Identification of novel HLA-B27 ligands derived from polymorphic regions of its own or other class I molecules based on direct generation by 20 S proteasome.

HLA-B27 is strongly associated with ankylosing spondylitis. Natural HLA-B27 ligands derived from polymorphic regions of its own or other class I HLA molecules might be involved in autoimmunity or provide diversity among HLA-B27-bound peptide repertoires from individuals. In particular, an 11-mer spanning HLA-B27 residues 169-179 is a natural HLA-B27 ligand with homology to proteins from Gram-negative bacteria. Proteasomal digestion of synthetic substrates demonstrated direct generation of the B27-(169-179) ligand. Cleavage after residue 181 generated a B27-(169-181) 13-mer that was subsequently found as a natural ligand of B*2705 and B*2704. Its binding to HLA-B27 subtypes in vivo correlated better than B27-(169-179) with association to spondyloarthropathy. Proteasomal cleavage generated also a peptide spanning B*2705 residues 150-158. This region is polymorphic among HLA-B27 subtypes and class I HLA antigens. The peptide was a natural B*2704 ligand. Since this subtype differs from B*2705 at residue 152, it was concluded that the ligand arose from HLA-B*3503, synthesized in the cells used as a source for B*2704-bound peptides. Thus, polymorphic HLA-B27 ligands derived from HLA-B27 or other class I molecules are directly produced by the 20 S proteasome in vitro, and this can be used for identification of such ligands in the constitutive HLA-B27-bound peptide pool.     

A major portion of DNA gyrase inhibitor microcin B17 undergoes an N,O-peptidyl shift during synthesis

Microcin B17 (McB) is a 43-amino acid antibacterial peptide targeting the DNA gyrase. The McB precursor is ribosomally produced and then post-translationally modified by the McbBCD synthase. Active mature McB contains eight oxazole and thiazole heterocycles. Here, we show that a major portion of mature McB contains an additional unusual modification, a backbone ester bond connecting McB residues 51 and 52. The modification results from an N → O shift of the Ser(52) residue located immediately downstream of one of McB thiazole heterocycles. We speculate that the N,O-peptidyl shift undergone by Ser(52) is an intermediate of post-translational modification reactions catalyzed by the McbBCD synthase that normally lead to formation of McB heterocycles.     

Augmentation of the affinity of HLA class I-binding peptides lacking primary anchor residues by manipulation of the secondary anchor residues

A direct binding assay has been used to investigate the effect of the secondary anchor residues on peptide binding to class I proteins of the major histocompatibility complex. Based on predictions from a previous chemometric approach, synthetic peptide analogues containing unnatural amino acids were synthesized and tested for B*2705 binding. Hydrophobic unnatural amino acids such as α-naphthyl- and cyclohexyl-alanine were found to be excellent substituents in the P3 secondary anchor position giving peptides with very high B*2705-binding affinity. The binding to B*2705 of peptides optimized for their secondary anchor residues, but lacking one of the P2 or P9 primary anchor residues was also investigated. Most such peptides did not bind, but one peptide, lacking the P2 Arg residue generally considered essential for binding to all B27 subtypes, was found to bind quite strongly. These findings demonstrate that peptide binding to class I proteins is due to a combination of all the anchor residues, which may be occupied also by unnatural amino acids–a necessary step towards the development of peptidic or non-peptidic antagonists for immunomodulation.     

A Molecular Basis for the Presentation of Phosphorylated Peptides by HLA-B Antigens

