Molecular mechanics analysis of inhibitor binding to HIV-1 protease

Crystallographic structures of HIV protease with three differentpeptide-mimetic inhibitors were subjected to energy minimizationusing molecular mechanics, the minimized structures analyzedand the inhibitor binding energies calculated. Partial chargeassignment for the hydrogen bonded catalytic aspartk acids,Asp25 and -25', was in good agreement with charge calculationsusing semi-empirical molecular orbital methods. Root mean squaredeviations on minimization were small and similar for both subunitsin the protease dimer. The surface loops, which had the largestB factors, changed most on minimization; the hydrophobic coreand the inhibitor binding site showed little change. The distance-dependentdielectric of D(r) = 4r was found to be preferable to D(r) =r. Distance restraints were applied for the intermolecular hydrogenbonds to maintain the conformation of the inhibitor bindingsite. Using the dielectric of D(r) = 4r, the calculated interactionenergy of the three inhibitors with the protease ranged from–53 to –56 kcal/mol. The groups of the inhibitorswere changed to add or remove a ‘transition state analogue’hydroxyl group, and the loss in energy on the removal of thisgroup was calculated to be 0.9–1.7 kcal/mol. This wouldrepresent 19–36% of the total measured difference in bindingenergy between the inhibitors JG365 and MVT-101.     

The application of hydrogen bonding analysis in X-ray crystallography to help orientate asparagine, glutamine and histidine side chains

Protein X-ray crystallography produces an electron density mapthat rarely detects individual hydrogen atoms or distinguishesbetween carbon, nitrogen and oxygen atoms in the electron density.This makes it difficult to orientate the side chains of Asn,Gln and His, which appear symmetrical in the electron density;their orientation is usually judged on the basis of hydrogenbonding. Based on the observation that almost all buried donorsand acceptors are satisfied, we have developed a simple algorithmto compare the alternative conformations of these residues and,where possible, identify the most favourable. In a cross-sectionof protein structures we found a few side chains (15.0% of Asnand Gln and 9.9% of His) which would be more favourable in thealternative orientation. We have also found that this proportionrises slightly with worsening resolution.     

Molecular mechanics calculations on HIV-1 protease with peptide substrates correlate with experimental data

Molecular models of HIV-1 protease and 21 peptide substrateswith single amino acid substitutions at positions from P4 toP3' were built and compared with kinetic measurements. The crystalstructure of HTV-1 protease with a peptidic inhibitor was modifiedto model the peptide substrate Pro-Ala-Val-Ser-Leu-Ala-Met-Thrfor the starting geometry. Models were built of two reactionintermediates, HIV protease with peptide substrate and withits tetrahedral intermediate. The energy minimization used anew algorithm that increased the speed and eliminated a cut-offfor non-bonded interactions. After minimization the models forsubstrate and tetrahedral intermediate both had root mean squaredeviations of 0.48 Å for all atoms of the HIV proteasecompared to the starting crystal structure. Differences in themodel structures and interaction energies for HIV protease withdifferent substrates were analyzed. The calculated interactionenergies for the 21 HIV protease-tetrahedral intermediate modelsgave a correlation coefficient of 0.64 with the kinetic measurements.The eight substrates with changes in the P1 and P1' residuesnext to the scissile bond gave the highest correlation of 0.93,while the 14 substrates with changes in P2-P2' gave a correlationcoefficient of 0.86. The catalytic mechanism and factors influencingthe catalytic efficiency of the different substrates are discussedin relation to the models. The predictive ability of molecularmechanics calculations is discussed in the context of the statisticalmechanics analysis of the differences in free energy.     

Structural analysis of the CD2 T lymphocyte antigen by site-directed mutagenesis to introduce a disulphide bond into domain 1

Many proteins have been predicted to contain domains with immunoglobulin-Iikefolds and hence to be members of the immunoglobulin superfamily(IgSF). However, several members lack the Cys residues capableof forming the disulphide bond that forms a characteristic bridgebetween the ß sheets in the Ig fold, e.g. domain 1of the lymphocyte antigen CD2. The assignment of ßstrands in CD2 by sequence analysis was tested by attemptingto introduce a disulphide bridge between the ß sheetsby mutating two residues in the relevant positions to Cys. Mutant,soluble forms of CD2 were expressed in Chinese hamster ovarycell lines and amino add sequencing showed that a disulphidebond was formed as predicted, but not in the control where oneCys residue was misplaced by four residues. Evidence that bothmutated molecules folded correctly is given by the indistinguishablebinding of three monoclonal antibodies recognising differentepftopes on CD2. The 3-D structure of rat CD2 domain 1 has beendetermined by NMR spectroscopy and X-ray crystallography, confirmingthe predictions from the sequence. Applications of this methodof insertion of disulphide residues for probing protein structuresare discussed, together with other structures of IgSF domainslacking the typical inter-sheet disulphide bond.     

