Free energy simulations of the HyHEL-10/HEL antibody-antigen complex

Free energy simulations are reported for the N31^L-D mutation,both in the HyHEL-10-HEL antibody-lysozyme complex and in theunliganded antibody, using the thermo-dynamic-cycle perturbationmethod. The present study suggests that the mutation would changethe free energy of binding of the complex by –5.6 kcal/mol(unrestrained free energy simulations), by –0.5 kcal/mol(free energy simulations with a restrained backbone) and by1.8 kcal/ mol (Poisson-Boltzmann calculations, which also usea restrained geometry model). A detailed structural analysishelps in estimating the contributions from various residuesand regions of the system. Enhanced recognition of HEL by themutant HyHEL-10 would arise from the combination of thermodynamicallymore favorable conformational changes of the CDR loops uponassociation and subsequent charge pairing with Lys96 in theantigen.     

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.     

Free energy changes associated with amino acid substitution in proteins

The estimation of free energy differences from computer simulationof macromolecular systems is important for rational strategiesfor drug design and for protein engineering. As an example ofone mutation, we have studied the free energy change resultingfrom the conversion of a polar group (OH) to an apolar group(CH₃) in aqueous solution. We have estimated the effect of variouslocal environments on the magnitude of the free energy differenceand find that significant environmental effects are found. Wehave also studied the reliability of the results in detail.     

Empirical free energy calculations of ligand-protein crystallographic complexes. I. Knowledge-based ligand-protein interaction potentials applied to the prediction of human immunodeficiency virus 1 protease binding affinity

The steadily increasing number of high-resolution human immunodeficiencyvirus (HIV) 1 protease complexes has been the impetus for theelaboration of knowledge-based mean field ligand-protein interactionpotentials. These potentials have been linked with the hydrophobicityand conformational entropy scales developed originally to explainprotein folding and stability. Empirical free energy calculationsof a diverse set of HIV-1 protease crystallographic complexeshave enabled a detailed analysis of binding thermodynamics.The thermodynamic consequences of conformational changes thatHIV-1 protease undergoes upon binding to all inhibitors, anda substantial concomitant loss of conformational entropy bythe part of HIV-1 protease that forms the ligand-protein interface,have been examined. The quantitative breakdown of the entropy-drivenchanges occurring during ligand-protein association, such asthe hydrophobic contribution, the conformational entropy termand the entropy loss due to a reduction of rotational and translationsaldegrees of freedom, of a system composed of ligand, proteinand crystallographic water molecules at the ligand-protein interfacehas been carried out The proposed approach provides reasonableestimates of distinctions in binding affinity and gives an insightinto the nature of enthalpy-entropy compensation factors detectedin the binding process.     

Highly effective protease inhibitors from variants of human pancreatic secretory trypsin inhibitor (hPSTI): an assessment of 3-D structure-based protein design

The results of a protein design project are used to comparedifferent predictive strategies with respect to proteinproteininteractions. We have been able to generate variants of humanpancreatic secretory trypsin inhibitor (hPSTI) optimized withrespect to the affinity and specificity for human leukocyteelastase relative to trypsin and chymotrypsin, and in particularchymotrypsin. The extremely strong and specific human leukocyteelastase inhibitors were thus developed in three rounds of mutagenesisand two rounds of 3-D modelling; only 24 variants in total weresynthesized, although variations at seven different amino acidpositions were involved (i.e. from 20⁷ possible variants). Anexcellent elastase inhibitor could be designed with the minimumof two amino acid exchanges. The value of structural modellingand actual structure determination is discussed in the lightof the experimental results of the designed protein variantsand the results of tertiary structure determinations of thefree variant and the inhibitorprotease complex. Particular referenceis given to the strategy to be followed in protein design projectsin general and to the development of protease inhibitors inparticular.     

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.     

Estimation of binding free energies for HIV proteinase inhibitors by molecular dynamics simulations

Absolute binding free energies for three inhibitors of HIV–1proteinase were estimated from molecular dynamics simulationsby a recently reported linear approximation procedure. The resultswere in fairly good agreement with experimental binding data.Two of the inhibitors were very similar and, for comparison,their relative free energies of binding were also calculatedby free energy perturbation methods, giving virtually the sameresult Effects of cutoff radii and charge states of the proteinmodel were examined. The effects of pH on binding of one ofthe inhibitors were predicted.     

