Noncontact Residues Research Articles

Potent, Selective, and Cell-Penetrating Inhibitors of Kallikrein-Related Peptidase 4 Based on the Cyclic Peptide MCoTI-II

Kallikrein-related peptidase 4 (KLK4) is a serine protease that has putative intracellular and extracellular functions in prostate cancer progression. Here we show that MCoTI-II, a 34-amino acid cyclic peptide found in the seeds of red gac (Momordica cochinchinensis), is an inhibitor of KLK4. By grafting a preferred KLK4 cleavage sequence into MCoTI-II, we produced a highly potent KLK4 inhibitor (K i = 0.1 nM) that displayed 100,000-fold selectivity over related KLKs and the ability to penetrate cells. Additionally, by substituting positively charged noncontact residues in this compound, we produced a potent and selective KLK4 inhibitor that does not penetrate cells. The inhibitors were shown to be nontoxic to human cells and stable in human serum. These KLK4 inhibitors provide useful chemical tools to further define the role(s) of both intracellular and extracellular KLK4 in prostate cancer cell lines and disease models.

RosettaAntibodyDesign (RAbD): A general framework for computational antibody design.

A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al., J. Mol. Biol., 406:228–256, 2011). It then performs sequence design according to amino acid sequence profiles of each cluster, and samples CDR backbones using a flexible-backbone design protocol incorporating cluster-based CDR constraints. Starting from an existing experimental or computationally modeled antigen-antibody structure, RAbD can be used to redesign a single CDR or multiple CDRs with loops of different length, conformation, and sequence. We rigorously benchmarked RAbD on a set of 60 diverse antibody–antigen complexes, using two design strategies—optimizing total Rosetta energy and optimizing interface energy alone. We utilized two novel metrics for measuring success in computational protein design. The design risk ratio (DRR) is equal to the frequency of recovery of native CDR lengths and clusters divided by the frequency of sampling of those features during the Monte Carlo design procedure. Ratios greater than 1.0 indicate that the design process is picking out the native more frequently than expected from their sampled rate. We achieved DRRs for the non-H3 CDRs of between 2.4 and 4.0. The antigen risk ratio (ARR) is the ratio of frequencies of the native amino acid types, CDR lengths, and clusters in the output decoys for simulations performed in the presence and absence of the antigen. For CDRs, we achieved cluster ARRs as high as 2.5 for L1 and 1.5 for H2. For sequence design simulations without CDR grafting, the overall recovery for the native amino acid types for residues that contact the antigen in the native structures was 72% in simulations performed in the presence of the antigen and 48% in simulations performed without the antigen, for an ARR of 1.5. For the non-contacting residues, the ARR was 1.08. This shows that the sequence profiles are able to maintain the amino acid types of these conserved, buried sites, while recovery of the exposed, contacting residues requires the presence of the antigen-antibody interface. We tested RAbD experimentally on both a lambda and kappa antibody–antigen complex, successfully improving their affinities 10 to 50 fold by replacing individual CDRs of the native antibody with new CDR lengths and clusters.

An Engineered Switch in T Cell Receptor Specificity Leads to an Unusual but Functional Binding Geometry

Utilizing a diverse binding site, Tcell receptors (TCRs) specifically recognize a composite ligand comprised of a foreign peptide and a major histocompatibility complex protein (MHC). To help understand the determinants of TCR specificity, we studied a parental and engineered receptor whose peptide specificity had been switched via molecular evolution. Altered specificity was associated with a significant change in TCR-binding geometry, but this did not impact the ability of the TCR to signal inan antigen-specific manner. The determinants of binding and specificity were distributed among contact and non-contact residues in germline and hypervariable loops, and included disruption of key TCR-MHC interactions that bias αβ TCRs toward particular binding modes. Sequence-fitness landscapes identified additional mutations that further enhanced specificity. Our results demonstrate that TCR specificity arises from the distributed action of numerous sites throughout the interface, with significant implications for engineering therapeutic TCRs with novel and functional recognition properties.