As aberrant protein phosphorylation is a hallmark of tumor cells, the display of tumor-specific phosphopeptides by Human Leukocyte Antigen (HLA) class I molecules can be exploited in the treatment of cancer by T-cell-based immunotherapy. Yet, the characterization and prediction of HLA-I phospholigands is challenging as the molecular determinants of the presentation of such post-translationally modified peptides are not fully understood. Here, we employed a peptidomic workflow to identify 256 unique phosphorylated ligands associated with HLA-B*40, -B*27, -B*39, or -B*07. Remarkably, these phosphopeptides showed similar molecular features. Besides the specific anchor motifs imposed by the binding groove of each allotype, the predominance of phosphorylation at peptide position 4 (P4) became strikingly evident, as was the enrichment of basic residues at P1. To determine the structural basis of this observation, we carried out a series of peptide binding assays and solved the crystal structures of HLA-B*40 in complex with a phosphorylated ligand or its nonphosphorylated counterpart. Overall, our data provide a clear explanation to the common motif found in the phosphopeptidomes associated to different HLA-B molecules. The high prevalence of phosphorylation at P4 is dictated by the presence of the conserved residue Arg62 in the heavy chain, a structural feature shared by most HLA-B alleles. In contrast, the preference for basic residues at P1 is allotype-dependent and might be linked to the structure of the A pocket. This molecular understanding of the presentation of phosphopeptides by HLA-B molecules provides a base for the improved prediction and identification of phosphorylated neo-antigens, as potentially used for cancer immunotherapy.     

Peptide presentation to an alloreactive CTL clone is modulated through multiple mechanisms involving polymorphic and conserved residues in HLA-B27

This study addressed the mechanisms by which HLA class I polymorphism modulates allorecognition. CTL 27S69 is an alloreactive clone raised against HLA-B*2705, with a known peptide epitope. This CTL cross-reacts with B*2702, which differs from B*2705 in the D77N, T80I, and L81A changes, but not with B*2701, which has D74Y, D77N, and L81A changes. To explain this differential recognition, B*2705 mutants mimicking subtype changes were used. The A81 mutant was not recognized, despite binding the natural epitope in vivo, suggesting that, when bound to this mutant, this peptide adopts an inappropriate conformation. The N77 and I80 mutations restored recognition in the N77A81 or I80A81 mutants. These compensatory effects explain the cross-reaction with B*2702. The Y74 and the Y74N77 mutants were weakly recognized or not recognized by CTL 27S69. This correlated with the absence or marginal presence of the peptide epitope in the Y74N77-bound pool. As with B*2701, exogenous addition of the peptide epitope sensitized Y74 and Y74N77 targets for lysis, indicating that failure to cross-react with B*2701 or these mutants was due to poor binding of the peptide in vivo and not to inappropriate presentation. The abrogating effect of Y74 was critically dependent upon the K70 residue, conserved among subtypes, as demonstrated with mutants at this position. Thus, HLA polymorphism affects allorecognition by modulating peptide binding or the conformation of bound peptides. Compensatory mutations and indirect effects of a polymorphic residue on residues conserved play a critical role.     

Modification of a tumor-derived peptide at an HLA-A2 anchor residue can alter the conformation of the MHC-peptide complex: probing with TCR-like recombinant antibodies

A common assumption about peptide binding to the class I MHC complex is that each residue in the peptide binds independently. Based on this assumption, modifications in class I MHC anchor positions were used to improve the binding properties of low-affinity peptides (termed altered peptide ligands), especially in the case when tumor-associated peptides are used for immunotherapy. Using a new molecular tool in the form of recombinant Abs endowed with Ag-specific MHC-restricted specificity of T cells, we show that changes in the identity of anchor residues may have significant effects, such as altering the conformation of the peptide-MHC complex, and as a consequence, may affect the TCR-contacting residues. We herein demonstrate that the binding of TCR-like recombinant Abs, specific for the melanoma differentiation Ag gp100 T cell epitope G9-209, is entirely dependent on the identity of a single peptide anchor residue at position 2. An example is shown in which TCR-like Abs can recognize the specific complex only when a modified peptide, G9-209-2 M, with improved affinity to HLA-A2 was used, but not with the unmodified natural peptide. Importantly, these results demonstrate, using a novel molecular tool, that modifications at anchor residues can dramatically influence the conformation of the MHC peptide groove and thus may have a profound effect on TCR interactions. Moreover, these results may have important implications in designing modifications in peptides for cancer immunotherapy, because most such peptides studied are of low affinity.     