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.     

A loop-mimetic inhibitor of the HCV-NS3 protease derived from a minibody

We have been interested for some time in establishing a strategyfor deriving lead compounds from macromolecule ligands suchas minibody variants. A minibody is a minimized antibody variabledomain whose two loops are amenable to combinatorial mutagenesis.This approach can be especially useful when dealing with `difficult'targets. One such target is the NS3 protease of hepatitis Cvirus (HCV), a human pathogen that is believed to infect about100 million individuals worldwide and for which an effectivetherapy is not yet available. Based on known inhibitor specificity(residues P6-P1) of NS3 protease, we screened a number of minibodiesfrom our collection and we were able to identify a competitiveinhibitor of this enzyme. We thus validated an aspect of recognitionby HCV NS3 protease, namely that an acid anchor is necessaryfor inhibitor activity. In addition, the characterization ofthe minibody inhibitor led to the synthesis of a constrainedhexapeptide mimicking the bioactive loop of the parent macromolecule.The cyclic peptide is a lead compound prone to rapid optimizationthrough solid phase combinatorial chemistry. We therefore confirmedthat the potential of turning a protein ligand into a low molecularweight active compound for lead discovery is achievable andcan complement more traditional drug discovery approaches.     

Calculations of antibody-antigen interactions: microscopic and semi-microscopic evaluation of the free energies of binding of phosphorylcholine analogs to McPC603

The study of antibody-antigen interactions should greatly benefitfrom the development of quantitative models for the evaluationof binding free energies in proteins. The present work addressesthis challenge by considering the test case of the binding freeenergies of phosphorylcholine analogs to the murine myelomaprotein McPC603. This includes the evaluation of the differentialbinding energy as well as the absolute binding energies andtheir corresponding electrostatic contributions. Four differentapproaches are examined: the Protein Dipoles Langevin Dipoles(PDLD) method, the semi-microscopic PDLD (PDLD/S) method, afree energy perturbation (FEP) method based on an adiabaticcharging procedure and a linear response approximation thataccelerates the FEP calculation. The PDLD electrostatic calculationsare augmented by estimates of the relevant hydrophobic and stericcontributions. The determination of the hydrophobic energy involvesan approach which considers the modification of the effectivesurface area of the solute by local field effects. The stericcontributions are analyzed in terms of the corresponding reorganizationenergies. This treatment, which considers the protein as a harmonicsystem, views the steric forces as the restoring forces forthe electrostatic interactions. The FEP method is found to giveunreliable results with regular cut-off radii and starts togive quantitative results only in very expensive treatment withvery large cut-off radii. The PDLD and PDLD/S methods are muchfaster than the FEP approach and give reasonable results forboth the relative and absolute binding energies. The speed andsimplicity of the PDLD/S method make it an effective strategyfor interactive docking studies and indeed such an option isincorporated in the program MOLARIS. A component analysis ofthe different energy contributions of the FEP treatment anda similar PDLD analysis indicate that electrostatic effectsprovide the largest contribution to the differential bindingenergy, while the hydrophobic and steric contributions are muchsmaller. This finding lends further support to the idea thatelectrostatic interactions play a major role in determiningthe antigen specificity of McPC603.     

The nature of the ion binding interactions in EF-hand peptide analogs: free energy simulation of Asp to Asn mutations