On the role of conserved proline residues in the structure and function of Clostridium pasteurianum 2[4Fe-4S] ferredoxin

The widespread occurrence of Pro residues adjacent to Cys ligandsin the sequences of [4Fe-4S] electron transfer proteins hasnot yet found a functional basis. The two such Pro of Clostridiumpasteurianum 2[4Fe-4S] ferredoxin have been probed by site-directedmutagenesis. Any one of them, but not both simultaneously, canbe substituted without impairing the proper folding of the protein.The reduction potentials of the ferredoxin variants fall ina narrow range of <20 mV above the potential of the nativeprotein. The biological activities with C.pasteurianum hydrogenaseand pyruvate-ferredoxin oxidoreductase do not change significantly,except when Lys replaces Pro. In these cases, the data suggestthat the two clusters of 2[4Fe-4S] ferredoxin may not alwaysbe equivalent in the interaction with the redox partners. Destabilizationof the structure has been observed as the consequence of theProl9 or Pro48 substitutions. Using 2-D NMR, this effect hasbeen associated with perturbations of both the hydrogen bondnetwork and one amino acid side chain around the [4Fe-4S] clusters.Thus, the conserved Pro found in the binding motif of [4Fe-4S]clusters in proteins strongly stabilizes the active site butdoes not play an essential role in the mechanism of electrontransfer.     

Free energy perturbation studies on binding of A-74704 and its diester analog to HIV-1 protease

Free energy simulations have been employed to rationalize thebinding differences between A-74704, a pseudo C₂- symmetricinhibitor of HIV-1 protease and its diester analog. The diesteranalog inhibitor, which misses two hydrogen bonds with the enzymeactive site, is surprisingly only 10-fold weaker. The calculatedfree energy difference of 1.7 ± 0.6 kcal/mol is in agreementwith the experimental result. Further, the simulations showthat such a small difference in binding free energies is dueto (1) weaker hydrogen bond interactions between the two (P₁and P₁) NH groups of A-74704 with Gly27/Gly27' carbonyls ofthe enzyme and (2) the higher desolvation free energy of A-74704compared with its ester analog. The results of these calculationsand their implications for design of HIV-1 protease inhibitorsare discussed.     

Correlation between occupancy and B factor of water molecules in protein crystal structures

An empirical relationship between occupancy and the atomic displacementparameter of water molecules in protein crystal structures hasbeen found by comparing a set of well refined sperm whale myoglobincrystal structures. The relationship agrees with a series ofindependent structural features whose impact on water occupancycan easily be predicted as well as with other known data andis independent of the protein fold. The estimation of the wateroccupancy in protein crystal structures may help in understandingthe physico-chemical properties of the protein–solventinterface and can allow the monitoring of the accuracy of theprotein crystal structure refinement.     

Identification and elimination by site-directed mutagenesis of thermolabile aspartyl bonds in Aspergillus awamori glucoamylase

Both Dative Aspergillus niger glucoamylase and wild-type Aspergillusawamori glucoamylase expressed in Saccharo-myces cerevisiae,which have identical primary structures, undergo hydrolysisat aspartyl bonds at low pH values and elevated temperatures.In native A.niger enzyme the Aspl26–Glyl27 bond was preferentiallycleaved at pH 3.5,while at pH 4.5 cleavage of the Asp257–Pro258and Asp293–Gly294 bonds was dominant. In wild-type A.awamoriglucoamylase, cleavage of the latter was dominant at both pH3.5 and 4.5. Site-directed mutations Aspl26Glu and Glyl27Alain wild-type enzyme decreased specific activities by 60 and30%, respectively, and increased irreversible thermoinactivationrates 3- to 4-fold at pH 4.5. Replacement of Asp257 with Gluand Asp293 with Glu or Gin decreased specific activities by20%, but greatly reduced cleavage of the Asp257–Pro258and Asp293–Gly294 bonds. The Asp257Glu mutant was producedvery slowly and was more thermostable than wild–type glucoamylaseat pH 4.5up to 70°C. Replacement of Asp293 with either Gluor Gln significantly raised protein production and slightlyincreased thermostability at pH 3.5 and 4.5, but not at pH5.     

