Side Chains Of Amino Acid Residues Research Articles

Crystal Structures of 8-Cl and 9-Cl TIBO Complexed with Wild-type HIV-1 RT and 8-Cl TIBO Complexed with the Tyr181Cys HIV-1 RT Drug-resistant Mutant

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is an important target for chemotherapeutic agents used in the treatment of AIDS; the TIBO compounds are potent non-nucleoside inhibitors of HIV-1 RT (NNRTIs). Crystal structures of HIV-1 RT complexed with 8-Cl TIBO (R86183, IC 50=4.6 nM) and 9-Cl TIBO (R82913, IC 50= 33 nM) have been determined at 3.0 Å resolution. Mutant HIV-1 RT, containing Cys in place of Tyr at position 181 (Tyr181Cys), is highly resistant to many NNRTIs and HIV-1 variants containing this mutation have been selected in both cell culture and clinical trials. We also report the crystal structure of Tyr181Cys HIV-1 RT in complex with 8-Cl TIBO (IC 50= 130 nM) determined at 3.2 Å resolution. Averaging of the electron density maps computed for different HIV-1 RT/NNRTI complexes and from diffraction datasets obtained using a synchrotron source from frozen (−165 °C) and cooled (−10 °C) crystals of the same complex was employed to improve the quality of electron density maps and to reduce model bias. The overall locations and conformations of the bound inhibitors in the complexes containing wild-type HIV-1 RT and the two TIBO inhibitors are very similar, as are the overall shapes and volumes of the non-nucleoside inhibitor-binding pocket (NNIBP). The major differences between the two wild-type HIV-1 RT/TIBO complexes occur in the vicinity of the TIBO chlorine substituents and involve the polypeptide segments around the β5-β6 connecting loop (residues 95 to 105) and the β13-β14 hairpin (residues 235 and 236). In all known structures of HIV-1 RT/NNRTI complexes, including these two, the position of the β12-β13 hairpin or the “primer grip” is significantly displaced relative to the position in the structure of HIV-1 RT complexed with a double-stranded DNA and in unliganded HIV-1 RT structures. Since the primer grip helps to position the template-primer, this displacement suggests that binding of NNRTIs would affect the relative positions of the primer terminus and the polymerase active site. This could explain biochemical data showing that NNRTI binding to HIV-1 RT reduces efficiency of the chemical step of DNA polymerization, but does not prevent binding of either dNTPs or DNA. When the structure of the Tyr181Cys mutant HIV-1 RT in complex with 8-Cl TIBO is compared with the corresponding structure containing wild-type HIV-1 RT, the overall conformations of Tyr181Cys and wild-type HIV-RT and of the 8-Cl TIBO inhibitors are very similar. Some positional changes in the polypeptide backbone of the β6-β10-β9 sheet containing residue 181 are observed when the Tyr181Cys and wild-type complexes are compared, particularly near residue Val179 of β. In the p51 subunit, the Cys181 side-chain is oriented in a similar direction to the Tyr181 side-chain in the wild-type complex. However, the electron density corresponding to the sulfur of the Cys181 side-chain in the p66 subunit is very weak, indicating that the thiol group is disordered, presumably because there is no significant interaction with either 8-Cl TIBO or nearby amino acid residues. In the mutant complex, there are slight reaarrangements of the side-chains of other amino acid residues in the NNIBP and of the flexible dimethylallyl group of 8-Cl TIBO; these conformational changes could potentially compensate for the interactions that were lost when the relatively large tyrosine at position 181 was replaced by a less bulky cysteine residue. In the corresponding wild-type complex, Tyr181 in the p66 subunit has significant interactions with the bound inhibitor and the position of the Tyr181 side-chain is well defined in both subunits. Apparently the Tyr181→Cys mutation eliminates favorable contacts of the aromatic ring of the tyrosine and the bound inhibitor, reducing the stability of NNRTI binding. This is consistent with the observation that the Tyr181Cys mutant HIV-1 RT is more resistant to NNRTIs that have extensive interactions with the Tyr181 side-chain. This interpretaton is supported by a recent biochemical study which found that an NNRTI dissociates from its complex with Tyr181 Cys HIV-1 RT faster than it does from a complex with wild-type HIV-1 RT.