The binding of the La³⁺ ion to a tridecapeptide, which is amodel for the EF-hand in calcium-binding proteins, is studiedhi solution by free energy simulations. The calculations analyzethe effect on the La³⁺ ion binding of the mutation of Asp toAsn for side chains that interact directly with the ion. Theresults are compared with the measurements of Marsden.B.J.,Hodges, R.S. and Sykes, B.D. (1989) Biochemistry, 28,8839, onthe same system. They found that the Asp to Asn mutation hasonly a small effect on the binding; the observed differencesin the free energies on changing one Asp to an Asn are between-0.3 and 1.8 kcal/ mol. This result is analyzed by alchemicalsimulations for the tridecapeptide in the bound Qoop) structureand free (extended) form. The free energy changes due to themutation of an Asp to an Asn are large and positive for boththe bound and free forms. However, since the values of the freeenergy changes are calculated to be similar hi the two forms,the difference in the binding free energy of Asp and Asn peptidesis found to be small, in agreement with experiment. By use ofthermodynamic integration, the various contributions to thefree energy changes are estimated. In the com-plexed form, theAsp to Asn mutation is favored by the reduction in the repulsiveinteraction with other charged residues of the peptide; it isdisfavored by the reduction of the stabilization of the ionand the surrounding water has a small effect. When the peptideadopts an extended conformation in the absence of the ion, themutation Asp to Asn is strongly disfavored by the interactionswith the water and is favored by the interactions within thepeptide. The results demonstrate the essential role of contributionsto the binding of EF-hands from interactions other than thosebetween the ion and the charged amino acid side chains. Theresults obtained from the simulations suggest, in accord withcrystal structures of La³⁺ bound to various ligands, that thecalcium-binding loop complexed with La³⁺ in solution has a significantlydifferent structure from that observed hi proteins.     

Effects of deletion in the flexible loop of the protease inhibitor SSI (Streptomyces subtilisin inhibitor) on interactions with proteases

The Streptomyces subtilisin inhibitor (SSI) is a proteinaceousprotease inhibitor which inhibits serine proteases by forminga stable Michaelis complex. The flexible loop region (Thr64–Val69)is a very flexible region in an SSI molecule and its importancein interactions with proteases has been suggested, since conformationalchange of this loop was found to occur for the smooth bindingof SSI with various proteases. In this study, mutated SSIs lackingone or two residues in this region were generated and the effectsof deletions on the interaction with proteases were investigated.Deletion was introduced into mutated SSI(Lys73) and SSI(Gly70Lys73)both known to be trypsin inhibitors, to examine the effectsof deletion on interactions with subtilisin BPN' or trypsin.The deletion of one residue (Gly66) caused increased inhibitoryactivity toward trypsin, indicating the protruding flexibleloop hinders binding with trypsin. Reduction of such hindranceby one-residue shortening in this loop is shown to be effectivefor the interaction of SSI(Lys73) with trypsin. In contrast,one-residue shortening had virtually no effect on inhibitiontoward subtilisin BPN'. Differences in the subsite structuresof these proteases may have been the reason for this contrast.The deletion of two residues (Thr64 and Gly66) in this regionconverted SSI into a temporary inhibitor. Structural analysisof the degradation intermediate showed that the peptide bondat the reactive site of doubly deleted SSI was cleaved by subtilisinBPN' after its binding with protease. Thus, the irreversibilityof the cleaved peptide bond at the reactive site of mutatedSSI in the complex with protease may possibly be the cause forits temporary inhibition. Irregular conformation around thereactive site caused by the deletion of two residues in theflexible loop would convert SSI into a temporary inhibitor.Thus, moderate flexibility in the flexible loop region may possiblybe a structural requirement for SSI to function.     

Binding free energy calculations for P450cam-substrate complexes

A recently proposed semi-empirical method for calculating bindingfree energies was used to examine the binding of a variety ofsubstrates to cytochrome P450cam. For a set of 11 differentpotential substrates of cytochrome P450cam, both the absoluteand relative binding free energies were generally well reproduced.The mean error in the calculated absolute binding free energyfor all 11 compounds is 0.55 kcal/mol. Forty-eight out of 55calculated relative binding free energies have the correct signand the mean unsigned error between calculated and experimentalrelative binding free energies is 0.77 kcal/mol. For one substrate,thiocamphor, the effect of substrate orientation on the calculatedbinding free energy was examined. The ability of this methodto predict the effect of active site mutations was also examinedin two cases.     