Computational characterization of residue couplings and micropolymorphism-induced changes in the dynamics of two differentially disease-associated human MHC class-I alleles

Human major histocompatibility complex class I (MHC I) – or human leukocyte antigen (HLA) – proteins present intracellularly processed peptides to cytotoxic T lymphocytes in the adaptive immune response to pathogens. A high level of polymorphism in human MHC I proteins defines the peptide-binding specificity of thousands of different MHC alleles. However, polymorphism as well as the peptide ligand can also affect the global dynamics of the complex. In this study, we conducted classical molecular dynamics simulations of two HLA alleles, the ankylosing spondylitis (AS) associated/tapasin-dependent HLA-B*27:05 and nondisease-associated/tapasin-independent HLA-B*27:09, both in peptide-free forms as well as complex with four different peptides ligands. Our results indicate that in peptide-free form, the single amino acid substitution distinguishing the two alleles (D116H), leads to a weaker dynamic coupling of residues in the tapasin-dependent HLA-B*27:05. In peptide-bound form, several residues of the binding-groove, mostly in A and B pockets, show hinge-like behavior in the global motion of the MHC. Moreover, allele-dependent changes are shown in residue interactions, affecting the B-pocket as well as the beta-2-microglobulin (β2m)-facing residues of the HLA chain.     

Loss of recognition by cross-reactive T cells and its relation to a C-terminus-induced conformational reorientation of an HLA-B*2705-bound peptide

The human major histocompatibility complex class I antigen HLA‐B*2705 binds several sequence‐related peptides (pVIPR, RRKWRRWHL; pLPM2, RRRWRRLTV; pGR, RRRWHRWRL). Cross‐reactivity of cytotoxic T cells (CTL) against these HLA‐B*2705:peptide complexes seemed to depend on a particular peptide conformation that is facilitated by the engagement of a crucial residue within the binding groove (Asp116), associated with a noncanonical bulging‐in of the middle portion of the bound peptide. We were interested whether a conformational reorientation of the ligand might contribute to the lack of cross‐reactivity of these CTL with a peptide derived from voltage‐dependent calcium channel α1 subunit (pCAC, SRRWRRWNR), in which the C‐terminal peptide residue pArg9 could engage Asp116. Analyses of the HLA‐B*2705:pCAC complex by X‐ray crystallography at 1.94 Å resolution demonstrated that the peptide had indeed undergone a drastic reorientation, leading it to adopt a canonical binding mode accompanied by the loss of molecular mimicry between pCAC and sequence‐related peptides such as pVIPR, pLMP2, and pGR. This was clearly a consequence of interactions of pArg9 with Asp116 and other F‐pocket residues. Furthermore, we observed an unprecedented reorientation of several additional residues of the HLA‐B*2705 heavy chain near the N‐terminal region of the peptide, including also the presence of double conformations of two glutamate residues, Glu63 and Glu163, on opposing sides of the peptide binding groove. Together with the Arg‐Ser exchange at peptide position 1, there are thus multiple structural reasons that may explain the observed failure of pVIPR‐directed, HLA‐B*2705‐restricted CTL to cross‐react with HLA‐B*2705:pCAC complexes.     

Crystal structures of HLA-A*0201 complexed with antigenic peptides with either the amino- or carboxyl-terminal group substituted by a methyl group

The crystal structures of class I major histocompatibility complex (MHC) molecules complexed with antigenic peptides revealed a network of hydrogen bonds between the charged amino- and carboxyl-termini of the peptides and conserved MHC residues at both ends of the peptide binding site. These interactions were shown to contribute substantially to the stability of class I MHC/peptide complexes by thermal denaturation studies using synthetic peptides in which either the amino- or carboxyl-terminal group is substituted by a methyl group. Here we report crystal structures of HLA-A*0201 complexed with these terminally modified synthetic peptides showing that they adopt the same bound conformation as antigenic peptides. A number of variations in peptide conformation were observed for the terminally modified peptides, including in one case, a large conformational difference in four central peptide residues that is apparently caused by the lattice contact. This is reminiscent of the way binding a T-cell receptor changed the conformation of central residues of an MHC-bound peptide. The structures determined identify which conserved hydrogen bonds are eliminated in terminally substituted peptides and suggest an increased energetic importance of the interactions at the peptide termini for MHC-peptide stability. Proteins 33:97–106, 1998. © 1998 Wiley-Liss, Inc.     