Naturally selected hepatitis C virus polymorphisms confer broad neutralizing antibody resistance.

For hepatitis C virus (HCV) and other highly variable viruses, broadly neutralizing mAbs are an important guide for vaccine development. The development of resistance to anti-HCV mAbs is poorly understood, in part due to a lack of neutralization testing against diverse, representative panels of HCV variants. Here, we developed a neutralization panel expressing diverse, naturally occurring HCV envelopes (E1E2s) and used this panel to characterize neutralizing breadth and resistance mechanisms of 18 previously described broadly neutralizing anti-HCV human mAbs. The observed mAb resistance could not be attributed to polymorphisms in E1E2 at known mAb-binding residues. Additionally, hierarchical clustering analysis of neutralization resistance patterns revealed relationships between mAbs that were not predicted by prior epitope mapping, identifying 3 distinct neutralization clusters. Using this clustering analysis and envelope sequence data, we identified polymorphisms in E2 that confer resistance to multiple broadly neutralizing mAbs. These polymorphisms, which are not at mAb contact residues, also conferred resistance to neutralization by plasma from HCV-infected subjects. Together, our method of neutralization clustering with sequence analysis reveals that polymorphisms at noncontact residues may be a major immune evasion mechanism for HCV, facilitating viral persistence and presenting a challenge for HCV vaccine development.

Protein-protein interfaces from cytochrome c oxidase I evolve faster than nonbinding surfaces, yet negative selection is the driving force.

Respiratory complexes are encoded by two genomes (mitochondrial DNA [mtDNA] and nuclear DNA [nDNA]). Although the importance of intergenomic coadaptation is acknowledged, the forces and constraints shaping such coevolution are largely unknown. Previous works using cytochrome c oxidase (COX) as a model enzyme have led to the so-called “optimizing interaction” hypothesis. According to this view, mtDNA-encoded residues close to nDNA-encoded residues evolve faster than the rest of positions, favoring the optimization of protein–protein interfaces. Herein, using evolutionary data in combination with structural information of COX, we show that failing to discern the effects of interaction from other structural and functional effects can lead to deceptive conclusions such as the “optimizing hypothesis.” Once spurious factors have been accounted for, data analysis shows that mtDNA-encoded residues engaged in contacts are, in general, more constrained than their noncontact counterparts. Nevertheless, noncontact residues from the surface of COX I subunit are a remarkable exception, being subjected to an exceptionally high purifying selection that may be related to the maintenance of a suitable heme environment. We also report that mtDNA-encoded residues involved in contacts with other mtDNA-encoded subunits are more constrained than mtDNA-encoded residues interacting with nDNA-encoded polypeptides. This differential behavior cannot be explained on the basis of predicted thermodynamic stability, as interactions between mtDNA-encoded subunits contribute more weakly to the complex stability than those interactions between subunits encoded by different genomes. Therefore, the higher conservation observed among mtDNA-encoded residues involved in intragenome interactions is likely due to factors other than structural stability.

Functional Differences of Invariant and Highly Conserved Residues in the Extracellular Domain of the Glycoprotein Hormone Receptors

Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in β-strands 2 (Lys(74)), 4 (Tyr(124), Asn(129), and Thr(130)), and 5 (Asp(150) and Asp(153)) of the FSHR ECD identified in the human FSH·FSHR ECD crystal structure as contact sites with the common glycoprotein hormone α-subunit, and on noncontact residues in β-strands 2 (Ser(78)) and 8 (Asp(224) and Ser(226)) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported α-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSH·FSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation.