A chimeric cysteine protease of Plasmodium berghei engineered to resemble the Plasmodium falciparum protease falcipain-2

The cysteine proteases falcipain-2 and falcipain-3 are hemoglobinases and potential targets for chemotherapy directed against Plasmodium falciparum, the most important human malaria parasite. Most in vivo evaluations of candidate antimalarials are conducted in murine malaria models, and falcipain homologs from rodent malaria parasites differ importantly from falcipain-2 and falcipain-3. We expressed berghepain-2, the single homolog of falcipain-2 and falcipain-3 of the rodent parasite P. berghei, in Escherichia coli, and characterized the refolded active enzyme. Berghepain-2 was biochemically very similar to the previously characterized rodent plasmodial protease vinckepain-2, but differed from falcipain-2 and falcipain-3 in its fine substrate and inhibitor specificity. We then used homology modeling and evolutionary trace analysis to predict key amino acids that mediate functional differences between falcipain-2 and berghepain-2. Thirteen amino acids were sequentially altered to replace berghepain-2 residues with those in falcipain-2. Mutant enzymes varied in activity and sensitivity to inhibitors. A berghepain-2 mutant with eight substitutions retained good activity and demonstrated fine substrate and inhibitor sensitivity more similar to that of falcipain-2 than berghepain-2. These results suggest that, to facilitate drug discovery, we can produce mutant animal model malaria parasites with biochemical properties more like those of the key drug target, P. falciparum.     

The binding mode of an E-64 analog to the active site of cathepsin B

Two binding modes of the isobutyl-NH-Eps-Leu-Pro inhibitor tocathepsin B have been proposed. Molecular docking using an empiricalforce field was carried out to distinguish between the two modes.The search began with manual docking, followed by random perturbationsof the docking conformation and cycles of Monte Carlo minimization.Finally, molecular dynamics was carried out for the most favorabledocking conformations. The present calculations predict thatthe isobutyl-NH-Eps-Leu-Pro inhibitor preferentially binds tothe S' rather than the S subsites of cathepsin B. The S' bindingmode prediction is supported by the X-ray crystal structureof cathepsin B bound to a closely related ethyl-O-Eps-Ile-Proinhibitor, which was found to bind in the S' subsite with theC-terminal epoxy ring carbon making a covalent bond to the sulfuratom of Cys29. This agreement, in turn, validates our dockingstrategy. Furthermore, the calculations provide evidence thatthe dominant contribution to the total stabilization energyof the enzyme–inhibitor complex stems from the strongelectrostatic interaction between the negatively charged C-terminalcarboxylate group of the ligand and the positively charged imidazoliumrings of His110 and His111. The latter are stabilized and heldin an optimal orientation for interactions with the C-terminalend of the ligand through a salt bridge between the side chainsof His110 and Asp22. By comparison with the crystal structure,some insight into the specificity of the epoxyldipeptide familytowards cathepsin B inhibition has been extracted. Both thecharacteristics of the enzyme (e.g. subsite size and hydrophobicity)as well as the nature of the inhibitor influence the selectivityof an inhibitor towards an enzyme.     

The interpretation of site-directed mutagenesis experiments by linear free energy relations

Fersht and co-workers have applied a linear free energy relation(Brønsted equation) to analyze site-directed mutagenesisexperiments involving the enzyme tyrosyl-tRNA synthetase andhave suggested that the Brønsted exponent is linearlycorrelated with the value of the reaction coordinate at thetransition state. We point out that when the mutants differsolely through the formation or deletion of a hydrogen bondaway from the reaction center, a linear free energy relationis expected only in limiting cases for which the Brønstedrelation exponent is 0, 1 or . The results may be correlatedwith a conformational coordinate but not with the developmentof the reaction coordinate per se.     

Can the stability of protein mutants be predicted by free energy calculations?

The use of free energy simulation techniques in the study ofprotein stability is critically evaluated. Results from twosimulations of the thermostability mutation Asn218 to Ser218in Subtilisin are presented. It is shown that components ofthe free energy change can be highly sensitive to the computationaldetails of the simulation leading to the conclusion that freeenergy calculations cannot currently be used to reliably predictprotein stability. The different factors that undermine thereliability are discussed.     

