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T cell receptor (TCR) recognition of peptide takes place in the context of the major histocompatibility complex (MHC) molecule, which accounts for approximately two-thirds of the peptide/MHC buried surface. Using the class I MHC HLA-A2 and a large panel of mutants, we have previously shown that surface mutations that disrupt TCR recognition vary with the identity of the peptide. The single exception is Lys66 on the HLA-A2 alpha1 helix, which when mutated to alanine disrupts recognition for 93% of over 250 different T cell clones or lines, independent of which peptide is bound. Thus, Lys66 could serve as a peptide-independent TCR binding determinant. Here, we have examined the role of Lys66 in TCR recognition of HLA-A2 in detail. The structure of a peptide/HLA-A2 molecule with the K66A mutation indicates that although the mutation induces no major structural changes, it results in the exposure of a negatively charged glutamate (Glu63) underneath Lys66. Concurrent replacement of Glu63 with glutamine restores TCR binding and function for T cells specific for five different peptides presented by HLA-A2. Thus, the positive charge on Lys66 does not serve to guide all TCRs onto the HLA-A2 molecule in a manner required for productive signaling. Furthermore, electrostatic calculations indicate that Lys66 does not contribute to the stability of two TCR-peptide/HLA-A2 complexes. Our findings are consistent with the notion that each TCR arrives at a unique solution of how to bind a peptide/MHC, most strongly influenced by the chemical and structural features of the bound peptide. This would not rule out an intrinsic affinity of TCRs for MHC molecules achieved through multiple weak interactions, but for HLA-A2 the collective mutational data place limits on the role of any single MHC amino acid side-chain in driving TCR binding in a peptide-independent fashion.

Mutational studies of T cell receptor (TCR) contact residues on the surface of the human class I major histocompatibility complex (MHC) molecule HLA-A2 have identified a "functional hot spot" that comprises Arg(65) and Lys(66) and is involved in recognition by most peptide-specific HLA-A2-restricted TCRs. Although there is a significant amount of functional data on the effects of mutations at these positions, there is comparatively little biochemical information that could illuminate their mode of action. Here, we have used a combination of fluorescence anisotropy, functional assays, and Biacore binding experiments to examine the effects of mutations at these positions on the peptide-MHC interaction and TCR recognition. The results indicate that mutations at both position 65 and position 66 influence peptide binding by HLA-A2 to various extents. In particular, mutations at position 66 result in significantly increased peptide dissociation rates. However, these effects are independent of their effects on TCR recognition, and the Arg(65)-Lys(66) region thus represents a true "hot spot" for TCR recognition. We also made the observation that in vitro T cell reactivity does not scale with the half-life of the peptide-MHC complex, as is often assumed. Finally, position 66 is implicated in the "dual recognition" of both peptide and TCR, emphasizing the multiple roles of the class I MHC peptide-binding domain.

Most peptides that bind to a particular major histocompatibility complex class I molecule share amino acid residues important for binding at one or two positions. Sequence analyses of peptides bound to HLA-B14 revealed at least four candidates for these so-called anchor residues: Arg at P2, Tyr at P3, Arg at P5, and Leu at P9. Combinations of any three of these amino acids sufficed for binding to HLA-B14 in vitro. Using this information, we identified an antigenic peptide critical for cytotoxic T lymphocyte recognition of virus-infected cells. Molecular models of HLA-B14 peptide complexes were constructed to investigate how the potential anchor residues might function. By using binding data to calculate the contribution to binding of each amino acid at anchor positions and predicting the stability of all possible nonapeptide complexes that could be formed from antigenic proteins, we estimate that three known antigenic nonapeptides are in the highest affinity cohort of peptides. Thus, even when multiple combinations of anchor residues contribute to binding, antigenic peptides are routinely identifiable.