Systematic Mutational Analysis of Peptide Inhibition of the p53–MDM2/MDMX Interactions

Inhibition of the interaction between the tumor suppressor protein p53 and its negative regulators MDM2 and MDMX is of great interest in cancer biology and drug design. We previously reported a potent duodecimal peptide inhibitor, termed PMI (TS FAEY WNL LSP), of the p53–MDM2 and –MDMX interactions. PMI competes with p53 for MDM2 and MDMX binding at an affinity roughly 2 orders of magnitude higher than that of 17–28p53 (ET FSDL WKL LPE) of the same length; both peptides adopt nearly identical α-helical conformations in the complexes, where the three highlighted hydrophobic residues Phe, Trp, and Leu dominate PMI or 17–28p53 binding to MDM2 and MDMX. To elucidate the molecular determinants for PMI activity and specificity, we performed a systematic Ala scanning mutational analysis of PMI and 17–28p53. The binding affinities for MDM2 and MDMX of a total of 35 peptides including 10 truncation analogs were quantified, affording a complete dissection of energetic contributions of individual residues of PMI and 17–28p53 to MDM2 and MDMX association. Importantly, the N8A mutation turned PMI into the most potent dual-specific antagonist of MDM2 and MDMX reported to date, registering respective K d values of 490 pM and 2.4 nM. The co-crystal structure of N8A–PMI– 25–109MDM2 was determined at 1.95 Å, affirming that high-affinity peptide binding to MDM2/MDMX necessitates, in addition to optimized intermolecular interactions, enhanced helix stability or propensity contributed by non-contact residues. The powerful empirical binding data and crystal structures present a unique opportunity for computational studies of peptide inhibition of the p53–MDM2/MDMX interactions.

Size-exclusion chromatography can identify faster-associating protein complexes and evaluate design strategies

BackgroundWe previously developed a set of rationally designed mutant MICA protein ligands for the NKG2D immunoreceptor in which MICA was mutated at residues that do not contact NKG2D. Some of these MICA mutants, predicted by RosettaDesign to be destabilized, bound NKG2D with affinities enhanced by more than an order of magnitude when evaluated by surface plasmon resonance (SPR).FindingsSmall-zone size-exclusion chromatography (SEC) detected persistent high-affinity MICA mutant-NKG2D complexes in solution as early-eluting peaks. The SEC binding assay used standard protein purification instrumentation to evaluate complex stability, qualitatively paralleled the SPR results, and successfully discriminated among complexes that differed only in on-rates. We used the SEC binding assay, along with SPR, to assess the results of a follow-up design strategy targeting the non-interfacial redesigned region. Both SEC and SPR agreed that these mutations did not enhance affinity as much as previous mutants. When the SEC binding assay was run in 1 M urea, only the highest affinity complex was detected.ConclusionThis SEC binding assay provides a correlation with SPR results for protein complex affinities, detecting changes in complex on-rates, and tunable to lower sensitivity with 1 M urea. The SEC binding assay is complementary to other protein design evaluation methods, can be adapted to the undergraduate research laboratory, and may provide additional structural information about changes in hydrodynamic radii from elution times. Our assay allowed us to conclude that further alteration of MICA at non-contacting residues is unlikely to further enhance NKG2D affinity.

Correlated evolution of nucleotide substitution rates and allelic variation in Mhc-DRB lineages of primates.

BackgroundThe major histocompatibility complex (MHC) is a key model of genetic polymorphism. Selection pressure by pathogens or other microevolutionary forces may result in a high rate of non-synonymous substitutions at the codons specifying the contact residues of the antigen binding sites (ABS), and the maintenance of extreme MHC allelic variation at the population/species level. Therefore, selection forces favouring MHC variability for any reason should cause a correlated evolution between substitution rates and allelic polymorphism. To investigate this prediction, we characterised nucleotide substitution rates and allelic polymorphism (i.e. the number of alleles detected in relation to the number of animals screened) of several Mhc class II DRB lineages in 46 primate species, and tested for a correlation between them.ResultsFirst, we demonstrate that species-specific and lineage-specific evolutionary constraints favour species- and lineage-dependent substitution rate at the codons specifying the ABS contact residues (i.e. certain species and lineages can be characterised by high substitution rate, while others have low rate). Second, we show that although the degree of the non-synonymous substitution rate at the ABS contact residues was systematically higher than the degree of the synonymous substitution rate, these estimates were strongly correlated when we controlled for species-specific and lineage-specific effects, and also for the fact that different studies relied on different sample size. Such relationships between substitution rates of different types could even be extended to the non-contact residues of the molecule. Third, we provide statistical evidence that increased substitution rate along a MHC gene may lead to allelic variation, as a high substitution rate can be observed in those lineages in which many alleles are maintained. Fourth, we show that the detected patterns were independent of phylogenetic constraints. When we used phylogenetic models that control for similarity between species, due to common descent, and focused on variations within a single lineage (DRB1*03), the positive relationship between different substitution rates and allelic polymorphisms was still robust. Finally, we found the same effects to emerge in the analyses that eliminated within-species variation in MHC traits by using strictly single population-level studies. However, in a set of contrasting analyses, in which we focused on the non-functional DRB6 locus, the correlation between substitution rates and allelic variation was not prevalent.ConclusionOur results indicate that positive selection for the generation of allelic polymorphism acting on the functional part of the protein has consequences for the nucleotide substitution rate along the whole exon 2 sequence of the Mhc-DRB gene. Additionally, we proved that an increased substitution rate can promote allelic variation within lineages. Consequently, the evolution of different characteristics of genetic polymorphism is not independent.