Structure and dynamics of a designed helix-loop-helix dimer in dilute aqueous trifluoroethanol solution. A strategy for NMR spectroscopic structure determination of molten globules in the rational design of native-like proteins

The overwhelming majority of engineered amino acid sequences designed to fold into well defined tertiary structures show the hallmarks of molten globules. Although imperfectly folded, the structures of these polypeptides are of considerable interest in assessing the predictive power of design strategies and in understanding the structural basis for the formation of proteins with native-like properties. This paper describes a strategy for the structural characterization of molten globules by NMR spectroscopy applied to the study of SA-42, a polypeptide with 42 amino acids that folds into a hairpin helix-loop-helix dimer. The 1H NMR spectrum of SA-42 was assigned in several mixtures of water and trifluoroethanol (TFE) (0-30 vol%) and small amounts of TFE were shown to have a significant effect on the spectrum. The secondary and supersecondary structures of SA-42 were determined. In aqueous solution a helix-loop-helix dimer is formed, but in 30 vol% of TFE the population of hairpin dimers are negligible and SA-42 is monomeric, folding into two non-interacting helical segments. In solutions containing less than 3 vol% of TFE the structure is very similar to that in water and the structural information may be used to develop the motif in aqueous solution. Less well ordered amino acid residue sidechains in the hydrophobic core were identified. Helix distortion in the tetrahelix bundle was found to be small. Detailed information about molten globule structures in aqueous solution can be obtained from NMR spectroscopy if the spectra are assigned in dilute TFE solution. On the basis of the NMR spectroscopic analysis, the solution structure of SA-42 was found to be close to the designed one. A route for developing native-like properties in SA-42 is suggested based on the identification by NMR spectroscopy of some less well ordered amino acid sidechains in the hydrophobic core and on the observed structural rigidity of the two helices.

Energetics of Zn2+ binding to a series of biologically relevant ligands: A molecular mechanics investigation grounded on ab initio self‐consistent field supermolecular computations

AbstractDetailed investigations are performed of the binding energetics of Zn2+ to a series of neutral and anionic ligands making up the sidechains of amino acid residues of proteins, as well as ligands which can be involved in Zn2+ binding during enzymatic activation: imidazole, formamide, methanethiol, methanethiolate, methoxy, and hydroxy. The computations are performed using the SIBFA molecular mechanics procedure (SMM), which expresses the interaction energy under the form of four separate contributions related to the corresponding ab initio supermolecular ones: electrostatic, short‐range repulsion, polarization, and charge transfer. Recent refinements to this procedure are first exposed. To test the reliability of this procedure in large‐scale simulations of inhibitor binding to metalloenzyme cavities, we undertake systematic comparisons of the SMM results with those of recent large basis set ab initio self‐consistent field (SCF) supermolecule computations, in which a decomposition of the total ΔE into its four corresponding components is done (N. Gresh, W. Stevens, and M. Krauss, J. Comp. Chem., 16, 843, 1995). For each complex, the evolution of each individual SMM energy component as a function of radial and in‐ and out‐of‐plane angular variations of the Zn2+ position reproduces with good accuracy the behavior of the corresponding SCF term. Computations performed subsequently on di‐ and oligoligated complexes of Zn2+ show that the SIBFA molecular mechanics (SMM) functionals, Epol and Ect, closely account for the nonadditive behaviors of the corresponding second‐order energy contributions determined from the ab initio SCF calculations on these complexes and their nonlinear dependence on the number of ligands. Thus, the total intermolecular interaction energies computed with this procedure reproduce, with good accuracy, the corresponding SCF ones without the need for additional, extraneous terms in the intermolecular potential of polyligated complexes of divalent cations. © 1995 by John Wiley & Sons, Inc.

PBM: a software package to create, display and manipulate interactively models of small molecules and proteins on IBM-compatible PCs.

