) was prepared and characterized: In comparison to the intact form, CMTI-V* exhibited markedly reduced inhibitory properties and binding affinities toward trypsin and human blood coagulation factor XIIa. The equilibrium constant of trypsin-catalyzed hydrolysis, Khyd, defined as [CMTI-V*]/[CMTI-V], was measured to be approximately 9.4 at 25 degrees C (delta G degrees = -1.3 kcal.mol-1). From the temperature dependence of delta G degrees, the following thermodynamic parameters were estimated: delta H degrees = 1.6 kcal.mol-1 and delta S degrees = 9.8 eu. In order to understand the functional and thermodynamic differences between the two forms, the three-dimensional solution structure of CMTI-V* was determined by a combined approach of NMR, distance geometry, and simulated annealing methods. Thus, following sequence-specific and stereospecific resonance assignments, including those of beta-, gamma-, delta-, and epsilon-hydrogens and valine methyl hydrogens, 809 interhydrogen distances and 123 dihedral angle constraints were determined, resulting in the computation and energy-minimization of 20 structures for CMTI-V*. The average root mean squared deviation in position for equivalent atoms between the 20 individual structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.15 A for the main chain atoms and 1.19 +/- 0.23 A for all the non-hydrogen atoms of residues 5-40 and residues 48-67.(ABSTRACT TRUNCATED AT 250 WORDS)