Structure of HIV-1 protease with KNI-272: a transition state mimetic inhibitor containing allophenylnorstatine.
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Amino Acid Sequence↗
Biomedical subjects
T N Bhat
Publications and source records attributed to T N Bhat.
Structural basis of drug resistance for the V82A mutant of HIV-1 proteinase.
A major problem in the development of antiviral therapies for AIDS has been the emergence of drug resistance. We report an analysis of the structure of a Val 82 to Ala mutant of HIV-1 proteinase complexed to A-77003, a C2 symmetry-based inhibitor. Modelling studies predicted that the V82A mutation would result in decreased van der Waals' interactions with the phenyl rings of A-77003 in both S1 and S1' subsites. Unexpected rearrangements of the protein backbone, however, resulted in favourable re-packing of inhibitor and enzyme atoms in the S1 but not the S1' subsite. This analysis reveals the importance of enzyme flexibility in accommodating alternate packing arrangements, and can be applied to the re-design of inhibitors targeted to drug resistant variants which emerge in the clinic.
Antiviral Agents↗
Selection and analysis of human immunodeficiency virus type 1 variants with increased resistance to ABT-538, a novel protease inhibitor.
Inhibitors of the human immunodeficiency virus protease represent a promising new class of antiretroviral drugs for the treatment of AIDS. We now report the in vitro selection of viral variants with decreased sensitivity to a symmetry-based protease inhibitor, ABT-538, currently being tested in clinical trials. Molecular characterization of the variants shows that an isoleucine-to-valine substitution at position 84 results in a substantial decrease in sensitivity to the drug. Moreover, an additional mutation at position 82, valine to phenylalanine, further decreases viral susceptibility to ABT-538. Three-dimensional analysis of the protease-drug complex provides a structural explanation for the relative drug resistance induced by these two mutations. These findings emphasize the importance of closely monitoring patients receiving ABT-538 for the emergence of viral resistance and provide information that may prove useful in designing the next generation of protease inhibitors.
Amino Acid Sequence↗
Three-dimensional structures of the free and the antigen-complexed Fab from monoclonal anti-lysozyme antibody D44.1.
The three-dimensional structures of the free and antigen-complexed Fabs from the mouse monoclonal anti-hen egg white lysozyme antibody D44.1 have been solved and refined by X-ray crystallographic techniques. The crystals of the free and lysozyme-bound Fabs were grown under identical conditions and their X-ray diffraction data were collected to 2.1 and 2.5 A, respectively. Two molecules of the Fab-lysozyme complex in the asymmetric unit of the crystals show nearly identical conformations and thus confirm the essential structural features of the antigen-antibody interface. Three buried water molecules enhance the surface complementarity at the interface and provide hydrogen bonds to stabilize the complex. Two hydrophobic buried holes are present at the interface which, although large enough to accommodate solvent molecules, are void. The combining site residues of the complexed FabD44.1 exhibit reduced temperature factors compared with those of the free Fab. Furthermore, small perturbations in atomic positions and rearrangements of side-chains at the combining site, and a relative rearrangement of the variable domains of the light (VL) and the heavy (VH) chains, detail a Fab accommodation of the bound lysozyme. The amino acid sequence of the VH domain, as well as the epitope of lysozyme recognized by D44.1 are very close to those previously reported for the monoclonal antibody HyHEL-5. A feature central to the FabD44.1 and FabHyHEL-5 complexes with lysozyme are three salt bridges between VH glutamate residues 35 and 50 and lysozyme arginine residues 45 and 68. The presence of the three salt bridges in the D44.1-lysozyme interface indicates that these bonds are not responsible for the 1000-fold increase in affinity for lysozyme that HyHEL-5 exhibits relative to D44.1.
Amino Acid Sequence↗
Solvent rearrangement in an antigen-antibody interface introduced by site-directed mutagenesis of the antibody combining site.