Beta-amino acid scan of a class I major histocompatibility complex-restricted alloreactive T-cell epitope

An HLA-B27-restricted self-octapeptide known to react with an alloreactive T-cell receptor has been modified by systematic substitution of a beta-amino acid for the natural alpha-amino acid residue, over the whole length of the parent epitope. All modified peptides were shown to bind to recombinant HLA-B*2705 and induce stable major histocompatibility complex-peptide complexes, but with some variation depending on the position of the beta-amino acid on the peptide sequence. Alteration of the natural peptide sequence at the two N-terminal positions (positions 1 and 2) decreases binding affinity and thermodynamic stability of the refolded complex, but all other positions (from position 3 to the C-terminal residue) were insensitive to the beta-amino acid substitution. All modified peptides were recognized by an alloreactive T-cell clone specific for the parent epitope with decreased efficiency, to an extent dependent of the position that was modified. Furthermore, the introduction of a single beta-amino acid at the first two positions of the modified peptide was shown to be sufficient to protect them against enzymatic cleavage. Thus, beta-amino acids represent new interesting templates for alteration of T-cell epitopes to design either synthetic vaccines of T-cell receptor antagonists.     

A novel form of neurotensin post-translationally modified by arginylation

A novel bioactive form of neurotensin post-translationally modified at a Glu residue was isolated from porcine intestine. Purification of the peptide was guided by detection of intracellular Ca2+ release in SK-N-SH neuroblastoma cells. Using high resolution accurate mass analysis on an ion trap Fourier transform mass spectrometer, the post-translational modification was identified as arginine linked to the gamma-carboxyl of Glu via an isopeptide bond, and we named the newly identified peptide "arginylated neurotensin" (R-NT, N-(neurotensin-C5-4-yl)arginine). Although arginylation is a known modification of N-terminal amino groups in proteins, its presence at a Glu side chain is unique. The finding places neurotensin among the few physiologically active peptides that occur both in post-translationally modified and unmodified forms. Pharmacologically, we characterized R-NT for its ligand activity on three known neurotensin receptors, NTR1, -2, and -3, and found that R-NT has similar pharmacological properties to those of neurotensin, however, with a slightly higher affinity to all three receptors. We expressed the intracellular receptor NTR3 as a soluble protein secreted into the cell culture medium, which allowed characterization of its R-NT and neurotensin binding properties. The creation of soluble NTR3 also provides a potential tool for neutralizing neurotensin action in vivo and in vitro. We have shown that SK-N-SH neuroblastoma cells express NTR1 and NTR3 but not NTR2, suggesting that the Ca2+ mobilization elicited by R-NT is via NTR1.     

Synthesis and ex vivo profiling of chemically modified cytomegalovirus CMVpp65 epitopes.

