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ABSTRACT: An approach towards accurate NMR measurements of deuterium isotope effects on the chemical shifts of all backbone nuclei in proteins ((15)N, (13)Cα, (13)CO, (1)Hα) and (13)Cβ nuclei arising from (1)H-to-D substitutions at amide nitrogen positions is described. Isolation of molecular species with a defined protonation/deuteration pattern at successive backbone nitrogen positions in the polypeptide chain allows quantifying all deuterium isotope shifts of these nuclei from the first to the fourth order. Some of the deuterium isotope shifts measured in the proteins ubiquitin and GB1 can be interpreted in terms of backbone geometry via empirical relationships describing their dependence on (φ; ψ) backbone dihedral angles. Because of their relatively large variability and notable dependence on the protein secondary structure, the two- and three-bond (13)Cα isotope shifts, (2)ΔCα(NiD) and (3)ΔCα(Ni+1D), and three-bond (13)Cβ isotope shifts, (3)ΔCβ(NiD), are useful reporters of the local geometry of the protein backbone.
Journal of Biomolecular NMR 04/2013; · 3.61 Impact Factor
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ABSTRACT: Thrombin is the pivotal serine protease enzyme in the blood cascade system and thus a target of drug design for control of its activity. The most efficient non-physiologic inhibitor of thrombin is hirudin, a naturally occurring small protein. Hirudin and its synthetic mimics employ a range of hydrogen bonding, salt bridging and hydrophobic interactions with thrombin to achieve tight binding with Ki values in the nano- to femtomolar range. The one-dimensional 1H NMR spectrum carried out at 600 MHz reveals a resonance at 15.33 ppm downfield from silanes in complexes between human α-thrombin and r-hirudin in pH 5.6-8.8 buffers and between 5 and 35 °C. There is also a resonance between 15.17 and 15.54 ppm seen in human α-thrombin complexes with hirunorm IV, hirunorm V, an Nα(Me)Arg-peptide, RGD-hirudin and Nα-2-naphthylsulfonyl-glycyl-DL-4-amidinophenylalanyl-piperidide acetate salt (NAPAP), while there is no such low-field resonance observed in a complex of porcine trypsin and NAPAP. The chemical shifts suggest that these resonances represented H-bonded environments. H-donor acceptor distances in the corresponding H-bonds are estimated to be <2.7 Å. Addition of Phe-Pro-Arg-Chloromethylketone (PPACK) to a complex of human α-thrombin with r-hirudin results in an additional signal at 18.03 ppm, which is 0.10 ppm upfield from one observed (Kovach, I. M. et al. Biochemistry 2009, 48, 7296-7304) for thrombin covalently modified with PPACK. In contrast, the peak at 15.33 ppm remains unchanged. The fractionation factors for the thrombin-hirudin type complexes are near 1.0 within 20% error. The most likely site of the short H-bond in thrombin complexes with the hirudin family of inhibitors is in the hydrophobic patch of the C-terminus of hirudin where Glu57' and Glu58' are embedded and interact with Arg75 and Arg77 and their solvate water (on thrombin). Glu57' and Glu58' present in the hirudin family of inhibitors is a key binding epitope of fibrinogen, thrombin's prime substrate, which lends substantial interest to the SHB as a binding element at the fibrinogen recognition site.
Biochemistry 03/2013; · 3.42 Impact Factor
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ABSTRACT: Ubiquitin (Ub) is a small protein highly conserved among eukaryotes and involved in practically all aspects of eukaryotic cell biology. Polymeric chains assembled from covalently-linked Ub monomers function as molecular signals in the regulation of a host of cellular processes. Our previous studies have shown that the predominant state of Lys48-linked di- and tetra-Ub chains at near-physiological conditions is a closed conformation, in which the Ub-Ub interface is formed by the hydrophobic surface residues of the adjacent Ub units. Because these very residues are involved in (poly)Ub interactions with the majority of Ub-binding proteins, their sequestration at the Ub-Ub interface renders the closed conformation of polyUb binding incompetent. Thus the existence of open conformation(s) and the interdomain motions opening and closing the Ub-Ub interface is critical for the recognition of Lys48-linked polyUb by its receptors. Knowledge of the conformational properties of a polyUb signal is essential for our understanding of its specific recognition by various Ub-receptors. Despite their functional importance, open states of Lys48-linked chains are poorly characterized. Here we report a crystal structure of the open state of Lys48-linked di-Ub. Moreover, using NMR, we examined interactions of the open state of this chain (at pH4.5) with a Lys48-linkage-selective receptor, the UBA2 domain of a shuttle protein hHR23a. Our results show that di-Ub binds UBA2 in the same mode and with comparable affinity as the closed state. Our data suggest a mechanism for polyUb signal recognition, whereby Ub-binding proteins select specific conformations out of the available ensemble of polyUb chain conformations. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.