The ability of amino acid side chains in the floor of the peptide binding groove of HLA-A2 to affect the presentation of a viral peptide to peptide-specific cytotoxic T lymphocytes (CTL) has been examined. HLA-A2 molecules with naturally occurring single amino acid substitutions of Phe to Tyr at position 9 (HLA-A2.4a, Tyr9) and Tyr to Cys at position 99 (HLA-A2.4b, Cys99) and a site directed mutant with a Val to Leu substitution at position 95 (Leu95) were examined for their ability to present the influenza virus matrix M1 55-73 peptide and several sequence variants of the M1 peptide to a panel of 36 M1 55-73-specific HLA-A2.1-restricted CTL lines. The Leu95 molecule demonstrated enhanced kinetics of M1 peptide presentation and the ability to be sensitized by lower concentrations of the M1 peptide than the A2.1 molecule. The Tyr9 and Cys99 molecules exposed to M1 peptide were not recognized by 33 out of 36 CTL lines. The Tyr9 and Cys99 HLA-A2 molecules could bind the M1 55-73 peptide because at least one CTL line was found that could recognize each of these molecules that were exposed to the M1 peptide. CTL recognition patterns of variant M1 peptides presented by the Tyr9 molecule demonstrated that the amino acid at position 9 can be a critical determinant of the conformation of the peptide-A2 complex, and indicated that a particular peptide can bind in the HLA-A2 peptide binding groove in more than one conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies have shown that individual TCRs are able to effectively recognize multiple peptide/MHC complexes that have varying degrees of structural diversity. These TCR cross-reactivities have usually been demonstrated by using peptides that have different amino acid sequences. To further examine the extent to which TCRs can accommodate structurally diverse ligands, we analyzed human TCR cross-reactivity to eight structurally distinct haptens that are coupled to the HLA-A2-binding Tax peptide with a lysine substitution at position 5 (Tax-5K, LLFG[K-hapten]PVYV). The results demonstrate that 71% percent of the haptenated-peptide-induced CTL lines could cross-react on at least one other hapten. We compared the effects of HLA-A2 mutants with substitutions at known TCR contact sites for recognition by hapten-cross-reactive CTL. Recognition of the A2 mutants was remarkably similar whether they were presenting the immunizing or the cross-reactive peptide, indicating that similar amino acid contacts are made by the TCR during recognition of both complexes. Thus, hapten cross-reactivity is apparently accomplished without major adjustments to the interaction between the TCR and the surface of the HLA-A2 molecule. Collectively, these results suggest that TCRs possess the molecular flexibility to accommodate very structurally diverse ligands while retaining conserved interactions with the surface of the MHC molecule.

The structures of αβ TCRs bound to complexes of class I MHC molecules and peptide show that the TCRs make multiple contacts with the α1 and α2 helixes of the MHC. Previously we have shown that the A6 TCR in complex with the HLA-A2/Tax peptide has 15 contact sites on HLA-A2. Single amino acid mutagenesis of these contact sites demonstrated that mutation of only three amino acids clustered on the α1 helix (R65, K66, A69) disrupted recognition by the A6 TCR. In the present study we have asked whether TCRs that recognize four other peptides presented by HLA-A2 interact with the MHC in identical, similar, or different patterns as the A6 TCR. Mutants K66A and Q155A had the highest frequency of negative effects on lysis. A subset of peptide-specific CTL also selectively recognized mutants K66A or Q155A in the absence of exogenous cognate peptides, indicating that these mutations affected the presentation of endogenous peptide/HLA-A2 complexes. These findings suggest that most HLA-A2-restricted TCRs recognize surfaces on the HLA-A2/peptide complex that are dependent upon the side chains of K66 and Q155 in the central portion of the peptide binding groove. Crystallographic structures of several peptide/HLA-A2 structures have shown that the side chains of these critical amino acids that make contact with the A6 TCR also contact the bound peptide. Collectively, our results indicate that the generalized effects of changes at these critical amino acids are probably due to the fact that they can be directly contacted by TCRs as well as influence the binding and presentation of the bound peptides.

Most peptides that bind to a particular MHC class I molecule share amino acid residues that are thought to physically "anchor" the peptide to polymorphic pockets within the class I binding site. Sequence analysis of endogenous peptides bound to HLA-B44 revealed two potential dominant anchor residues: Glu at P2 and Tyr, or occasionally Phe, at P9. In vitro assembly assays employing synthetic peptides and recombinant HLA-B44 produced by Escherichia coli revealed that an acidic amino acid at P2 was necessary for promoting stable peptide binding to HLA-B44. Surprisingly, although Tyr was almost exclusively found at P9 of the endogenous peptide sequences, a wide variety of amino acid residues such as Leu, Ala, Arg, Lys, His, and Phe could be tolerated at this position. Using this information, we identified antigenic peptides from the influenza virus components nonstructural protein 1 and nucleoprotein that are presented by HLA-B44 to antiinfluenza type A cytotoxic T lymphocytes. In addition, cytotoxic T lymphocytes induced by these antigenic peptides were shown to be capable of recognizing endogenously processed peptides from influenza-infected cells, indicating a potential use for these peptides in vaccine development. Finally, molecular models were created to investigate the possible ways in which the anchor residues might function to stabilize the binding of peptides to HLA-B44, and these models indicate that the acidic residue at P2 most likely interacts primarily with Lys 45 of the HLA-B44 heavy chain and makes additional contacts with Ser 67 and Tyr 9.