The three-dimensional structure of a site-directed mutant of the bacterially expressed Fv fragment from monoclonal antibody D1.3, complexed to the specific antigen lysozyme has been determined to a nominal resolution of 1.8 A using X-ray diffraction data. The replacement of VL Trp92 by Asp allows two water molecules to occupy space taken by Trp92 in the wild-type complex, in agreement with a previous observation that water molecules play an important role in stabilizing this antigen-antibody complex. The equilibrium constant for the binding of the mutant Fv to the antigen decreases by three orders of magnitude (from 2.3 x 10(8) M-1 to 2.6 x 10(5) M-1). Titration calorimetry shows that this results from a smaller negative binding enthalpy (delta delta H = -16 kJ mol-1 at 24 degrees C), whereas the value of the binding entropy is not affected. Since in the complex between the mutated Fv and antigen the buried area has decreased relative to that of the wild-type Fv by about 150 A2, the contribution of the buried unit area to the decrease in free energy (delta Gzero) is approximately 117 J mol-1 (28 cal mol-1) per A2. The loss of interatomic contacts in replacing Trp by Asp permits an approximate calculation for the contribution of van der Waals interactions made by Trp92 in this complex, which gives an average of 2.1 kJ mol-1 (0.5 kcal mol-1) for contacts between carbon atoms.
Antibodies, Monoclonal↗
Bound water molecules and conformational stabilization help mediate an antigen-antibody association.
We report the three-dimensional structures, at 1.8-A resolution, of the Fv fragment of the anti-hen egg white lysozyme antibody D1.3 in its free and antigen-bound forms. These structures reveal a role for solvent molecules in stabilizing the complex and provide a molecular basis for understanding the thermodynamic forces which drive the association reaction. Four water molecules are buried and others form a hydrogen-bonded network around the interface, bridging antigen and antibody. Comparison of the structures of free and bound Fv fragment of D1.3 reveals that several of the ordered water molecules in the free antibody combining site are retained and that additional water molecules link antigen and antibody upon complex formation. This solvation of the complex should weaken the hydrophobic effect, and the resulting large number of solvent-mediated hydrogen bonds, in conjunction with direct protein-protein interactions, should generate a significant enthalpic component. Furthermore, a stabilization of the relative mobilities of the antibody heavy- and light-chain variable domains and of that of the third complementarity-determining loop of the heavy chain seen in the complex should generate a negative entropic contribution opposing the enthalpic and the hydrophobic (solvent entropy) effects. This structural analysis is consistent with measurements of enthalpy and entropy changes by titration calorimetry, which show that enthalpy drives the antigen-antibody reaction. Thus, the main forces stabilizing the complex arise from antigen-antibody hydrogen bonding, van der Waals interactions, enthalpy of hydration, and conformational stabilization rather than solvent entropy (hydrophobic) effects.
Animals↗
Crystal structure of a tethered dimer of HIV-1 proteinase complexed with an inhibitor.
HIV-1 proteinase (HIV PR) is a dimeric enzyme composed of two identical polypeptide chains that associate with twofold symmetry. We have determined to 1.8 A the crystal structure of a covalently tethered dimer of HIV PR. The tethered dimer:inhibitor complex is identical in nearly every respect to the complex of the same inhibitor with the wild type dimeric molecule, except for the linker region. Our results suggest that the tethered dimer may be a useful surrogate enzyme for studying the effects of single site mutations on substrate and inhibitor binding as well as on enzyme asymmetry, and for simulating independent mutational drift of the two domains which has been proposed to have led to the evolution of modern day, single-chain aspartic proteinases.
Alcohols↗
Three-dimensional structure of a heteroclitic antigen-antibody cross-reaction complex.
Although antibodies are highly specific, cross-reactions are frequently observed. To understand the molecular basis of this phenomenon, we studied the anti-hen egg lysozyme (HEL) monoclonal antibody (mAb) D11.15, which cross-reacts with several avian lysozymes, in some cases with a higher affinity (heteroclitic binding) than for HEL. We have determined the crystal structure of the Fv fragment of D11.15 complexed with pheasant egg lysozyme (PHL). In addition, we have determined the structure of PHL, Guinea fowl egg lysozyme, and Japanese quail egg lysozyme. Differences in the affinity of D11.15 for the lysozymes appear to result from sequence substitutions in these antigens at the interface with the antibody. More generally, cross-reactivity is seen to require a stereochemically permissive environment for the variant antigen residues at the antibody-antigen interface.
Amino Acid Sequence↗
Crystal structures of native and inhibited forms of human cathepsin D: implications for lysosomal targeting and drug design.