Biochimica et Biophysica Acta 04/2012; 1823(11):2046-56. · 4.66 Impact Factor
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ABSTRACT: Substrates tagged with (poly)ubiquitin for degradation can be targeted directly to the 26 S proteasome where they are proteolyzed. Independently, ubiquitin conjugates may also be delivered by bivalent shuttles. The majority of shuttles attach to the proteasome through a ubiquitin-like domain (UBL) while anchoring cargo at a C-terminal polyubiquitin-binding domain(s). We found that two shuttles of this class, Rad23 and Dsk2, dock at two different receptor sites embedded within a single subunit of the 19 S proteasome regulatory particle, Rpn1. Their association/dissociation constants and affinities for Rpn1 are similar. In contrast, another UBL-containing protein, the deubiquitinase Ubp6, is also anchored by Rpn1, yet it dissociates slower, thus behaving as an occasional proteasome subunit that is distinct from the transiently associated shuttles. Two neighboring subunits, Rpn10 and Rpn13, show a marked preference for polyubiquitin over UBLs. Rpn10 attaches to the central solenoid portion of Rpn1, although this association is stabilized by the presence of a third subunit, Rpn2. Rpn13 binds directly to Rpn2. These intrinsic polyubiquitin receptors may compete with substrate shuttles for their polyubiquitin-conjugate cargos, thereby aiding release of the emptied shuttles. By binding multiple ubiquitin-processing factors simultaneously, Rpn1 is uniquely suited to coordinate substrate recruitment, deubiquitination, and movement toward the catalytic core. The broad range of affinities for ubiquitin, ubiquitin-like, and non-ubiquitin signals by adjacent yet nonoverlapping sites all within the base represents a hub of activity that coordinates the intricate relay of substrates within the proteasome, and consequently it influences substrate residency time and commitment to degradation.
Journal of Biological Chemistry 02/2012; 287(18):14659-71. · 4.77 Impact Factor
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ABSTRACT: As a signal for substrate targeting, polyubiquitin meets various layers of receptors upstream to the 26S proteasome. We obtained structural information on two receptors, Rpn10 and Dsk2, alone and in complex with (poly)ubiquitin or with each other. A hierarchy of affinities emerges with Dsk2 binding monoubiquitin tighter than Rpn10 does, whereas Rpn10 prefers the ubiquitin-like domain of Dsk2 to monoubiquitin, with increasing affinities for longer polyubiquitin chains. We demonstrated the formation of ternary complexes of both receptors simultaneously with (poly)ubiquitin and found that, depending on the ubiquitin chain length, the orientation of the resulting complex is entirely different, providing for alternate signals. Dynamic rearrangement provides a chain-length sensor, possibly explaining how accessibility of Dsk2 to the proteasome is limited unless it carries a properly tagged cargo. We propose a mechanism for a malleable ubiquitin signal that depends both on chain length and combination of receptors to produce tetraubiquitin as an efficient signal threshold.
Molecular cell 12/2009; 36(6):1018-33. · 14.61 Impact Factor
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ABSTRACT: The ubiquitin-like domain (UBL) of yeast protein Dsk2p is widely believed to recognize and bind to ubiquitin receptors on the proteasome and, as part of Dsk2p, to bridge polyubiquitinated substrates and proteasomal degradation machinery. Here we report NMR resonance assignment for (1)H, (15)N, and (13)C nuclei in the backbone and side chains of the UBL domain of Dsk2p. This assignment will aid in NMR studies focused on understanding of Dsk2's interactions with proteasomal receptors and its role as a polyubiquitin shuttle in the ubiquitin-dependent proteasomal degradation as well as other cellular pathways.
Biomolecular NMR Assignments 01/2009; 2(2):147-9. · 0.72 Impact Factor
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ABSTRACT: A sensitive 2D NMR experiment for simultaneous time-shared TROSY-type detection of amide and methyl groups in high-molecular-weight proteins is described. The pulse scheme is designed to preserve the slowly decaying components of both 1H-15N and methyl 13CH3 spin systems in the course of indirect evolution and acquisition periods. The proposed methodology is applied to the study of substrate binding to {U-[15N,2H]; Ile-[13CH3]; Leu,Val-[13CH3/12CD3]}-labeled 82-kDa enzyme Malate Synthase G and is expected to accelerate NMR-based screening of large proteins labeled with 15N and selectively labeled with 13CH3 at methyl sites.