The Most Common Dasatinib-Resistant BCR-ABL Kinase Domain Mutations in Patients with Chronic Myeloid Leukemia Are Sensitive to VX-680: Rationale for Early Combination Kinase Inhibitor Therapy.

Selection for CML cells with BCR-ABL kinase domain mutations represents the predominant molecular mechanism responsible for loss of response to imatinib. Similarly, we have found acquired resistance to dasatinib to be associated with kinase domain mutations in 100% of cases (n=15). However, unlike the multitude of imatinib resistant mutations, which to a large extent occur at non-contact residues and destabilize the inactive confirmation to which imatinib binds, only two mutations appear to be responsible for nearly all cases of dasatinib resistance, T315I and V299L (Shah et al, submitted, ASH 2006). Both of these mutations occur at critical contact residues between the ABL kinase domain and dasatinib. Successful treatment of dasatinib-resistant cases will therefore require strategies to successfully eliminate cells that harbor these mutations.Use of a combination of kinase inhibitors with the ability to collectively suppress all BCR-ABL kinase domain mutants would be predicted to lead to profoundly minimize disease resistance and relapse on targeted therapy. Although the combination of imatinib and dasatinib may prevent selection of the dasatinib-resistant V299L mutation, these agents share many targets, and their combination may therefore result in substantial toxicity. Moreover, the combination of imatinib and dasatinib is not predicted to effectively inhibit the growth of cells harboring BCR-ABL/T315I.We previously have shown that the Aurora kinase inhibitor VX-680 can bind to the ABL kinase domain and inhibit the kinase activity of the T315I mutation at low micromolar concentration. VX-680 is showing early signs of efficacy in CML cases associated with the T315I mutation. Interestingly, the co-crystal structure of VX-680 reveals that V299 is one of 14 contact residues within the ABL kinase domain. Substitution of leucine at this residue might therefore be expected to diminish the potential affinity of VX-680 for BCR-ABL/V299L. However, analysis of the amino acid sequence of Aurora-A revealed divergence from native BCR-ABL at this the corresponding amino acid position due to the presence of a leucine in Aurora-A. BCR-ABL/V299L is therefore a mimetic of Aurora-A, and as a result, predicted to retain sensitivity to VX-680.We therefore assessed VX-680 for its ability to inhibit the kinase activity of BCR-ABL/V299L in Ba/F3 cells and found effective inhibition of the kinase activity at low micromolar concentration. Consistent with predictions based upon structural considerations, the V299L mutation is somewhat more sensitive to VX-680 than BCR-ABL/T315I. We confirmed these results in an analysis of primary human PBMCs obtained from a dasatinib-resistant patient who had evolved the V299L mutation on therapy.Our findings suggest that early combination therapy with two kinase inhibitors, dasatinib and VX-680, may successfully suppress resistant disease by collectively eliminating BCR-ABL kinase domain mutation as a mechanism of resistance, and thereby achieve effective long-term disease control in the vast majority of patients.