The effect of substituting unnatural hydrophobic amino acids into the critical MHC binding residues of an HLA-A*0201-restricted cytomegalovirus CMVpp65 epitope, NLVPMVATV, has been investigated. A new set of peptides containing the amino acids tert-butyl glycine (Tgl), cyclohexyl glycine (Chg), neo-pentyl glycine (Npg), cyclohexyl alanine (Cha) and cyclo leucine (Cyl), at either position 2, to mimic Leu, or position 9, to mimic Val, have been synthesised. Immunological profiling using class I MHC stabilisation assays to assess MHC binding affinity, and enzyme-linked immunospot (ELISPOT) assays to assess the ability of the modified peptides to re-stimulate a specific cytotoxic T-lymphocyte (CTL) response, compared to the native epitope, have been performed. It was found that the majority of the unnatural substitutions resulted in a decrease in either HLA-A*0201 binding affinity or cytotoxic T-cell activity. However, the HLA-A*0201 binding affinity was unrelated to the ability to re-stimulate a T-cell response. Minimisation and molecular dynamics studies proved helpful in dissecting the ELISPOT responses. Two principal peptide binding modes were found by minimisation, designated kinked and straight. Peptides that bound in a kinked conformation were poor at re-stimulating a T-cell response. Of the peptides that bound in a straight conformation, molecular dynamics (MD) simulations revealed that those capable of re-stimulating the strongest responses had the greatest degree of flexibility (as determined by RMSD values across the MD simulation) around the P6 residue, one of the residues important for T-cell receptor recognition.     

A Viral,Transporter Associated with Antigen Processing (TAP)-independent,High Affinity Ligand with Alternative Interactions Endogenously Presented by the Nonclassical Human Leukocyte Antigen E Class I Molecule

The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8⁺ lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.     

Identification of the peptide-binding motif recognized by the pigtail macaque class I MHC molecule Mane-A1*082:01 (Mane A*0301)

Rhesus and pigtail macaques have proven to be valuable animal models for several important human diseases, including HIV, where they exhibit similar pathology and disease progression. Because rhesus macaques have been extensively characterized in terms of their major histocompatibility complex (MHC) class I alleles, their demand has soared, making them increasingly difficult to obtain for research purposes. This problem has been exacerbated by a continued export ban in place since 1978. Pigtail macaques represent a potential alternative animal model. However, because their MHC class I alleles have not been characterized in detail, their use has been hindered. To address this, in the present study, we have characterized the peptide binding specificity of the pigtail macaque class I allele Mane-A1*082:01 (formerly known as Mane A*0301), representative of the second most common MHC class I antigen detected across several cohorts. The motif was defined on the basis of binding studies utilizing purified MHC protein and panels of single amino acid substitution analog peptides, as well as sequences of peptide ligands eluted from Mane-A1*082:01. Based on these analyses, Mane-A1*082:01 was found to recognize a motif with H in position 2 and the aromatic residues F and Y, or the hydrophobic/aliphatic residue M, at the C-terminus. Finally, analysis of the binding of a combinatorial peptide library allowed the generation of a detailed quantitative motif that proved effective in the prediction of a set of high-affinity binders derived from chimeric SIV/HIV, an important model virus for studying HIV infection in humans.     

The resistance to tryptic hydrolysis of peptide bonds adjacent to N epsilon,N-dimethyllysyl residues

A peptide from sperm whale myoglobin, residues 132-153, and a chromogenic substrate, H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide diacetate, were selected to investigate the susceptibility of peptide bonds adjacent to N epsilon,N-dimethyllysyl residues to tryptic hydrolysis. The peptides were exhaustively methylated using formaldehyde and sodium cyanoborohydride (N. Jentoft and D. G. Dearborn (1979) J. Biol. Chem. 254, 4359-4365). Unmodified and methylated peptides were digested with trypsin or submaxillary protease, an enzyme that catalyzes the hydrolysis of only arginyl bonds in proteins. Trypsin catalyzed the hydrolysis of the methylated apomyoglobin peptide only at the single arginyl residue and not at any of the four N epsilon,N-dimethyllysyl residues. Trypsin also failed to catalyze the hydrolysis of reductively methylated H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide. Even a 17-fold molar excess of the methylated substrate did not appear to alter the rate of tryptic hydrolysis of the unmodified substrate. These results are discussed with regard to the interactions of substrates within the specificity site of trypsin.     