Cathepsin D (EC 3.4.23.5) is a lysosomal protease suspected to play important roles in protein catabolism, antigen processing, degenerative diseases, and breast cancer progression. Determination of the crystal structures of cathepsin D and a complex with pepstatin at 2.5 A resolution provides insights into inhibitor binding and lysosomal targeting for this two-chain, N-glycosylated aspartic protease. Comparison with the structures of a complex of pepstatin bound to rhizopuspepsin and with a human renin-inhibitor complex revealed differences in subsite structures and inhibitor-enzyme interactions that are consistent with affinity differences and structure-activity relationships and suggest strategies for fine-tuning the specificity of cathepsin D inhibitors. Mutagenesis studies have identified a phosphotransferase recognition region that is required for oligosaccharide phosphorylation but is 32 A distant from the N-domain glycosylation site at Asn-70. Electron density for the crystal structure of cathepsin D indicated the presence of an N-linked oligosaccharide that extends from Asn-70 toward Lys-203, which is a key component of the phosphotransferase recognition region, and thus provides a structural explanation for how the phosphotransferase can recognize apparently distant sites on the protein surface.
Amino Acid Sequence↗
Crystallographic refinement of the three-dimensional structure of the FabD1.3-lysozyme complex at 2.5-A resolution.
The three-dimensional crystal structure of the complex between the Fab from the monoclonal anti-lysozyme antibody D1.3 and the antigen, hen egg white lysozyme, has been refined by crystallographic techniques using x-ray intensity data to 2.5-A resolution. The antibody contacts the antigen with residues from all its complementarity determining regions. Antigen residues 18-27 and 117-125 form a discontinuous antigenic determinant making hydrogen bonds and van der Waals interactions with the antibody. Water molecules at or near the antigen-antibody interface mediate some contacts between antigen and antibody. The fine specificity of antibody D1.3, which does not bind (K alpha less than 10(5) M-1) avian lysozymes where Gln121 in the amino acid sequence is occupied by His, can be explained on the basis of the refined model.
Amino Acid Sequence↗
Small rearrangements in structures of Fv and Fab fragments of antibody D1.3 on antigen binding.
The potential use of monoclonal antibodies in immunological, chemical and clinical applications has stimulated the protein engineering and expression of Fv fragments, which are heterodimers consisting of the light and heavy chain variable domains (VL and VH) of antibodies. Although Fv fragments exhibit antigen binding specificity and association constants similar to their parent antibodies or Fab moieties, similarity in their interactions with antigen at the level of three-dimensional structure has not been investigated. We have determined the high-resolution crystal structure of the genetically engineered FvD1.3 fragment of the anti-hen egg-white lysozyme (HEL) monoclonal antibody D1.3, and of its complex with HEL. On comparison with the crystallographically refined FabD1.3-HEL complex, we find that FvD1.3 and FabD1.3 make, with minor exceptions, very similar contacts with the antigen. Furthermore, a small but systematic rearrangement of the domains of FvD1.3 occurs on binding HEL, bringing the contacting residues closer to the antigen by a mean value of about 0.7 A for VH (aligning on VL) or of 0.5 A for VL (aligning on VH). This is indicative of an induced fit rather than a 'lock and key' fit to the antigen.
Antibodies, Monoclonal↗
Crystallization and preliminary X-ray diffraction study of the bacterially expressed Fv from the monoclonal anti-lysozyme antibody D1.3 and of its complex with the antigen, lysozyme.
The associated heavy (VH) and light (VL) chain variable domains (Fv) of the monoclonal anti-lysozyme antibody D1.3, secreted from Escherichia coli, have been crystallized in their antigen-bound and free forms. FvD1.3 gives tetragonal crystals, space group P4(1)2(1)2 (or P4(3)2(1)2), with a = 90.6 A, c = 56.4 A. The FvD1.3-lysozyme complex crystallizes in space group C2, with a = 129.2 A, b = 60.8 A, c = 56.9 A and beta = 119.3 degrees. The crystals contain one molecule of Fv or of the Fv-lysozyme complex in their asymmetric units and diffract X-rays to high resolution, making them suitable for X-ray crystallographic studies.
Amino Acid Sequence↗
Structure of 5-hydroxy-5-phenyl-7-azatricyclo[7.4.0.02,7]trideca-2,9(1), 10,12-tetraen-8-one by the consistent electron density approach.