Action‐at‐a‐distance interactions enhance protein binding affinity

The identification of protein mutations that enhance binding affinity may be achieved by computational or experimental means, or by a combination of the two. Sources of affinity enhancement may include improvements to the net balance of binding interactions of residues forming intermolecular contacts at the binding interface, such as packing and hydrogen-bonding interactions. Here we identify noncontacting residues that make substantial contributions to binding affinity and that also provide opportunities for mutations that increase binding affinity of the TEM1 beta-lactamase (TEM1) to the beta-lactamase inhibitor protein (BLIP). A region of BLIP not on the direct TEM1-binding surface was identified for which changes in net charge result in particularly large increases in computed binding affinity. Some mutations to the region have previously been characterized, and our results are in good correspondence with this results of that study. In addition, we propose novel mutations to BLIP that were computed to improve binding significantly without contacting TEM1 directly. This class of noncontacting electrostatic interactions could have general utility in the design and tuning of binding interactions.

Peptide Mapping of a Novel Discontinuous Epitope of the Major Surface Adhesin from Streptococcus mutans

Guy's 13 is a mouse monoclonal antibody that specifically recognizes the major cell-surface adhesion protein SA I/II of Streptococcus mutans, one of the major causative agents of dental caries. Passive immunization with Guy's 13 prevents bacterial colonization in humans. To help elucidate the mechanism of prevention of colonization conferred by this antibody, the SA I/II epitope recognized by Guy's 13 was investigated. It was previously established that the epitope is conformational, being assembled from two non-contiguous regions of SA I/II. In the current study, using recombinant fragments of SA I/II and, ultimately, synthetic peptides, the discontinuous epitope was localized to residues 170-218 and 956-969. This work describes the mapping of a novel discontinuous epitope that requires an interaction between each determinant in order for epitope assembly and recognition by antibody to take place. Guy's 13 binds to the assembled epitope but not to these individual epitope fragments. The assembled epitope results from the interaction between the individual antigenic determinants and can be formed by mixing together determinants present on separate polypeptide chains. The data are consistent with one of the epitope fragments adopting a polyproline II-like helical conformation.

Evolution of nuclear- and mitochondrial-encoded subunit interaction in cytochrome c oxidase.

Mitochondrial DNA (mtDNA)-encoded proteins function in eukaryotes as subunits of respiratory complexes that also contain nuclear DNA (nDNA)-encoded subunits. The importance of functional interactions between mtDNA- and nDNA-encoded proteins was previously demonstrated by testing the survivability of cybrid cells or individuals containing nDNA and mtDNA from different populations or species. This report focuses on the multisubunit respiratory complex cytochrome c oxidase (COX), made up of both mtDNA-encoded and nDNA-encoded subunits. A combination of evolutionary and crystallographic data is employed to determine whether rates of nonsynonymous substitutions have been higher, the same, or lower for residues in close proximity that are encoded by a different genome (nDNA or mtDNA). This determination is performed by simply taking the ratio, called the interaction ratio i, of the nonsynonymous substitution rate of the close-contact residues to the nonsynonymous substitution rate of the noncontact residues. We assume that the close-contact residues (which are more likely to interact) are functionally important and that, therefore, amino acid replacements among these residues cannot escape the scrutiny of natural selection. i = 1 indicates that the close-contact residues have been under neither greater purifying selection nor greater positive selection than the noncontact residues as a specific consequence of their being encoded by separate genomes. i < 1 indicates that the close-contact residues have been under greater purifying selection but less positive selection than have the noncontact residues. Conversely, i > 1 indicates that the close-contact residues have been under less purifying but greater positive selection than have the noncontact residues. i < 1 may be referred to as a constraining interaction; i.e., the close-contact residues compared with the noncontact residues appear to be under greater structural-functional constraints. On the other hand, i > 1 may be referred to as an optimizing interaction; i.e., apparently many different amino acid replacements are required to optimize this subunit's interaction with the other subunit. A major finding is that the nDNA-encoded residues in close physical proximity to mtDNA-encoded residues evolve more slowly than the other nuclear-encoded residues (and thus display a constraining interaction), whereas the mtDNA-encoded residues in close physical proximity to nDNA-encoded residues evolve more rapidly than the other mitochondrial-encoded residues (and thus display an optimizing interaction). A possible reason for this striking difference between the nuclear- and mitochondrial-encoded COX subunits in how their functional interaction evolves is discussed.