Recombinant pro-regions from papain and papaya proteinase IV are selective high affinity inhibitors of the mature papaya enzymes

Proteolytic enzymes require the presence of their proregionsfor correct folding. Of the four proteolytic enzymes from Caricapapaya, papain and papaya proteinase IV (PPIV) have 68% sequenceidentity. We find that their proregions are even more similar,exhibiting 73.6% identity. cDNAs encoding the pro-regions ofthese two proteinases have been expressed in Escherichia coliindependently from their mature enzymes. The recombinant pro-regionsof papain and PPIV have been shown to be high affinity inhibitorsof all four of the mature native papaya cysteine proteinases.Their inhibition constants are in the range 10^–6–;10^–;9M. PPIV was inhibited two to three orders of magnitude lesseffectively than papain, chymopapain and caricain. The pro-regionof PPIV, however, inhibited its own mature enzyme more effectivelythan did the proregion of papain. Alignment of the sequencesof the four papaya enzymes shows that there is a highly variablesection towards the C-terminal of the pro-region. This regionmay therefore confer selectivity to the pro-regions for theindividual proteolytic enzymes.     

Model structures and action of interleukin 1 and its antagonist

A comparison has been made between the homology and hydrophobkityprofiles of six interleukin amino add sequences and that ofthe human interleukin 1ß (IL-lß) for whicha crystal structure exists. The resulting sequence alignmentwas used to build model structures for the sequences for threeIL-l, two IL-1ß and an interleukin receptor antagonist.Analysis of these structures demonstrates that the interleukinmolecule has a strong electric dipole which is generated bythe topological position of the amino acids in the sequence.Electrostatic surface calculations implicate a particular residues(Lysl45) as being fundamental to interleukin activity and thissupports site-directed mutation evidence that this residue isrequired for activity.     

Molecular dynamics simulations of HIV-1 protease with peptide substrate

Molecular dynamics simulations of human immunodeficiency virus(HIV)-l protease with a model substrate were used to test ifthere is a stable energy minimum for a proton that is equidistantfrom the four delta oxygen atoms of the two catalytic asparticacids. The crystal structure of HIV-1 protease with a peptidicinhibitor was modified to model the peptide substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val-Glnfor the starting geometry. A proton was positioned between thetwo closest oxygen atoms of the two catalytic aspartic acids,and close to the carbonyl oxygen of the scissile bond in thesubstrate. All crystallographic water molecules were included.Two molecular dynamics simulations were run: 30 ps with united-atompotentials and 40 ps using the more accurate all-atom potentials.The molecular dynamics used a new algorithm that increased thespeed and allowed the elimination of a cut-off for non-bondedinteractions and the inclusion of an 8 shell of water moleculesin the calculations. The overall structure of the protease dimer,including the catalytic aspartic acids, was stable during thecourse of the molecular dynamics simulations. The substrateand a water molecule, that is an important component of thebinding site, were stable during the simulation using all-atompotentials, but more mobile when united-atom potentials wereused. A Poincare map representation showed that the positionsof the proton and its coordinating oxygen atoms were stablefor 93% of both simulations, although many of the buried andpoorly accessible water molecules exchanged with solvent. Theproton has a stable minimum energy position and maintains coordinationwith all four delta oxygen atoms of the two catalytic asparticacids and the carbonyl oxygen of the scissile bond of the substrate.Therefore, a loosely bound hydrogen ion at this position willnot be rapidly exchanged with solvent, and will rebond to eithera catalytic aspartic acid or possibly the substrate. The implicationsfor the reaction mechanism are discussed.     

Two-dimensional surface display of functional groups on a beta-helical antifreeze protein scaffold

We tested a disulfide-rich antifreeze protein as a potential scaffold for design or selection of proteins with the capability of binding periodically organized surfaces. The natural antifreeze protein is a beta-helix with a strikingly regular two-dimensional grid of threonine side chains on its ice-binding face. Amino acid substitutions were made on this face to replace blocks of native threonines with other amino acids spanning the range of beta-sheet propensities. The variants, displaying arrays of distinct functional groups, were studied by mass spectrometry, reversed-phase high performance liquid chromatography, thiol reactivity and circular dichroism and NMR spectroscopies to assess their structures and stabilities relative to wild type. The mutants are well expressed in bacteria, despite the potential for mis-folding inherent in these 84-residue proteins with 16 cysteines. We demonstrate that most of the mutants essentially retain the native fold. This disulfide bonded beta-helical scaffold, thermally stable and remarkably tolerant of amino acid substitutions, is therefore useful for design and engineering of macromolecules with the potential to bind various targeted ordered material surfaces.     