C18H15NO2, Mr = 277.3, monoclinic, P21/n, a = 7.408 (2), b = 22.311 (7), c = 8.613 (2) A, beta = 103.53 (3) degrees, V = 1384 (1) A3, Z = 4, Dx = 1.33 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 0.94 cm-1, F(000) = 594, T = 293 K, 2229 unique diffractometer data, 963 with I greater than 3 sigma(I), R = 0.050. Structure solution was by the consistent electron density approach which combines the concepts of the OMIT map and density modification procedures. The benzolactam portion of the molecule is highly planar; most bond lengths and angles have typical values.
Bridged-Ring Compounds↗
Structure of N,N,N',N'-tetrakis(2-fluoro-2,2-dinitroethyl)oxamide by the consistent electron density approach.
C10H8F4N10O18, Mr = 632.22, monoclinic, Pc, a = 7.8885 (6), b = 6.7787 (4), c = 21.595 (2) A, beta = 108.21 (1) degree, V = 1096 (1) A3, Z = 2, Dx = 1.914 g cm-3, lambda(Cu K alpha) = 1.5418 A, mu = 18.7 cm-1, F(000) = 636, T = 293 K, 1791 unique data, 1733 with I greater than 3 sigma (I), R = 0.047. The structure was solved by the consistent electron density approach (CEDA), in which a small starting set of random phases was refined and expanded by the application of restraints to the electron density. A refinable preliminary structure was obtained by fitting a model to a 3 A map, calculated with 27 reflections (all data with d greater than 3 A, and magnitude of Fo greater than 170) whose phases had been determined by the CEDA. The molecule has an extended, open conformation; the two pairs of fluorodinitroethyl substituents located across the molecular center from each other show i and C2 pseudo-symmetry respectively.
Amino Acids↗
Structure of tyrosyl-tRNA synthetase refined at 2.3 A resolution. Interaction of the enzyme with the tyrosyl adenylate intermediate.
The crystal structure of tyrosyl-tRNA synthetase (EC 6.1.1.1) from Bacillus stearothermophilus has been refined to a crystallographic R-factor of 22.6% at 2.3 A resolution using a restrained least-squares procedure. In the final model the root-mean-square deviation from ideality for bond distances is 0.018 A and for angle distances is 0.044 A. Each monomer consists of three domains: an alpha/beta domain (residues 1 to 220) containing a six-stranded beta-sheet, an alpha-helical domain (248 to 318) containing five helices, and a disordered C-terminal domain (319 to 418) for which the electron density is very weak and where it has not been possible to trace the polypeptide chain. Complexes of the enzyme with the catalytic intermediate tyrosyl adenylate and the inhibitor tyrosinyl adenylate have also been refined to R-factors of 23.9% at 2.8 A resolution and 21.0% at 2.7 A resolution, respectively. Formation of the complexes results in some crystal cracking, but there is no significant difference in the conformation of the polypeptide chain of the three structures described here. The relative orientation of the alpha/beta and alpha-helical domains is similar to that previously observed for the "A" subunit of a deletion mutant lacking the C-terminal domain. Differences between these structures are confined to surface loops that are involved in crystal packing. Tyrosyl adenylate and tyrosinyl adenylate bind in similar conformations within a deep cleft in the alpha/beta domain. The tyrosine moiety is in the equivalent position to that occupied by tyrosine in crystals of the truncated mutant and makes similar strong polar interactions with the enzyme. The alpha-phosphate group interacts with the main-chain nitrogen of Asp38. The two hydroxyl groups of the ribose form strong interactions with the protein. The 2'-hydroxyl group interacts with the carboxylate of Asp194 and the main-chain nitrogen of Gly192 while the 3'-hydroxyl interacts with a tightly bound water molecule (Wat326). The adenine moiety appears to make no significant polar interactions with the protein. The results of site-directed mutagenesis studies are examined in the light of these refined structures.
Adenine↗
Correlation between occupancy and temperature factors of solvent molecules in crystal structures of proteins.
Correlation between occupancy and temperature factors during the refinement of a disordered molecule is discussed on the basis of their effects on an atomic scattering factor curve. When the resolution of the data is limited, occupancy and temperature factors affect scattering-factor curves in a similar fashion, and therefore they are correlated. An examination of this correlation suggests that small deviations (0.1) of occupancy factors from their tentative values may be compensated by suitable changes in temperature factors when the data are limited to medium (2.5 A) resolution.
Chemical Phenomena↗
Immunochemical and crystallographic studies of antibody D1.3 in its free, antigen-liganded, and idiotope-bound states.
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Animals↗
Improving crystallographic macromolecular images: the real-space approach.
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Crystallography↗