Thermodynamic Binding Studies of Lectins from the Diocleinae Subtribe to Deoxy Analogs of the Core Trimannoside of Asparagine-linked Oligosaccharides

Lectins from seven different species of the Diocleinae subtribe have been recently isolated and characterized in terms of their carbohydrate binding specificities (Dam, T. K., Cavada, B. S., Grangeiro, T. B., Santos, C. F., de Sousa, F. A. M., Oscarson, S., and Brewer, C. F. (1998) J. Biol. Chem. 273, 12082-12088). The lectins included those from Canavalia brasiliensis, Cratylia floribunda, Dioclea rostrata, Dioclea virgata, Dioclea violacea, and Dioclea guianensis. All of the lectins exhibited specificity for Man and Glc residues, but much higher affinities for the branched chain trimannoside, 3,6-di-O-(alpha-d-mannopyranosyl)-d-mannose, which is found in the core region of all asparagine-linked carbohydrates. In the present study, isothermal titration microcalorimetry is used to determine the binding thermodynamics of the above lectins, including a new lectin from Canavalia grandiflora, to a complete series of monodeoxy analogs of the core trimannoside. From losses in the affinity constants and enthalpies of binding of certain deoxy analogs, assignments are made of the hydroxyl epitopes on the trimannoside that are involved in binding to the lectins. The pattern of binding of the deoxy analogs is similar for all seven lectins, and similar to that of concanavalin A which is also a member of the Diocleinae subtribe. However, differences in the magnitude of the thermodynamic binding parameters of the lectins are observed, even though the lectins possess conserved contact residues in many cases, and highly conserved primary sequences. The results indicate that non-contact residues in the lectins, even those distant from the binding sites, modulate their thermodynamic binding parameters.

Antibody-antigen Interactions: Contact Analysis and Binding Site Topography

We have analysed antigen-contacting residues and combining site shape in the antibody Fv and Fab crystal structures now available from the Protein Data Bank. Antigen-contacting propensities are presented for each antibody residue, allowing a new definition for the complementarity determining regions (CDRs) to be proposed based on observed antigen contacts. Contacts are more common at CDR residues which are located centrally within the combining site; some less central CDR residues are only contacted by large antigens. Non-contacting residues within the CDRs coincide with residues identified by Chothia and co-workers as important in defining “canonical” conformations. An objective means of classifying protein surfaces by gross topography has been developed and applied to the antibody combining site surfaces. The surfaces have been clustered into four topographic classes: concave and moderately concave (mostly hapten binders), ridged (mostly peptide binders) and planar (mostly protein binders). We have determined the topographic classes for ten pairs of complexed and uncomplexed antibody-antigen crystal structures; four change topographic class on complexation. The results will be of use in antibody engineering, antigen docking and in clinical immunology. To demonstrate one application, we show how the data can be used to locate the antigen binding pocket on antibody structures.

Characterization by serial deletion competition ELISAs of HIV-1 V3 loop epitopes recognized by monoclonal antibodies

Characterization of the sites recognized by antibody on the V3 loop of the envelope glycoprotein gp120 of HIV-1 was done by competition ELISAs on a series of four mouse mAbs, a human mAb and a human Fab. The solid-phase antigen consisted of biotin-YNKRKRIHIGPGRAFYTTKN, a sequence from the center of the V3 loop of gp120 MN, applied to streptavidin-coated wells. Competing antigens were two series of peptides with the HIV-1 MN sequence each serially deleted at either the N or C terminus but kept constant at the other terminus. For each series, the amino acid at the deleting end needed to give a minimum K D was identified. The epitope was defined as the sequence including both of the identified amino acids as terminal amino acids. For the six antibodies reported, the epitope length ranged from seven to 14 amino acids. Use of a cyclic peptide as competing fluid-phase antigen suggested the influence of conformational constraints on presumed “linear” epitopes. The operationally-defined epitope was longer than the contact residues in one of two instances in which the X-ray crystallographic structure had been determined. The longer estimates of epitope length in the current study based on competition ELISAs with serial deletions suggest that non-contact residues are significant both in epitope definition and in functional applications including immunogen design.