Journal of the American Chemical Society 08/2008; 130(33):10872-3. · 9.91 Impact Factor
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ABSTRACT: Hydrolytic reactions of oligopeptide 4-nitroanilides catalyzed by human-alpha-thrombin, human activated protein C and human factor Xa were studied at pH 8.0-8.4 and 25.0+/-0.1 degrees C by the progress curve method and individual rate constants were calculated mostly within 10% internal error using DYNAFITV. A systematic strategy has been developed for fitting a three-step consecutive mechanism to eighteen hundred to six thousand time-course data points polled from two to four independent kinetic experiments. Enzyme and substrate concentrations were also calculated. Individual rate constants well reproduce published values obtained under comparable conditions and the Michaelis-Menten kinetic parameters calculated from these elementary rate constants are also within reasonable limits of published values. For comparison, the integrated Michaelis-Menten equation was also fitted to data from twelve sets. Both the k(cat) and k(cat)/K(m) values are within 15% agreement with those calculated using the elementary rate constants obtained with DYNAFITV. Rate constants for the second and third consecutive steps are within 3-4 fold indicating that both determine the overall rate. The Factor Xa-catalyzed hydrolysis of N-alpha-Z-D-Arg-Gly-Arg-pNA.2HCl at pH 8.4 in a series of buffers containing increasing fractions of deuterium at 25.0+/-0.1 degrees C shows a very strong dependence of k(3) and a moderate dependence of k(2) on D content in the buffer: the fractionation factors are: 0.49+/-0.03 for K(1,) 0.70+/-0.05 for k(2), and (0.32+/-0.03)(2) for k(3).
Biochimica et Biophysica Acta 06/2008; 1784(5):827-33. · 4.66 Impact Factor
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ABSTRACT: Ubiquilin/PLIC proteins belong to the family of UBL-UBA proteins implicated in the regulation of the ubiquitin-dependent proteasomal degradation of cellular proteins. A human presenilin-interacting protein, ubiquilin-1, has been suggested as potential therapeutic target for treating Huntington's disease. Ubiquilin's interactions with mono- and polyubiquitins are mediated by its UBA domain, which is one of the tightest ubiquitin binders among known ubiquitin-binding domains. Here we report the three-dimensional structure of the UBA domain of ubiquilin-1 (UQ1-UBA) free in solution and in complex with ubiquitin. UQ1-UBA forms a compact three-helix bundle structurally similar to other known UBAs, and binds to the hydrophobic patch on ubiquitin with a K(d) of 20 microM. To gain structural insights into UQ1-UBA's interactions with polyubiquitin chains, we have mapped the binding interface between UQ1-UBA and Lys48- and Lys63-linked di-ubiquitins and characterized the strength of UQ1-UBA binding to these chains. Our NMR data show that UQ1-UBA interacts with the individual ubiquitin units in both chains in a mode similar to its interaction with mono-ubiquitin, although with an improved binding affinity for the chains. Our results indicate that, in contrast to UBA2 of hHR23A that has strong binding preference for Lys48-linked chains, UQ1-UBA shows little or no binding selectivity toward a particular chain linkage or between the two ubiquitin moieties in the same chain. The structural data obtained in this study provide insights into the possible structural reasons for the diversity of polyubiquitin chain recognition by UBA domains.
Journal of Molecular Biology 04/2008; 377(1):162-80. · 4.00 Impact Factor
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ABSTRACT: Due to their RNA-N-glycosidase activity, ribosome-inactivating proteins (RIPs) are attractive candidates as antitumor and antiviral agents in biomedical and agricultural research. We have isolated and characterized two such proteins, foetidissimin II and texanin, from two Cucurbitaceae species. Foetidissimin II, obtained from the roots of Cucurbita foetidissima, was identified as a type-2 RIP, with a molecular weight of 61 kDa, as estimated by gel electrophoresis. It is composed of two chains, a 29-kDa chain A, and a 32-kDa chain B. Texanin, isolated from the fruits of Cucurbita texana, is a type-I RIP, with a single chain of molecular weight 29.7 kDa, as estimated by MALDI-TOF-MS. Both proteins exhibit RNA-N-glycosidase activity, with aniline playing a critical role in rRNA cleavage. The IC50 value of foetidissimin II, determined by cell-free protein-synthesis inhibition, was 0.251 muM. In an in vitro cytotoxicity assay, foetidissimin II exhibited IC50 values of ca. 70 nM to both adenocarcinoma and erythroleukemia cells. Texanin exhibited a weaker anticancer activity against erythroleukemia cells, with an IC50 value of 95 microM, but no activity against adenocarcinoma cells. The N-terminal sequences of both proteins were compared with those of reported RIPs.