The PBM package was developed to create, display and conveniently manipulate protein and small molecule structures on IBM-compatible microcomputers. It consists of four modules: CREATE, SPHERE, RIBBON and CONVERT. CREATE includes commands to create or alter ('mutate') the primary and subsequently the tertiary structure of a given peptide or protein by defining phi and psi angles of residues at will, options to add, delete or alter atoms in a structure, utilities to choose easily between the most common rotamers of amino acid residue sidechains and options to analyse in various ways a protein conformation. SPHERE provides for an interactive manipulation of structures containing up to 2700 atoms which can belong up to six different molecules. All manipulations can be made with the use of an ordinary mouse, by choosing from a variety of pull-down menus. Three types of models can be implemented to display molecules on the computer screen or the plotter: skeletal, solid space-filling and wireframe space-filling models. RIBBON creates ribbon models of proteins and allows for a limited variety of interactive manipulations. CONVERT is a file converter, which is capable of converting files of atom coordinates of literally any format to Brookhaven Data Bank format files. The package produces very good results for protein molecules of reasonable sizes, both in terms of graphics quality and speed of operations, on an 80486 IBM PC-compatible machine equipped with a 1 MByte VGA display card and a colour VGA monitor, which is a recommended configuration.

Binding and interaction of the primary and the secondary electron acceptor quinones in bacterial photosynthesis: An infrared spectroelectrochemical study of Rhodobacter sphaeroides reaction centers

The vibrational modes of the primary and secondary electron acceptors Q A and Q B, their semiquinone anions and their respective protein environment in Rhodobacter sphaeroides reaction centers have been characterized using combinations of electrochemically-induced and light-induced Fourier-transform infrared (IR) difference spectroscopy. Q − A/Q A and Q − B/Q B IR difference spectra without contributions of other cofactors were generated by three different methods: (1) electrochemically, by reduction of Q A to Q − A; (2) photochemically, with flash-induced formation of P +Q − A or P +Q − B, rereduction of P + by cytochrome (cyt) c 2, and electrochemical rereduction of cyt c 2; (3) photochemically and electrochemically, by subtraction of redox-induced P +/P difference spectra from light-induced P +Q − A/PQ A and P +Q − B/PQ B difference spectra. Although Q − A was generated by completely different methods, and in one case (3) a charge-separated state is involved, almost identical Q −A/Q A and only slightly different Q − B/Q B difference spectra have been obtained. Bands at 1630 cm −1 and 1640 cm −1 are proposed as candidates for the C=O modes of Q A and Q B, respectively. The C-O modes of Q − A and Q − B are assigned to bands at 1462 cm −1 and 1478-88 cm −1, respectively. Difference bands at 1668 cm −1 and 1652 cm −1 in Q − A/Q A difference spectra are more likely to arise from amide-I modes or side chain vibrations of amino acids to which Q A is hydrogen-bonded. A number of difference bands between 1520 cm −1 and 1560 cm −1 possibly arise from amide-II vibrations and aromatic amino-acid side chain residues in the vicinity of Q A and Q B. A differential feature at 1734 cm −1 1726 cm −1 in Q − A/Q A difference spectra probably arises from changes in the protonation state or environment of distant carboxyl groups. An alternative explanation in terms of changes in the environment of the 10a ester C=O group of bacteriopheophytin L, however, cannot be excluded. Bands between 1724 cm −1 and 1740 cm −1 in Q − B/Q B difference spectra are tentatively assigned to a protonation of ASP L213 and/or a change in the environment of GLU L212, both being located in the vicinity of Q B and involved in the proton transfer to Q B (Okamura, M.Y. and Feher, G. (1992) Annu. Rev. Biochem. 61, 861–896).

Additive and synergistic pharmacologic inhibition of equine fibrinoligase (factor XIIIa*-like) biochemical activity

