Class II major histocompatibility complex (MHC) proteins bind peptides and present them at the cell surface for interaction with CD4؉ T cells as part of the system by which the immune system surveys the body for signs of infection. Peptide binding is known to induce conformational changes in class II MHC proteins on the basis of a variety of hydrodynamic and spectroscopic approaches, but the changes have not been clearly localized within the overall class II MHC structure. To map the peptideinduced conformational change for HLA-DR1, a common human class II MHC variant, we generated a series of monoclonal antibodies recognizing the ␤ subunit that are specific for the empty conformation. Each antibody reacted with the empty but not the peptide-loaded form, for both soluble recombinant protein and native protein expressed at the cell surface. Antibody binding epitopes were characterized using overlapping peptides and alanine scanning substitutions and were localized to two distinct regions of the protein. The pattern of key residues within the epitopes suggested that the two epitope regions undergo substantial conformational alteration during peptide binding. These results illuminate aspects of the structure of the empty forms and the nature of the peptide-induced conformational change. Major histocompatibility complex (MHC)1 molecules are heterodimeric cell-surface proteins that play an important role in the initiation of antigen-specific immune responses. Class II MHC proteins bind peptides derived from extracellular, endosomal, and internalized cell-surface antigens, and present them at the cell surface for inspection by CD4 ϩ T cells (1). Three-dimensional structures have been determined for peptide complexes of several polymorphic variants of both human and murine class II MHC molecules (reviewed in Ref.2). Both the MHC ␣ and ␤ chains contribute to the peptide binding site, which is made up of a ␤ sheet floor topped by two roughly parallel ␣ helical regions. Each subunit contributes an immunoglobulin-like domain below the peptide binding site, as well as short transmembrane and cytoplasmic domains. Peptides bind in an extended conformation in the groove between the two helices, with ϳ10 residues able to interact with the MHC protein, and the peptide termini extending from the binding site. This conformation, similar to a polyproline type II helix, has a 2.7-residue repeat and appears to be dictated by a network of conserved hydrogen bonding interactions between the MHC and bound peptide (3). The conformation places 4 -6 of the peptide side chains into pockets within the overall groove. The residues lining these pockets vary between allelic variants, providing different peptide-sequence binding specificity. Overall the interaction buries ϳ70% of the peptide surface area in the central region of a bound peptide, leaving the remainder available for interaction with antigen receptors on T cells (4).Although the canonical structure visualized by x-ray crystallography is relatively stereotyped, a number of studies h...

Serine and thiol proteases react with peptide substrates to form an acyl-enzyme. We have synthesized inhibitors which are pseudo-substrates and react with the proteases to generate acyl-enzymes which hydrolize slowly. This is achieved by incorporating an electron-donating group near the carbonyl group of inhibitors I [Ac-Phe--C(O)NH--NH--C(O)X] and II [benzyl-O-C(O)-psiAla-Leu-ArgOMe]. The acyl-enzymes derived from the reaction of I with papain and II with chymotrypsin hydrolyze with t1/2 of 12 and 1 h, respectively. The increased electron density on the carbonyl group of the inhibitor also reduces the rate of acyl-enzyme formation. Components were incorporated into the inhibitor which interact with the leaving group binding site (S' subsite) and which accelerate the rate of reaction of inhibitor with enzyme. For inhibitor I, X = NH(CH3), k(on) < 0.13 M(-1) s(-1) for the reaction papain, but if X = psiLeu(CH3)2,k(on) =10(5) M(-1) s(-1). Similar results were obtained with II and chymotrypsin. Concomitant with acyl-enzyme formation, X is released and a slowly hydrolyzing acyl-enzyme remains.