The conversion of isopentenyl pyrophosphate to phytoene in Neurospora crassa requires both a soluble and a particulate fraction. Soluble and particulate enzyme fractions obtained from light-treated and dark-grown wild type, albino-1, albino-2, albino-3, and white collar-1 strains were mixed in various combinations, and the activity for conversion of [1-(14)C]isopentenyl pyrophosphate to phytoene was assayed. From such experiments it can be concluded that: (a) albino-3 is defective in the soluble fraction; (b) albino-2 is defective in the particulate fraction; (c) the in vivo light treatment increases the enzyme activity in the particulate fraction; (d) this light effect occurs in wild type, albino-1, and albino-3 strains; and (e) enzyme activity is present in the particulate fraction obtained from the white collar-1 mutant, but the in vivo light treatment does not cause an increase in this activity. To measure directly the level of particulate enzyme activity, [(14)C]geranylgeranyl pyrophosphate was used as a substrate. This compound, which is not available commercially, was synthesized enzymically using extracts of pea cotyledons. Particulate enzyme fractions obtained from wild type, albino-1, and albino-3 strains incorporate [(14)C]geranylgeranyl pyrophosphate into phytoene, and this activity is higher in extracts obtained from light-treated cultures. The particulate fraction obtained from the white collar-1 mutant also incorporates [(14)C]geranylgeranyl pyrophosphate into phytoene, but the in vivo light treatment does not cause an increase in this activity. No incorporation occurs when particulate fractions obtained from either dark-grown or light-treated albino-2 cultures are assayed. The soluble enzyme fraction obtained from the albino-3 mutant was shown to be almost totally defective in enzyme activity required for the biosynthesis of [(14)C]geranylgeranyl pyrophosphate from [1-(14)C]isopentenyl pyrophosphate. An in vivo light treatment increases the level of this activity in wild type, albino-1, albino-2, and albino-3 strains, but not in the white collar-1 mutant. A model is presented to account for all of the results obtained in this investigation. It is proposed that the white collar-1 strain is a regulatory mutant blocked in the light induction process, whereas the albino-1, albino-2, and albino-3 strains are each defective for a different enzyme in the carotenoid biosynthetic pathway.

An enzyme system catalyzing the conversion of isopentenyl pyrophosphate to phytoene has been isolated from Neurospora crassa mycelia. This enzyme system shows an absolute requirement for Mg−, but no other cofactors. Cultures of N. crassa exhibit a low level of phytoene synthesizing activity when grown in the dark. A 2-min in vivo blue light irradiation results in a ninefold increase in activity after 24 h. This increase is dependent on the duration of the light treatment and is inhibited by cycloheximide. A similar blue light-induced elevation of phytoene synthesizing activity was demonstrated in an albino-1 mutant. This enzyme activity was not found in either dark-grown or irradiated cultures of an albino-2 or an albino-3 mutant.

Association constants, enthalpies, and stoichiometries of Bowman-Birk soybean inhibitor for trypsin and alpha-chymotrypsin were measured in the pH range 4-8 at 25 degrees, 0.01 M Ca2+. The results are quoted in terms of moles of protease active sites, from active site titration. Enthalpies were obtained from calorimetry. The inhibitor was modified by carboxyl group modification, and by tryptic and chymotryptic attack. Association thermodynamics and stoichiometries of the modified inhibitors with both proteases were also determined. There is one independent site for each protease on the inhibitor protein. Modification decreases association to some extent, but does not appear to change stoichiometry or protease binding site independency. In the pH 4 region the association enthalpies are endothermic, of the order 6 kcal/mol for both trypsin and chymotrypsin. With increasing pH, the enthalpies decrease and become exothermic at pH 8 for chymotrypsin. Positive entropies, 50 cal mol-1 deg-1, occur at pH 4-5. They decrease as pH increases, but are always positive in sign. The observed to accompany the overall reaction, such as H+ transfer steps. The enthalpies and entropies probably compensate over the pH range 4-8, with a characteristic temperature of 390 plus or minus 30 degrees K. Estimates were made of the macromolecular Coulomb charge products in inhibitor-protease interaction. These range from about +5 to -60, over pH range 4-8, depending on the protease. Although intermolecular Coulombic forces cannot be easily delineated at the specific side chain level, they may operate at the macromolecule level.