Summary A selected group of pharmaceutical compounds were evaluated for the ability to inhibit the biochemical activity of fibrinoligase (coagulation factor XIIIa*) in pooled equine plasma. Criteria for the pharmaceuticals selected were based on the mechanism of the transglutamination biochemical reaction mediated by coagulation factor XIIa*. These criteria were complemented by recognition of the molecular configuration and chemical composition of amino acid residue side chains involved in the process of covalent fibrin monomer polymerization (cross-linking, transglutamination) mediated by this enzyme. Each pharmaceutical was evaluated individually and in combination with other potential coagulation factor XIIIa* inhibitors in an effort to detect additive and synergistic phenomenon. In this context, pharmaceuticals with a carbonylamide (eg, cefuroxime, Girard's reagent-P, prolinamide) were applied in concert with compounds with a terminal amine (eg, d-arginine, l-lysine) functional group. In concept, this method theoretically served to competitively simulate glutamine and lysine amino acid residues within strands of fibrin monomer substrate involved in phase I (carbonylamide) and phase II (terminal amine) of the transglutamination reaction (covalent fibrin monomer cross-linking). Halogen-dinitro and ethylene compounds were also evaluated because of their reported ability to inactivate enzyme systems dependent on an intact sulfhydryl group located at their biochemically active site (eg, cystine amino acid residue). This group of pharmaceutical compounds failed to inhibit the biochemical activity mediated by coagulation factor XIIIa* in equine plasma.

From short-time molecular dynamics to long-time stochastic dynamics of proteins

To extend the time scales of descriptions of protein dynamics, beyond those accessible by the molecular dynamics method, theories of stochastic processes are utilized for both short- and long-time dynamics. A first step is the bridging from short-time fluctuations in conformational states to transitions between conformational states. Stochastic short-time dynamics of a reaction coordinate of a conformational transition is deduced starting from the classical equations of motion of a molecular system. The coupling strength between the reaction coordinate and the bath, that remaining degrees of freedom constitute, is determined by an analysis of the short-time fluctuations in molecular dynamics trajectories. An effective potential energy function of the reaction coordinate is obtained by an energy minimization method. The required transition rates are determined from the nonstationary solutions of the Fokker–Planck equation for Brownian motion. As a first application of this approach, dihedral transitions in the sidechain of an aromatic amino acid residue in an α-helix are studied. The rate constants of elementary conformational transitions constitute the basic parameters of a stochastic model of protein conformational relaxation dynamics. This model is useful for descriptions of the coupling between protein conformational dynamics and reactions involved in the functions of proteins.

Raman and infrared studies on the conformation of porcine pancreatic and Crotalus durissus terrificus phosphlipases A 2

Raman and infrared spectroscopies were used to investigate conformational features of Crotalus durissus terrificus and porcine pancreatic phospholipases A 2, as well as the proenzyme of the latter. The results indicate that conformational changes occur for the phospholipase molecules as a consequence of different experimental conditions such as change of physical state, presence of certain ionic species and interaction with a model substrate analog. Amorphous and crystalline solid phospholipase present discrepant conformational features. Conformational transitions were detected for the pancreatic zymogen → phospholipase A 2 transformation and different secondary contents were observed for a toxic and a nontoxic form of the phospholipase molecule. All those structural changes have been shown to involve primarily the architecture of the polypeptide backbone rather than the conformation of amino acid residue side-chains. Disulfide bridges have shown consistently a gauche-gauche geometry which has not been disturbed by any of the experimental conditions employed. The external occurrence of tryptophan residues has been a common feature for the systems assayed, as well as the predominant localization of tyrosine residues in hydrophilic environment, probably at the molecular surface.

Use of mutants to analyze regions on the H-2Kb molecule for interaction with immune receptors.

MHC variants isolated both in vivo (by tissue graft rejection) and in vitro (by antibody selection) were utilized to study sites on the H-2Kb molecule involved in interaction with antibodies and with the TCR. Kb mutants selected by antibodies were found to have single point mutations, which when analyzed in the context of the three-dimensional structure of a Kb molecule modeled from HLA-A2 coordinates showed that the altered residues were localized mostly to the alpha 1 and alpha 2 helices. The side chains of the variant amino acid residues pointed upward and away from the antigenic site. Analysis of the altered amino acids in the previously described tissue-graft-selected Kb mutants showed that the side chains of the variant residues occur either in the alpha-helical regions or in the beta-pleated-sheet floor of the antigen groove, but every mutant contained at least one and sometimes several acid side chains projecting into the antigen-binding groove. Monoclonal antibody studies showed that the available monoclonal antibodies mapped to discrete domain-specific sites. Analysis of CTL recognition sites using cloned mutant anti-parent or allogeneic combinations showed that all CTL clones examined interacted with amino acid side-chain residues on both the alpha 1 and alpha 2 helices. Thus, we concluded that the CTLs must simultaneously interact with the amino acid residues in both the alpha-helical stretches of the alpha 1 and alpha 2 domains. Our analyses with the point mutants imply that the TCR must interact with the MHC molecule over a relatively large surface area in such an orientation that it interfaces with the two alpha helices, as well as with the foreign or self-peptide in the antigen-binding site between the helices. These findings, together with the observation that several of the in vivo Kb mutants induce strong alloreactions yet have changes only in the bottom of the antigen-binding groove and no alterations in the alpha-helical residues, are consistent with the hypothesis that in some cases alloreaction can be the result of T-cell recognition of an altered pattern on the MHC molecule due to a changed peptide in the antigen groove.