The mRNA display approach to in vitro protein selection is based upon the puromycin-mediated formation of a covalent bond between an mRNA and its gene product. This technique can be used to identify peptide sequences involved in macromolecular recognition, including those identical or homologous to natural ligand epitopes. To demonstrate this approach, we determined the peptide sequences recognized by the trypsin active site, and by the anti-c-Myc antibody, 9E10. Here we describe the use of two peptide libraries of different diversities, one a constrained library based on the trypsin inhibitor EETI-II, where only the six residues in the first loop were randomized (6.4 x 10(7) possible sequences, 6.0 x 10(11) sequences in the library), the other a linear-peptide library with 27 randomized amino acids (1.3 x 10(35) possible sequences, 2 x 10(13) sequences in the library). The constrained library was screened against the natural target of wild-type EETI, bovine trypsin, and the linear library was screened against the anti-c-myc antibody, 9E10. The analysis of selected sequences revealed minimal consensus sequences of PR(I,L,V)L for the first loop of EETI-II and LISE for the 9E10 epitope. The wild-type sequences, PRILMR for the first loop of EETI-II and QKLISE for the 9E10 epitope, were selected with the highest frequency, and in each case the complete wild-type epitope was selected from the library.

Class II major histocompatibility complex proteins bind peptides for presentation to T-cells as part of the immune response process. Monoclonal antibody MEM-265 recognizes the peptide-free conformation of the major histocompatibility complex class II protein HLA-DR1 through specific binding to an epitope contained between residues 50 -67 of the ␤-chain. In previous work using alanine scanning (1), we identified residues Leu-53, Asp-57, Tyr-60, Trp-61, Ser-63, and Leu-67 as essential for specific recognition by MEM-265. The spacing of these residues approximates a 3.5-residue repeat, suggesting that MEM-265 may recognize the epitope in an ␣-helical conformation. In the folded, peptide-loaded DR1 structure, the ␤-chain residues 50 -67 contain a kinked ␣-helical segment spanning Glu-52-Ser-63 (2). However, the conformation of this segment in the peptide-free form is unknown. We have used a new surface plasmon resonance approach in a SpotMatrix format to compare the kinetic rates and affinities for 18 alanine scanning mutants comprising epitope residues 50 -67. In addition to the six essential residues described previously, we found two additional residues, Glu-52 and Gln-64, that contribute by enhancing MEM-265 binding. By contrast, mutation of either Gly-54 or Pro-56 to an alanine actually improved binding to MEM-265. In essentially all cases peptide substitutions that either improve or reduce MEM-265 recognition could be traced to differences in the dissociation rate (k off ). The kinetic details of the present study support the presence of a structural component in the antigenic epitope recognized by MEM-265 in the peptide-free form of major histocompatibility complex II DR1 ␤-chain.Research on factors influencing molecular recognition is an area of vibrant activity. The molecular basis of specificity for antigen-antibody recognition can involve numerous factors and is not limited to the primary amino acid sequence of the antigenic epitope. The epitope can have a complex structural component in the context of the entire tertiary structure of the antigen. In such a case, an analogous linear variant of the original antigen, for instance, a short peptide, would not be expected to retain much structural information, particularly in the absence of any disulfide bridges. However, antibodies that can recognize short peptides that appear to adopt a defined conformation have previously been described (3, 4).MEM-265 is a mouse monoclonal antibody that recognizes the empty conformation of the human class II MHC protein HLA-DR1. Although the antibody was raised against the denatured ␤ subunit, it appears to recognize a conformational epitope present in the native ␣-␤ heterodimer, which becomes unavailable upon peptide binding (1). Preliminary characterization has mapped the epitope to residues 50 -67 of the ␤ subunit, with residues Leu-53, Asp-57, Tyr-60, Trp-61, Ser-63, and Leu-67 being essential for binding. To assess the individual contributions of nonessential residues to binding, we used SpotMatrix SPR 1 to obtain a co...

Deubiquitinating enzymes (DUBs) have emerged as promising drug targets, but few small molecule DUB inhibitors have been identified. Herein we introduce carbonates as a novel class of mechanism‐based DUB inhibitors. Activity profiling was used to determine inhibitor potency and specificity in cell lysates. The most potent inhibitor, C17, has a preference for USP9x and USP7. In K562 cells, C17 (50 micromolar) causes the degradation of Bcr‐Abl kinase, as expected when USP9x is inhibited. In MCF7 cells, C17 (25 micromolar) causes the degradation of Mdm2 and upregulation of P53, as expected when USP7 is inhibited. We believe that carbonates will be useful tools for studying the ubiquitination pathways and provide a new strategy for the design of cysteine protease inhibitors. Grant Funding Source: Supported by the NIH R01 GM100921(LH), HHMI International Student Fellowship (MJCL).