Chemistry & Biodiversity 04/2007; 4(3):431-42. · 1.80 Impact Factor
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ABSTRACT: Kinetic solvent isotope effects (KSIEs) for the factor Xa (FXa)-catalyzed activation of prothrombin in the presence and absence of factor Va (FVa) and 5.0 x 10(-5) M phospholipid vesicles are slightly inverse, 0.82-0.93, when substrate concentrations are at 0.2 Km. This is consistent with the rate-determining association of the enzyme-prothrombin assembly, rather than the rate-limiting chemical transformation. FVa is known to effect a major conformational change to expose the first scissile bond in prothrombin, which is the likely event triggering a major solvent rearrangement. At prothrombin concentrations > 5 Km, the KSIE is 1.6 +/- 0.3, when FXa is in a 1:1 ratio with FVa but becomes increasingly inverse, 0.30 +/- 0.05 and 0.19 +/- 0.04, when FXa/FVa is 1:4, with an increasing FXa and substrate concentration. The rate-determining step changes with the conditions, but the chemical step is not limiting under any circumstance. This corroborates the proposed predominance of the meizothrombin pathway when FXa is well-saturated with the prothrombin complex. In contrast, the FXa-catalyzed hydrolysis of N-alpha-Z-D-Arg-Gly-Arg-pNA.2HCl (S-2765) and H-D-Ile-L-Pro-L-Arg-pNA.HCl (S-2288) is most consistent with two-proton bridges forming at the transition state between Ser195 OgammaH and His57 N(epsilon)2 and His57 Ndelta1 and Asp102 COObeta- at the active site, with transition-state fractionation factors of phi1 = phi2 = 0.57 +/- 0.07 and phiS = 0.78 +/- 0.16 for solvent rearrangement for S-2765 and phi1 = phi2 = 0.674 +/- 0.001 for S-2288 under enzyme saturation with the substrate at pH 8.40 and 25.0 +/- 0.1 degrees C. The rate-determining step(s) in these reactions is most likely the cleavage of the C-N bond and departure of the leaving group.
Biochemistry 11/2006; 45(47):14175-82. · 3.42 Impact Factor
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ABSTRACT: Proton inventory studies of the thrombin-catalyzed fibrinogen activation to fibrinopeptide A are most consistent with a two-proton bridge forming at the transition state probably between Ser195 OgammaH and His57 Nepsilon2 and His57 Ndelta1 and Asp102 COObeta- at the active site, with fractionation factors 0.66 +/- 0.03 under enzyme saturation with substrate and 0.64 +/- 0.03 at fibrinogen concentration at 0.2 Km, at pH 8.0, pD 8.6, and 25.0 +/- 0.1 degrees C. Strongly inverse solvent isotope effects (SIEs) result from inverse lag times and maximal slopes of blood clotting plots, which are also anion and cation dependent. The blood clot is much coarser in D2O, as indicated in clotting curves with 3-9 times shorter lag time and steeper slopes with respect to H2O. The finer the particles, the weaker the H-bonds interlocking the fibrin mesh and/or in water structure around fibrin. Proton inventories of inverse lag times and maximal slopes of blood clotting curves in buffers containing Na+ and Cl- ions give the best fit to an exponential dependence on deuterium content in the buffer and give fractionation factors 5.6 +/- 0.5 and 7.8 +/- 0.6 at pH 8.0 and 25.0 +/- 0.1 degrees C. The thrombin-catalyzed activation of protein C (PC) to APC is associated with inverse kinetic SIEs (KSIEs) of 0.75 +/- 0.09 and 1.02 +/- 0.06 in 0.3 M NaCl and 0.3 M choline chloride, respectively, at substrate concentrations = 0.2 Km. In comparison, thrombin-catalyzed hydrolysis of chromogenic substrates gives greater KSIEs (Enyedy, E. I.; Kovach. I. M J. Am. Chem. Soc. 2004, 126, 6017-6024) and more complex proton inventories than the ones reported here for the first time for natural substrates. The present study illuminates differences in the character of the rate-determining transition state for the initial phase of the two physiological reactions catalyzed by thrombin.
Journal of the American Chemical Society 04/2005; 127(11):3760-6. · 9.91 Impact Factor
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Daoning. Zhang
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ABSTRACT: Thesis (Ph. D.)--Chemistry and Biochemistry Dept., South Dakota State University, 2004. Includes bibliographical references (leaves 117-135).
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ABSTRACT: Cucurbita foetidissima was analyzed for ribosome-inactivating proteins, which potentially could be used as specific anticancer and antiviral agents, and in plant protection. A novel type II RIP, foetidissimin, was found and characterized in this study. Foetidissimin was isolated from the roots of C. foetidissima by size exclusion and ion exchange chromatographic methods. A non-radioactive protein synthesis inhibition assay was used to guide the separation. The molecular weight of the dimeric protein is 63 kDa and the two chains are held together by non-covalent interactions. Using a non-radioactive protein synthesis inhibition assay, foetidissimin inhibited cell-free translation in rabbit reticulocyte lysate with an LC50 of 25.9 nM. The N-terminal sequence analysis of the first 14 amino acids showed that the A chain of foetidissimin shares 88% similarity with α-trichosanthin, a type I RIP, and 72% similarity with A chain of nigrin F, a type II RIP.
Plant Science.