Labelling and exploration of the active site of enkephalinase (EC 3.4.24.11) in kidney membranes with [ 3H]thiorphan as ligand

[ 3H]Thiorphan, a potent inhibitor of enkephalinase (EC 3.4.24.11), was used to label the enzyme in membranes from rat kidney cortex an to explore its specificity at the active site. [ 3H]Thiorphan binding occurred reversibly, with low non-specific binding and to a single class of sites. The dissociation constant, determined by either kinetics or saturation studies was ∼ 0.4 nM. The ratio of the maximal velocity of enkephalinase with enkephalins as substrates to the maximal binding of [ 3H]thiorphan was consistent with the catalytic constant of the enzyme. Enkephalinase inhibitors competed with [ 3H]thiorphan and had inhibitory constants in agreement with the corresponding values derived from measurement of the enzyme catalytic activity, whereas inhibitors of other metallopeptidases were ineffective. The inhibitory potencies of a series of systematically varied oligopeptides regarding [ 3H]thiorphan binding and enkephalinase activity were also highly correlated. Structure-activity relationships among competitors indicated that the main subsites of enkephalinase are: (1) the hydrophobic pocket in P′ 1, the requirements of which are best satisfied by aromatic amino acid side chain residues (2) the P′ 2 subsite, the requirements of which are best satisfied by amino acids with a short, uncharged side chain and a free terminal carboxyl group. This novel binding assay should facilitate the exploration of the active site of enkephalinase and the development of new inhibitors.

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra : Computation of sterically allowed proton-proton distances and statistical analysis of proton-proton distances in single crystal protein conformations

Two different, theoretical studies of intramolecular proton-proton distances in polypeptide chains are described. Firstly, the distances between amide, C α and C β protons of neighbouring residues in the amino acid sequence, which correspond to the sterically allowed values for the dihedral angles φ i, ψ i and χ i 1, were computed. Secondly, the frequency with which short distances occur between amide, C α and C β protons of neighbouring and distant residues in the amino acid sequence were statistically evaluated in a representative sample of globular protein crystal structures. Both approaches imply that semi-quantitative measurements of short, non-bonding proton-proton distances, e.g. by nuclear Overhauser experiments, should present a reliable and generally applicable method for sequential, individual resonance assignments in protein 1H nuclear magnetic resonance spectra. Similar calculations imply that corresponding distance measurements can be used for resonance assignments in the side-chains of the aromatic amino acid residues, asparagine and glutamine, where the complete spin systems cannot usually be identified from through-bond spin-spin coupling connectivities.

Circular dichroism studies of glutathione reductase.