Study of antibody-antigen interaction through site-directed mutagenesis of the VH region of a hybrid phage-antibody fragment.

An important aspect of the study of antibody structure-function relationships involves analysis of natural or synthetic mutations of antigen-combining sites. The anti-hen egg lysozyme monoclonal antibody HyHEL-10 has been a focus for antibody structure-function studies. We have displayed on bacteriophage of a hybrid single chain Fv, containing the light chain variable region of HyHEL-10 and the heavy chain variable region of a structurally related but functionally distinct antibody, AS32. By using a combination of site-directed mutagenesis, complementary determining region grafting and molecular modeling, we have identified a number of contact and non-contact residues that are important in the affinity of HyHEL-10 for lysozyme. In particular, the heavy chain variable region framework residue at position 94 was shown to be an important determinant of high-affinity binding. The phage display approach eliminates the need for purification of antibodies and, when used in combination with polymerase chain reaction for variable region sequence mutagenesis, facilitates the rapid generation and characterization of mutant antibodies.

Structural factors governing hemin dissociation from metmyoglobin.

Rates of hemin dissociation from approximately 100 different metmyoglobin mutants were measured to determine which amino acid residues are important for retaining the prosthetic group. Most of the amino acids examined are within 4 A of the porphyrin ring, but replacements of a number of noncontact residues were also made. Mutations of His93(F8) and Leu89(F4) can result in > 100-fold increases in the rate of hemin loss at pH 5 and 7. Some replacements of the contact residues His64(E7), Val68(E11), His97(FG3), Ile99(FG5), Thr39(C4), and Tyr103(G4) cause > 10-fold changes in the rate of hemin dissociation. Substitutions of the noncontact residues Leu29(B10), Phe46(CD4), and Gly65(E8) can also increase the rate of hemin loss > 10-fold. The key structural factors stabilizing bound hemin in myoglobin are (1) hydrophobic interactions between apolar residues in the heme pocket and the porphyrin ring, (2) the covalent bond between His93(F8) and the Fe3+ atom, and (3) hydrogen bonding between distal residues and coordinated water. Specific electrostatic interactions between the heme propionates and amino acids at the surface of the protein appear to be less important. Loss of these polar interactions can be compensated by increasing the apolar character of either the heme group by esterification of the propionates or replacement of charged surface residues with large apolar side chains [e.g., replacing His97(FG3) with Phe].

Two binding orientations for peptides to the Src SH3 domain: development of a general model for SH3-ligand interactions.

Solution structures of two Src homology 3 (SH3) domain-ligand complexes have been determined by nuclear magnetic resonance. Each complex consists of the SH3 domain and a nine-residue proline-rich peptide selected from a large library of ligands prepared by combinatorial synthesis. The bound ligands adopt a left-handed polyproline type II (PPII) helix, although the amino to carboxyl directionalities of their helices are opposite. The peptide orientation is determined by a salt bridge formed by the terminal arginine residues of the ligands and the conserved aspartate-99 of the SH3 domain. Residues at positions 3, 4, 6, and 7 of both peptides also intercalate into the ligand-binding site; however, the respective proline and nonproline residues show exchanged binding positions in the two complexes. These structural results led to a model for the interactions of SH3 domains with proline-rich peptides that can be used to predict critical residues in complexes of unknown structure. The model was used to identify correctly both the binding orientation and the contact and noncontact residues of a peptide derived from the nucleotide exchange factor Sos in association with the amino-terminal SH3 domain of the adaptor protein Grb2.