Biological heterogeneity of the peptide-binding motif of the 70-kDa heat shock protein by surface plasmon resonance analysis

70-kDa heat shock protein family is a molecular chaperone that binds to a variety of client proteins and peptides in the cytoplasm. Several studies have revealed binding motifs between 70-kDa heat shock protein family and cytoplasmic proteins by conventional techniques such as phage display library screening. However, little is known about the binding motif based on kinetic parameters determined by surface plasmon resonance analysis. We investigated the major inducible cytosolic 70-kDa heat shock protein (Hsp70)-binding motif with the human leukocyte antigen B*2702-derived peptide Bw4 (RENLRIALRY) by using a Biacore system based on surface plasmon resonance analysis. The K(D) value of Hsp70-Bw4 interaction was 1.8 x 10(-6) m. Analyses with truncated Bw4 variant peptides showed the binding motif of Hsp70 to be seven residues, LRIALRY. To further study the characteristics of this motif, 126 peptides derived from Bw4, each with single amino acid substitution, were synthesized and analyzed for Hsp70 binding affinity. Interestingly, the Hsp70 binding affinity was abrogated when the residues were substituted for by acidic (Asp and Glu) ones at any position. In contrast, if the substitute residue was aromatic (Trp, Tyr, and Phe) or an Arg residue at any position, Hsp70 binding affinity was maintained. Thus, this study presents a new binding motif between Hsp70 and peptides derived from the natural protein human leukocyte antigen B*2702 and may also elucidate some characteristics of the Hsp70 binding characteristic, enhancing our understanding of Hsp70-binding determinants that may influence diverse cellular and physiological processes.     

Large scale mass spectrometric profiling of peptides eluted from HLA molecules reveals N-terminal-extended peptide motifs

The majority of >2000 HLA class I molecules can be clustered according to overlapping peptide binding specificities or motifs recognized by CD8(+) T cells. HLA class I motifs are classified based on the specificity of residues located in the P2 and the C-terminal positions of the peptide. However, it has been suggested that other positions might be relevant for peptide binding to HLA class I molecules and therefore be used for further characterization of HLA class I motifs. In this study we performed large-scale sequencing of endogenous peptides eluted from K562 cells (HLA class I null) made to express a single HLA molecule from HLA-B*3501, -B*3502, -B*3503, -B*3504, -B*3506, or -B*3508. Using sequence data from >1,000 peptides, we characterized novel peptide motifs that include dominant anchor residues extending to all positions in the peptide. The length distribution of HLA-B35-bound peptides included peptides of up to 15 residues. Remarkably, we determined that some peptides longer than 11 residues represented N-terminal-extended peptides containing an appropriate HLA-B35 peptide motif. These results provide evidence for the occurrence of endogenous N-terminal-extended peptide-HLA class I configurations. In addition, these results expand the knowledge about the identity of anchor positions in HLA class I-associated peptides that can be used for characterization of HLA class I motifs.     

DNA binding domain of Escherichia coli DNA polymerase I: identification of arginine-841 as an essential residue

To identify the DNA binding site(s) in Escherichia coli DNA polymerase I (pol I) (Klenow fragment), we have used an active-site-directed reagent, phenylglyoxal (PG), which specifically reacts with arginine residues. Preincubation of DNA pol I with PG resulted in the loss of polymerase, 3'-5'-exonuclease, and DNA binding functions. Furthermore, the presence of DNA but not deoxynucleoside triphosphates protected the enzyme from inactivation. Labeling studies with [7-14C]PG indicated that two arginine residues were modified per mole of enzyme. In order to locate the site of PG modification, we digested the PG-treated enzyme with trypsin and V-8 protease. The resulting peptides from each digest were then resolved on reverse-phase hydrophobic columns. An appearance of a new peptide peak was observed in both tryptic and V-8 protease digests. Since inclusion of template-primer during PG modification of enzyme blocks the appearance of these peaks, these peptides were concluded to represent the template-primer binding domain of pol I. Indeed, the extent of inactivation of enzyme by PG treatment correlated very well with the quantitative increase in the new tryptic peptide peak. Amino acid composition analysis of both tryptic peptide and V-8 peptide revealed that the two peptides were derived from the same general region; tryptic peptide spanned between residues 837 and 857 while V-8 peptide spanned between residues 841 and 870 in the primary sequence of pol I. Sequence analysis of tryptic peptide further identified arginine-841 as the site of PG modification, which implicates this residue in the DNA binding function of pol I.     