The circular dichroic spectrum of glutathione reductase was studied. The far‐ultraviolet region of the spectrum indicated a low content of helix and β structures, as calculated on tire basis of commonly used spectral parameters of secondary structure. The fractions of amino acid residues present in helix and β structures, respectively, constitute approximately 30% each of the total number in crystals of the enzyme from human erythrocytes [G. E. Schulz, R. H. Schirmer, W. Sachsenheimer, and E. F. Pai (1978) Nature (Lond.) 273, 120–124]. This discrepancy between the estimates may originate from the presence of supersecondary structures in the enzyme, which invalidate the use of parameters defined for circular dichroic spectra of simple secondary structures. Nevertheless, the similarities between the spectra of glutathione reductase from calf liver, porcine erythrocytes, and human erythrocytes strongly suggest that the secondary and supersecondary structures of the three enzymes are very similar. Furthermore, the far‐ultraviolet circular dichroic spectrum of the apoenzyme is not significantly different from that of the holoenzyme, indicating lack of major structural differences between the two forms of the protein. The circular dichroism of the near‐ultraviolet region indicates changes in the interaction of FAD with the protein matrix as well as local conformational changes involving aromatic side‐chains of amino acid residues during redox changes of the holoenzyme. Differences between near‐ultraviolet spectra of apoenzyme and holoenzyme likewise indicate reversible local conformational changes upon binding and release of the prosthetic group FAD. The reconstituted holoenzyme had a spectrum indistinguishable from the native enzyme. The 300–600‐nm region of the spectrum could be resolved into six Gaussian functions corresponding to the vibronic bands identified in other flavin‐dependent dehydrogenases. The rotational strength of the Cotton effects in the 300–600‐nm region increased and the positions of the maxima in the circular dichroism spectrum shifted upon reduction of the enzyme The increase in the 400–510‐nm region indicates a stronger asymmetric interaction between the isoalloxazine ring of FAD and the protein than that observed in the oxidized form of the enzyme. The enhancement may be effected by the dithiol, which is formed upon reduction of the enzyme.

Buoyant densities of dissolved and crystalline lactate dehydrogenase from pig heart and pig muscle

Buoyant densities of the H4 and M4 isoenzymes of lactate dehydrogenase from pig, in both dissolved and crystalline cross-linked states, have been determined at neutral pH in concentrated CsCl solutions by equilibrium centrifugations in an ultracentrifuge. In both states, buoyant densities of the M4-LDH were lower than those of the H4-LDH, presumably due to the binding of counterions and hydration water by ten additional charged amino acid residues present on the M4-LDH. The cross-linked crystals had lower buoyant densities than enzymes in dissolved state consistent with the lower density of the cross-linking reagent. The variations in buoyant densities observed in CsCl solutions are in agreement with a previously described model for the water-and ion-binding to proteins which assigns almost all of the bound water to the charged amino acid side chain residues. In Cs2SO4 solutions the buoyant densities of the H4-LDH in both physical states were less than in CsCl, probably because strongly hydrated sulphate ions bind to the charged amino acid residues.

Circular dichroism and sedimentation studies on the reconstitution of tobacco mosaic virus

Reconstitution of tobacco mosaic virus from its constituents, the coat protein and RNA, was investigated by means of ultracentrifugation and circular dichroism measurement. Tobacco mosaic virus protein forms a 20S double-layer disc under conditions favorable for tobacco mosaic virus reconstitution. Dibromination of the tyrosine 139 residue of tobacco mosaic virus protein prevents formation of the 20S disc. Acidification of the tobacco mosaic virus protein solution causes 20S discs to polymerize into long helical rods. Changes in the CD spectra of tobacco mosaic virus protein in the near-ultraviolet region suggest that stacking of the aromatic sidechains of amino acid residues stabilizes the helical rod. The dibrominated tobacco mosaic virus protein also has the ability of rod elongation under acidic condition. CD studies reveal that assembly of tobacco mosaic virus particles from its constituents is stabilized by the stacking effect between the base residues of RNA and the aromatic residues of tobacco mosaic virus protein. Cucumber green mottle mosaic virus protein, which acts as a substituent for tobacco mosaic virus protein in tobacco mosaic virus reconstitution, was also investigated.

Chemical properties of a high molecular weight spleen extract with permeability activity.

The extract of proteins from spleen (SPF) described in the foregoing paper has been subjected to chromatographic and electrophoretic separation procedures. Although different protein fractions are obtained, most possess considerable permeability activity and no localisation of activity has been observed. Treatment of SPF with chemical reagents to modify certain amino acid side chain residues shows that primary amino groups are essential for activity. Conversion of carboxylate groups to electroneutral derivatives results in an increase in the specific activity of SPF. It is proposed that permeability activity, as observed in SPF and protein fractions derived therefrom, probably resides in certain features of primary structure which are shared by different proteins.