Mapping of residues involved in the interaction between the Bacillus subtilis xylanase A and proteinaceous wheat xylanase inhibitors

The Bacillus subtilis xylanase A was subjected to site-directed mutagenesis, aimed at changing the interaction with Triticum aestivum xylanase inhibitor, the only wheat endogenous proteinaceous xylanase inhibitor interacting with this xylanase. The published structure of Bacillus circulans XynA was used to target amino acids surrounding the active site cleft of B.subtilis XynA for mutation. Twenty-two residues were mutated, resulting in 62 different variants. The catalytic activity of active mutants ranged from 563 to 5635 XU/mg and the interaction with T.aestivum xylanase inhibitor showed a similar variation. The results indicate that T.aestivum xylanase inhibitor interacts with several amino acid residues surrounding the active site of the enzyme. Three different amino acid substitutions in one particular residue (D11) completely abolished the interaction between T.aestivum xylanase inhibitor and B.subtilis xylanase A.     

Rapid crystallization of T4 lysozyme by intermolecular disulfide cross-linking

In an attempt to facilitate crystallization, engineered cysteineswere used to promote formation of a ‘back–to–back’dimer that occurs in different crystal forms of wild–typeand mutant T4 lysozymes. The designed double mutant, N68C/A93C,in which the surface residues Asn68 and Ala93 were replacedby cysteines, formed dimers in solution and crystallized isomorphouslyto wild–type, but at a much faster rate. Overall, themutant structure remained very similar to wildtype despite theformation of two intermolecular disulfide bridges. The crystalsof cross–linked dimers had thermal factors somewhat lowerthan wild–type, indicating reduced mobility, but did notdiffract to noticeably higher resolution. Introduction of thesame cross-links was also used to obtain crystals in a differentspace group of a T4 lysozyme mutant that could not be crystallizedpreviously. The results suggest that the formation of the lysozymedimer is a critical intermediate in the formation of more thanone crystal form and that covalent cross–Unking of theintermediate accelerates nucleation and facilitates crystalgrowth. The disulfide crosslinks are located on the ‘back’of the molecule and formation of the cross–linked dimerappears to leave the active sites completely unobstructed. Nevertheless,the cross–linked dimer is completely inactive. One explanationfor this behavior is that the disulfide bridges prevent hinge-bendingmotion that may be required for catalysis. Another possibilityis that the formation of the dimer increases the overall bulkof the enzyme and prevents its access to the susceptible glycosidkbonds within the cell wall substrate     

Escherichia coli aspartate carbamoyltransferase: the probing of crystal structure analysis via site-specific mutagenesis

Crystal structures are known for aspartate carbamoyltransferase(ATCase) in the T and R states, with and without the allostericactivator adenosine triphosphate (ATP) or inhibitor cytidinetriphosphate (CTP). Visual inspection of X-ray crystal structuresdoes not provide all of the information necessary for the determinationof structure-function relationships in protein molecules. Thisproblem is compounded because the crystalline states of themolecule may introduce effects due to crystal packing, restrictedflexibility and less than optimum enzymatic conditions. Therefore,alternative techniques are required to test mechanisms conjecturedfrom three-dimensional crystal structures of proteins. The techniqueof site-specific mutagenesis allows the researcher to test structure- function models based on threedimensional structures and toobtain further insight into characteristics of the enzyme. Site-specificmutagenesis has been used to probe residues believed to be criticalin the structure and function of ATCase. Selection of residuesto be mutated has depended extensively on three-dimensionalcrystal structures of the enzyme. To date, 48 site-specificmutations at 37 different amino acid sites have been published.Although a total of 118 mutants at 58 different sites has beencommunicated to our laboratory, only published mutants willbe considered in this review. In this paper, we compile forthe first time, review, and analyze the site-specific mutantsof ATCase. Site-specific mutagenesis of proteins has becomea powerful technique in modern-day molecular biology, especiallyin studying a molecule as large as aspartate carbamoyltransferase.In this review, the role of site-specific mutagenesis of ATCaseis discussed and improvements in the analysis are suggested.     

Can three-dimensional contacts in protein structures be predicted by analysis of correlated mutations?

A method has been developed to detect pairs of positions withcorrelated mutations in protein multiple sequence alignments.The method is based on reconstruction of the phylogenetic treefor a set of sequences and statistical analysis of the distributionof mutations in the branches of the tree. The database of homology-derivedprotein structures (HSSP) is used as the source of multiplesequence alignments for proteins of known three-dimensionalstructure. We analyse pairs of positions with correlated mutationsin 67 protein families and show quantitatively that the presenceof such positions is a typical feature of protein families.A significant but weak tendency is observed for correlated residuepairs to be close in the three-dimensional structure. With furtherimprovements, methods of this type may be useful for the predictionof residue-residue contacts and subsequent prediction of proteinstructure using distance geometry algorithms. In conclusion,we suggest a new experimental approach to protein structuredetermination in which selection of functional mutants afterrandom mutagenesis and analysis of correlated mutations providesufficient proximity constraints for calculation of the proteinfold     

Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering

Recognition by ribonuclease T₁ of guanine bases via multidentatehydrogen bonding and stacking interactions appears to be mediatedmainly by a short peptide segment formed by one stretch of aheptapeptide, Tyr⁴²-Asn⁴³-Asn⁴⁴-Tyr⁴⁵-Gly⁴⁶-Gly⁴⁷-Phe⁴⁸. Thesegment displays a unique folding of the polypeptide chain—consistingof a reverse turn, Asn⁴⁴-Tyr⁴⁵-Glu⁴⁶-Gly⁴⁷, stabilized by ahydrogen-bond network involving the side chain of Asn⁴⁴, themain-chain atoms of Asn⁴⁴, Gly⁴⁷ and Phe⁴⁸ and one water molecule.The segment is connected to the C terminus of a ß-strandand expands into a loop region between Asn⁴³ and Ser⁵⁴. Lowvalues for the crystallographic thermal parameters of the segmentindicate that the structure has a rigidity comparable to thatof a ß-pleated sheet. Replacement of Asn⁴⁴ with alanineleads to a far lower enzymatic activity and demonstrates thatthe side chain of Asn⁴⁴ plays a key role in polypeptide foldingin addition to a role in maintaining the segment structure.Substitution of Asn⁴³ by alanine to remove a weak hydrogen bondto the guanine base destabilized the transition state of thecomplex by 6.3 kJ/mol at 37°C. In contrast, mutation ofGlu⁴⁶ to alanine to remove a strong hydrogen bond to the guaninebase caused a destabilization of the complex by 14.0 kJ/mol.A double-mutant enzyme with substitutions of Asn⁴³ by a histidineand Asn⁴⁴ by an aspartic acid, to reproduce the natural substitutionsfound in ribonuclease M_s, showed an activity and base specificitysimilar to that of the wild-type ribonuclease M_s. The segmenttherefore appears to be well conserved in several fungal ribonucleases.     

Activity of linked HIV-1 proteinase dimers containing mutations in the active site region

Mutations were introduced into the active site triplet (Asp–Thr–Gly)of one or both subunits of a linked dimer of human immunodeficiencyvirus type 1 proteinase. Mutation of Thr to Ser in one or bothsubunits did not alter the activity of the enzyme substantially,whereas its mutation to Asn in one subunit caused a dramaticdecrease in catalytic efficiency. Mutation of Gly to Val inone subunit also yielded an enzyme with very low activity. Theenzymes containing Thr Asn and Gly Val mutations in both subunitsresulted in inactive enzymes, based on their inability to self-processand on assay with an oligopeptide substrate. The dramatic decreasein enzyme efficiency of the mutants was interpreted using molecularmodels of the enzymes.     

Sequence divergence analysis for the prediction of seven-helix membrane protein structures: I. Comparison with bacteriorhodopsin

A method using protein sequence divergence to predict the three-dimensionalstructure of the transmembrane domain of seven-helix membraneproteins is described. The key component in the multistep procedureis the calculation of a hydrophilic and lipophilic variabilityindex for each amino acid in an alignment of a family of homologousproteins. The variability profile, a plot of the calculatedvariability index versus alignment position, can be used topredict a tertiary model of the backbone conformation of thetransmembrane domain. This method was applied to bacteriorhodopsin(BR) and the model obtained was compared with the known structureof this protein. Using an alignment of the amino acid sequencesof BR and closely related (20% identity) proteins, the boundariesof the transmembrane regions, their secondary structures andorientations inside the membrane bilayer were predicted basedon the variability profile. Additional information about theshape of the helix bundle was also obtained from the averagevariability of each transmembrane helix with the assumptionthat the helices are packed sequentially and form a closed helixbundle. Correct features of the known structure of BR were foundin the model structure, suggesting that a similar strategy canbe used to predict transmembrane helices and the packing shapeof other membrane proteins with seven transmembrane helices,such as the opsins and other G-protein coupled receptors.     

An assessment of the effect of the helix dipole in protein structures

The locations of the cations bound to the peptide group at theC-termini and the anions attached to the main-chain NH groupat the N-termini of helices are analysed. The ions are hardlyfound along the helical axis, where the effect due to the helixmacrodipole is likely to be the maximum. The disposition ofthe ions appears to be controlled more by the stereoelectronicrequirements of the ligand group rather than any long distanceelectric field. This and other related structural observationscall for some circumspection in assigning a role for the helixdipole in protein structure and function.