Peptide and peptide mimetic inhibitors of antigen presentation by HLA-DR class II MHC molecules. Design, structure-activity relationships, and X-ray crystal structures.

Molecular features of ligand binding to MHC class II HLA-DR molecules have been elucidated through a combination of peptide structure-activity studies and structure-based drug design, resulting in analogues with nanomolar affinity in binding assays. Stabilization of lead compounds against cathepsin B cleavage by N-methylation of noncritical backbone NH groups or by dipeptide mimetic substitutions has generated analogues that compete effectively against protein antigens in cellular assays, resulting in inhibition of T-cell proliferation. Crystal structures of four ternary complexes of different peptide mimetics with the rheumatoid arthritis-linked MHC DRB10401 and the bacterial superantigen SEB have been obtained. Peptide-sugar hybrids have also been identified using a structure-based design approach in which the sugar residue replaces a dipeptide. These studies illustrate the complementary roles played by phage display library methods, peptide analogue SAR, peptide mimetics substitutions, and structure-based drug design in the discovery of inhibitors of antigen presentation by MHC class II HLA-DR molecules.

Peptidomimetic compounds that inhibit antigen presentation by autoimmune disease-associated class II major histocompatibility molecules.

We have identified a heptapeptide with high affinity to rheumatoid arthritis-associated class II major histocompatibility (MHC) molecules. Using a model of its interaction with the class II binding site, a variety of mimetic substitutions were introduced into the peptide. Several unnatural amino acids and dipeptide mimetics were found to be appropriate substituents and could be combined into compounds with binding affinities comparable to that of the original peptide. Compounds were designed that were several hundred-fold to more than a thousand-fold more potent than the original peptide in inhibiting T-cell responses to processed protein antigens presented by the target MHC molecules. Peptidomimetic compounds of this type could find therapeutic use as MHC-selective antagonists of antigen presentation in the treatment of autoimmune diseases.

Different modes of peptide interaction enable HLA-DQ and HLA-DR molecules to bind diverse peptide repertoires.

The role of HLA-DQ molecules in Ag presentation has, thus far, remained elusive. Here we report that two DQ allotypes, DQ7 (DQA1*0501/B1*0301) and DQ9 (DQA1*0201/B1*0303), are capable of binding peptide repertoires in complementarity with DR molecules. The results reflect fundamental differences in the binding modes of these two HLA class II isotypes, in that DQ7 and DQ9 but not DR molecules appear to have the capacity to bind peptide structures without type 1-like anchor residues. Consistent with this is our observation that none of the amino acid side chains of the class II-associated invariant chain peptides (CLIP) are required for association with DQ7 and DQ9, even though many of them are essential for CLIP-DR interaction. Together, these data reveal a functional complementarity of HLA-DR and -DQ molecules in Ag presentation.

T cell repertoire: genomic or somatic bias toward recognition of major histocompatibility complex molecules?

The prevailing concept about a major influence of thymic positive selection on shaping the T cell repertoire during ontogeny is confronted with an old idea emphasizing a dominant role for genetic (evolutionary) factors in molding the recognition potential of mature T cells. Our recent results are not readily interpreted without introducing a new version of the old concept, according to which complementarity to the major histocompatibility complex peptide-binding site is a major evolutionary selective pressure on T cell antigen receptor variable genes, with alloreactivity being a reflection of this fact.

The helper T-cell repertoire of mice expressing class II major histocompatibility complex beta chains in the absence of alpha chains.

Mutant mice generated by disrupting the H2-Aab major histocompatibility complex (Mhc) gene are demonstrated here to express Abetab chains in the absence of alpha chains. These mice possess a CD4(+) helper T cell (Th) repertoire that uses predominantly the Vbeta7 T-cell antigen receptor (Tcr) segment for recognition of any protein antigen presented by the alpha-free Abeta molecule. As an alloantigen, the Aalpha-free Abeta molecule is recognized very poorly by T cells from a series of class II disparate mouse strains, indicating that it is grossly different from normal alpha/beta heterodimers. Indeed, molecular modeling suggests a beta/beta homodimer arrangement with an altered geometry of the Tcr contact area. Interestingly, the mutant mice exhibit normal alloreactivity, without a restricted Vbeta usage, toward a series of foreign alpha/beta class II heterodimers, although their T cells developed in the absence of such heterodimers. Thus, the complementarity of Tcr to normal alpha/beta heterodimers, and thereby also alloreactivity, appears to be an ontogeny independent (i. e., germline-encoded) feature.

Peptide binding specificity of HLA-DR4 molecules: correlation with rheumatoid arthritis association.

We have investigated whether sequence 67 to 74 shared by beta chains of rheumatoid arthritis (RA)-associated HLA-DR molecules imparts a specific pattern of peptide binding. The peptide binding specificity of the RA-associated molecules, DRB1*0401, DRB1*0404, and the closely related, RA nonassociated DRB1*0402 was, therefore, determined using designer peptide libraries. The effect of single key residues was tested with site-directed mutants of DRB1*0401. The results have demonstrated striking differences between RA-linked and unlinked DR allotypes in selecting the portion of peptides that interacts with the 67-74 area. Most differences were associated with a single amino acid exchange at position 71 of the DR beta chain, and affected the charge of residues potentially contacting position 71. The observed binding patterns permitted an accurate prediction of natural protein derived peptide sequences that bind selectively to RA-associated DR molecules. Thus, the 67-74 region, in particular position 71, induces changes of binding specificity that correlate with the genetic linkage of RA susceptibility. These findings should facilitate the identification of autoantigenic peptides involved in the pathogenesis of RA.

