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.     

Glycine 85 of the trp-repressor of E.coli is important in forming the hydrophobic tryptophan binding pocket: experimental and computational approaches

Experimental and computational analyses were performed on thecorepressor (L-tryptophan) binding site of the trp-repressorof Escherichia coli to investigate the ligandprotein interactions.Gly 85, one of the residues forming the hydrophobic pocket ofthe binding site, was systematically replaced with Ala, Val,Leu and Trp by cassette mutagenesis. Biochemical characterizationshowed that all these mutations caused significant decreasesin tryptophan binding activity. Free energy perturbation calculationswere performed for the mutants and were consistent with theexperimental results. The lack of a side chain at position 85was concluded to be essential for binding the corepressor; thestructure of the binding pocket was suggested to be tight inthe vicinity of Gly85.     

Contribution of a salt bridge to binding affinity and dUMP orientation to catalytic rate: mutation of a substrate-binding arginine in thymidylate synthase

Invariant arginine 179, one of four arginines that are conservedin all thymidylate synthases (TS) and that bind the phosphatemoiety of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP),can be altered even to a negatively charged glutainic acid withlittle effect on k_cat. In the mutant structures, ordered wateror the other phosphate binding arginines compensate for thehydrogen bonds made by Arg179 in the wild-type enzyme and thereis almost no change in the conformation or binding site of dUMP.Correlation of dUMP K_ds for TS R179A and TS R179K with the structuresof their binary complexes shows that the positive charge onArg179 contributes significantly to dUMP binding affinity. k_cat/Kmfor dUMP measures the rate of dUMP binding to TS during theordered bi-substrate reaction, and in the ternary complex dUMPprovides a binding surface for the cofactor. k_cat/Km reflectsthe ability of the enzyme to accept a properly oriented dUMPfor catalysis and is less sensitive than is K_d to the changesin electrostatics at the phosphate binding site.     

Engineering of protein epitopes: a single deletion in a snake toxin generates full binding capacity to a previously unrecognized antibody

Structural features associated with the ability of a monoclonalantibody (mAb) to discriminate between protein variants areidentified and engineered. The variants are the curaremimetictoxin from Naja nigricollis and erabutoxin a or b from Laticaudasemifasciata which differ from each other by 16 substitutionsand one insertion. The neutralizing mAb M1 recognizes with highaffinity a topographical epitope on the surface of toxin , butfails to recognize the erabutoxins although they possess mostof the residues forming the presumed epitope. Examinations ofthe toxin and erabutoxin 3-D structures and molecular dynamicssimulations reveal several differences between the variants.In particular, the region involving the ß-turn 17–24is organized differently. Analysis of the differences foundin this region suggests that the insertion (or deletion) atposition 18 of the variant amino add sequences is particularlyimportant in determining the differential cross-reactivity.To test this proposal, residue 18 was deleted in one erabutoxinusing sitedirected mutagenesis, and the biological propertiesof the resulting mutant were examined. We found that full antigenicitywas restored in the previously unrecognized variant. The implicationsof this finding are discussed.     

Structural effects induced by removal of a disulfide-bridge: the X-ray structure of the C30A/C51A mutant of basic pancreatic trypsin inhibitor at 1.6 A

The X-ray structure of a variant of basic pancreatic trypsininhibitor (BPTI) has been analyzed to determine the structuralaccommodation resulting from removal of a disulfide crosslinkin a protein. The disulfide removed, Cys30–Cys51, hasbeen implicated in both the folding pathway of the protein andits overall thermal stability. In the variant studied, C30A/C51A,the disulfide cysteines were replaced by less bulky alanines.The atomic displacements observed for C30A/C51A indicate a setof concerted shifts of two segments of chain, which togethersignificantly diminish a packing defect at the site of the removedcysteine sulfur atoms. The observed structural changes are distributedasymmetrically around the sites of mutation, indicating thatthe adjacent ß-sheet is more resistant to the perturbationthan the -helix on the opposite side of the disulfide bond.The thermal parameters of groups involved in the structuralaccommodation are not significantly altered. A comparison ofthe X-ray structures reported for native BPTI determined inthree different crystal forms indicates that the magnitude ofits conformational variability exceeds that of the structuralchanges caused by the disulfide removal. This emphasizes thenecessity of using isomorphous crystal systems to determinethe relatively small effects due to mutation.     

Structure and function of an antifreeze polypeptide from ocean pout, Macrozoarces americanus: role of glutamic acid residues in protein stability and antifreeze activity by site-directed mutagenesis

The successful expression and purification of the recombinantocean pout antifreeze polypeptide (rAFP) in Escherichia colihave enabled the study of its structure-function relationshipby site-directed mutagenesis. The role of carboxyl groups atGlu23 and Glu36 of the rAFP was probed by replacing these residueswith either glutamine or alanine residues as both single anddouble mutants. The AFP mutants were expressed, purified andcharacterized in terms of primary and secondary structures,thermal stability and antifreeze activities. The propertiesof these mutants were compared with those of the rAFP. Threedistinct functions are identified for the carboxyl groups: (i)the negative charges at positions 23 and 36 are involved inthe thermal stability of the polypeptide; (ii) the negativecharges at positions 23 and 36 contribute to the thermal hystereticactivities of the polypeptide; and (iii) the negative chargeat position 23 and hydrogen-bonding ability at position 36 contributeto the ice-binding activity of the polypeptide.