The Cys-67 residue of HLA-B27 influences cell surface stability, peptide specificity, and T-cell antigen presentation.

Cys-67 of HLA-B27 is located in the B pocket, which determines peptide-binding specificity. We analyzed effects of the Cys-67 --> Ser mutation on cell surface expression, peptide specificity, and T-cell recognition of HLA-B*2705. Surface expression was assessed with antibodies recognizing either native or unfolded HLA proteins. Whereas native B*2705 molecules predominated over unfolded ones, this ratio was reversed in the mutant, suggesting lower stability. Comparison of B*2705- and Cys-67 --> Ser-bound peptides revealed that the mutant failed to bind approximately 15% of the B*2705 ligands, while binding as many novel ones. Two peptides with Gln-2 found in both B*2705 and Cys-67 --> Ser are the first demonstration of natural B*2705 ligands lacking Arg-2. Other effects of the mutation on peptide specificity were: 1) average molecular mass of natural ligands higher than for B*2705, 2) bias against small residues at peptide position (P) 1, and 3) increased P2 permissiveness. The results suggest that the Cys-67 --> Ser mutation weakens B pocket interactions, leading to decreased stability of the mutant-peptide complexes. This may be partially compensated by interactions involving bulky P1 residues. The effect of the mutation on allorecognition was consistent with that on peptide specificity. Our results may aid understanding of the pathogenetic role of HLA-B27 in spondyloarthropathy.     

Myristoyl CoA:protein N-myristoyltransferase activities from rat liver and yeast possess overlapping yet distinct peptide substrate specificities

A variety of eukaryotic viral and cellular proteins possesses an NH2-terminal N-myristoylglycine residue important for their biological functions. Recent studies of the primary structural requirements for peptide substrates of the enzyme responsible for this modification in yeast demonstrated that residues 1, 2, and 5 play a critical role in enzyme: ligand interactions (Towler, D. A., Adams, S. P., Eubanks, S. R., Towery, D. S., Jackson-Machelski, E., Glaser, L., and Gordon J. I. (1987b) Proc. Natl. Acad. Sci. U. S. A. 84, 2708-2812). This was determined by examining as substrates a series of synthetic peptides whose sequences were systematically altered from a "parental" peptide derived from the known N-myristoylprotein bovine heart cyclic AMP-dependent protein kinase (A kinase) catalytic subunit. We have now extended these studies in order to examine structure/activity relationships in the COOH-terminal regions of octapeptide substrates of yeast N-myristoyltransferase (NMT). The interaction between yeast NMT and the side chain of residue 5 in peptide ligands is apparently sterically constrained, since Thr5 is unable to promote the very high affinity binding observed with a Ser5 substitution. A substrate hexapeptide core has been defined which contains much of the information necessary for recognition by this lower eukaryotic NMT. Addition of COOH-terminal basic residues to this hexapeptide enhances peptide binding, while COOH-terminal acidic residues destabilize NMT: ligand interactions. Based on the results obtained from our in vitro studies of over 80 synthetic peptides and yeast NMT, we have identified a number of potential N-myristoylproteins from searches of available protein databases. These include hepatitis B virus pre-S1, human SYN-kinase, rodent Gi alpha, and bovine transducin-alpha. Peptides corresponding to the NH2-terminal sequences of these proteins and several known N-myristoylproteins were assayed using yeast NMT as well as partially purified rat liver NMT. While a number of the synthetic peptides exhibited similar catalytic properties with the yeast and mammalian enzymes, surprisingly, the SYN-kinase, Gi alpha, and transducin-alpha peptides were N-myristoylated by rat NMT but not by yeast NMT. This suggests that either multiple NMT activities exist in rat liver or the yeast and rodent enzymes have similar but distinct peptide substrate specificities.