A negative electrostatic determinant mediates the association between the Escherichia coli trp repressor and its operator DNA.

The electrostatic potential surfaces were characterized for trp repressor models that bind to DNA with sequence specificity, without specificity, and not at all. Comparisons among the surfaces were used to isolate protein surface features likely to be important in DNA binding. Models that differ in protein conformation and tryptophan-analogue binding consistently showed positive potential associated with the protein surfaces that interact with the DNA major groove. However, negative potential is associated with the trp repressor surface that contacts the DNA minor groove. This negative potential is significantly neutralized in the protein conformation that is bound to DNA. Positive potential is also associated with the tryptophan binding-site surface, a consequence of the tryptophan- or tryptophan analogue-induced allosteric change. This protein region is complementary to the strongest negative potential associated with the DNA phosphate backbone and is also present in the isolated protein structure from the protein-DNA complex. The effects of charge-change mutation, pH dependence, and salt dependence on the electrostatic potential surfaces were also examined with regard to their effects on protein-DNA binding constants. A consistent model is formed that defines a role for long-range electrostatics early in the protein-DNA association process and complements previous structural, molecular association, and mutagenesis studies.

Molecular dynamics studies of a DNA-binding protein: 2. An evaluation of implicit and explicit solvent models for the molecular dynamics simulation of the Escherichia coli trp repressor.

Although aqueous simulations with periodic boundary conditions more accurately describe protein dynamics than in vacuo simulations, these are computationally intensive for most proteins. Trp repressor dynamic simulations with a small water shell surrounding the starting model yield protein trajectories that are markedly improved over gas phase, yet computationally efficient. Explicit water in molecular dynamics simulations maintains surface exposure of protein hydrophilic atoms and burial of hydrophobic atoms by opposing the otherwise asymmetric protein-protein forces. This properly orients protein surface side chains, reduces protein fluctuations, and lowers the overall root mean square deviation from the crystal structure. For simulations with crystallographic waters only, a linear or sigmoidal distance-dependent dielectric yields a much better trajectory than does a constant dielectric model. As more water is added to the starting model, the differences between using distance-dependent and constant dielectric models becomes smaller, although the linear distance-dependent dielectric yields an average structure closer to the crystal structure than does a constant dielectric model. Multiplicative constants greater than one, for the linear distance-dependent dielectric simulations, produced trajectories that are progressively worse in describing trp repressor dynamics. Simulations of bovine pancreatic trypsin were used to ensure that the trp repressor results were not protein dependent and to explore the effect of the nonbonded cutoff on the distance-dependent and constant dielectric simulation models. The nonbonded cutoff markedly affected the constant but not distance-dependent dielectric bovine pancreatic trypsin inhibitor simulations. As with trp repressor, the distance-dependent dielectric model with a shell of water surrounding the protein produced a trajectory in better agreement with the crystal structure than a constant dielectric model, and the physical properties of the trajectory average structure, both with and without a nonbonded cutoff, were comparable.

Variability of conformations at crystal contacts in BPTI represent true low-energy structures: correspondence among lattice packing and molecular dynamics structures.

The structures of five basic pancreatic trypsin inhibitor (BPTI) molecules are compared to establish the extent and nature of the conformational variability resulting from crystal packing effects. BPTI is an ideal system to evaluate such factors because of the availability of high resolution X-ray models of five different BPTI structures, each in a different crystal packing environment. Differences observed among the structures are found to be distributed throughout the molecule, although the regions that display most variability are associated with the loop structures (residues 14-17 and 24-29). The regions of structure that show the largest rms deviations from the mean of the five packing motifs correlate well with the presence of intermolecular contacts in the crystal lattice. For most of the molecules there is also a correspondence between a larger number of intermolecular contacts and systematically higher B-factors, although it is not apparent whether this is induced by the crystal contact or results from the fact that the contacts are made predominantly through surface loops. The conformational differences seen among the X-ray models constitute more than local shifts at the lattice contact surfaces, and in fact involve in some cases the making and breaking of intramolecular H-bonds. The magnitudes of the differences among packing models are significantly larger than those usually associated with changes induced by mutagenesis; for instance; the structural differences at the site of mutation observed on removing an internal disulfide from the molecule are significantly less than those associated with lattice contact effects. The crystal packing conformations are compared to representative structures of BPTI generated during a 96-psec molecular dynamics (MD) simulation. This comparison shows a high level of correspondence between the protein flexibility indicated by the X-ray and MD analyses, and specifically between those regions that are most variable. This suggests that the regions that show most variability among the crystal packing models are not artifacts of crystallization, but rather represent true low-energy conformers that have been preferentially selected by crystallization factors.

Assimilation of 3H in photosynthesizing leaves exposed to HTO.

Potato and wine grape plants were exposed for a period of 4 h to tritiated water vapor, and simultaneously to 14CO2 which served as a tracer for photosynthetic assimilation. During and after exposure, foliar samples were collected in which the exchangeable and nonexchangeable fractions of organic 3H were determined, taking care that the exchangeable fraction should not be lost to atmosphere. It was demonstrated that about 20% of the organic H in vegetation could be exchanged with 3H of tissue water, and that nonexchangeable 3H was fixed by photosynthesis. The kinetic behavior of the 2 forms was very different.