Synthesis of a squash-type protease inhibitor by gene engineering and effects of replacements of conserved hydrophobic amino acid residues on its inhibitory activity

Cucurbita maxima trypsin inhibitor I (CMTI-I), a member of thesquash-type protease inhibitor family, is composed of 29 aminoacids and shows strong inhibition of trypsin by its compactstructure. To study the structure–function relationshipof this inhibitor using protein engineering methods, we constructedan expression system for CMTI-I as a fused protein with porcineadenylate kinase (ADK). A Met residue was introduced into thejunction of ADK and CMTI-I to cleave the fusion protein withCNBr, whereas a Met at position 8 of authentic CMTI-I was replacedby Leu. Escherichia coli JM109 transformed with the constructedplasmid expressed the fused protein as an inclusion body. Aftercleavage of the expressed protein with CNBr, fully reduced speciesof CMTI-I were purified by reversed-phase HPLC and then oxidizedwith air by shaking. For efficient refolding of CMTI-I, we used50 mM NH₄HCO₃ (pH 7.8) containing 0.1% PEG 6000 at higher proteinconcentration. Strong inhibitory activity toward trypsin wasdetected only in the first of three HPLC peaks. The inhibitorconstant of CMTI-I thus obtained, in which Met8 was replacedby Leu, was 1.4x10-¹⁰ M. The effect of replacement of Met withLeu at position 8 was shown to be small by comparison of theinhibitor constant of authentic CMTI-III bearing Lys at position9 (8.9x10-¹¹ M) with that of its mutant bearing Leu at position8 and Lys at position 9 (1.8x10-¹⁰ M). To investigate the roleof the well conserved hydrophobic residues of CMTI-I in itsinteraction with trypsin, CMTI-I mutants in which one or allof the four hydrophobic residues were replaced by Ala were prepared.The inhibitor constants of these mutants indicated that thosewith single replacements were 5–40 times less effectiveas trypsin inhibitors and that the quadruple mutant was –450times less effective, suggesting that the hydrophobic residuesin CMTI-I contribute to its tight binding with trypsin. However,each mutant was not converted to a temporary inhibitor.     

Probing the nature of substrate binding in Humicola lanuginosa lipase through X-ray crystallography and intuitive modelling

The catalytic triad of the neutral lipase from Humicola lanuginosais buried by a short helix under aqueous conditions renderingthe enzyme inactive. Upon adsorption to a lipid substrate interfacethis helix is displaced, thereby exposing the active site (interfacialactivation). By covalently linking inhibitors to the activeserine, it is possible to crystallize the enzyme in an interfaciallyactivated state. Two such structures are reported here whichmimic the tetrahedral transition states of lipolysis. To date,no crystal structures of a lipase–triglyceride complexexist for this enzyme. Therefore, possible interactions betweenthis lipase and its substrate have been analysed through molecularmodelling.     

Relative binding free energy calculations of inhibitors to two mutants (Glu46-- Ala/Gln) of ribonuclease T1 using molecular dynamics/free energy perturbation approaches

We present free energy perturbation calculations on the complexesof Glu46— Ala46 (E46A) and Glu46— Gln46 (E46Q) mutantsof ribonuclease T1 (RNaseT1) with inhibitors 2‘-guanosinemonophosphate (GMP) and 2’adenosine monophosphate (AMP)by a thermodynamic perturbation method implemented with moleculardynamics (MD). Using the available crystal structure of theRNaseT1–GMP complex, the structures of E46A-GMP and E46Q-GMPwere model built and equilibrated with MD simulations. The structuresof E46A-AMP and E46Q-AMP were obtained as a final structureof the GMP—AMP perturbation calculation respectively.The calculated difference in the free energy of binding (Gbind)was 0.31 kcal/mol for the E46A system and —1.04 kcal/molfor the E46Q system. The resultant free energies are much smallerthan the experimental and calculated value of 3 kcal/mol forthe native RNase T1, which suggests that both mutants have greaterrelative adenine affinities than native RNaseT1. EspeciallyE46Q is calculated to have a larger affinity for adenine thanguanine, as we suggested previously from the calculation onthe native RNaseT1. Thus, the molecular dynamics/free energyperturbation method may be helpful in protein engineering, directedtoward increasing or changing the substrate specificity of enzymes.