The human CD8 coreceptor effects cytotoxic T cell activation and antigen sensitivity primarily by mediating complete phosphorylation of the T cell receptor zeta chain.

Recognition of antigen by cytotoxic T lymphocytes (CTL) is determined by interaction of both the T cell receptor and its CD8 coreceptor with peptide-major histocompatibility complex (pMHC) class I molecules. We examine the relative roles of these receptors in the activation of human CTL using mutations in MHC class I designed to diminish or abrogate the CD8/pMHC interaction. We use surface plasmon resonance to determine that point mutation of the alpha3 loop of HLA A2 abrogates the CD8/pMHC interaction without affecting the affinity of the T cell receptor/pMHC interaction. Antigen-presenting cells expressing HLA A2 which does not bind to CD8 fail to activate CTL at any peptide concentration. Comparison of CTL activation by targets expressing HLA A2 with normal, abrogated, or diminished CD8/pMHC interaction show that the CD8/pMHC interaction enhances sensitivity to antigen. We determine that the biochemical basis for coreceptor dependence is the activation of the 23-kDa phosphoform of the CD3zeta chain. In addition, we produce mutant MHC class I multimers that specifically stain but do not activate CTL. These reagents may prove useful in circumventing undesirable activation-related perturbation of intracellular processes when pMHC multimers are used to phenotype antigen-specific CD8+ lymphocytes.

Peripheral membrane proteins: FYVE sticky fingers.

The recently determined structure of the lipid-binding 'FYVE' domain provides several clues to the mode of interaction for this class of peripheral membrane proteins. However, the application of traditional modes of structural analysis to diffusible membrane-binding proteins exposes some limitations of these techniques.

NMR analysis of CYP1(HAP1) DNA binding domain-CYC1 upstream activation sequence interactions: recognition of a CGG trinucleotide and of an additional thymine 5 bp downstream by the zinc cluster and the N-terminal extremity of the protein.

The DNA binding domain of the yeast transcriptional activator CYP1(HAP1) contains a zinc-cluster structure. The structures of the DNA binding domain-DNA complexes of two other zinc-cluster proteins (GAL4 and PPR1) have been studied by X-ray crystallography. Their binding domains present, besides the zinc cluster, a short linker peptide and a dimerization element. They recognize, as homodimers, two rotationally symmetric CGG trinucleotides, the linker peptide and the dimerization element playing a crucial role in binding specificity. Surprisingly, CYP1 recognizes degenerate forms of a direct repeat, CGGnnnTAnCGGnnnTA, and the role of its linker is under discussion. To better understand the binding specificity of CYP1, we have studied, by NMR, the interaction between the CYP1(55-126) peptide and two DNA fragments derived from the CYC1 upstream activation sequence 1B. Our data indicate that CYP1(55-126) interacts with a CGG and with a thymine 5 bp downstream. The CGG trinucleotide is recognized by the zinc cluster in the major groove, as for GAL4 and PPR1, and the thymine is bound in the minor groove by the N-terminal region, which possesses a basic stretch of arginyl and lysyl residues. This suggests that the CYP1(55-126) N-terminal region could play a role in the affinity and/or specificity of the interaction with its DNA targets, in contrast to GAL4 and PPR1.

1H, 15N resonance assignment and three-dimensional structure of CYP1 (HAP1) DNA-binding domain.

CYP1(HAP1) is a transcriptional activator involved in the aerobic metabolism of the yeast Saccharomyces cerevisiae. The amino acid sequence of its DNA-binding domain suggests that it belongs to the "zinc cluster" class. This region is indeed characterized by a pattern known to form a bimetal thiolate cluster where two zinc ions are coordinated by six cysteine residues. Structures of two such domains, those from GAL4 and PPR1, have been solved as complexes with DNA. These domains consist of the zinc cluster connected to a dimerization helix by a linker peptide. They recognize, as a dimer, an inverted repeat of a CGG motif that is separated by a specific number of bases. Interestingly, the specificity of that interaction seems not to be due to the interaction between the cluster region and the DNA but rather to a fine tune between the structure of the linker peptide and the number of base-pairs separating the two CGGs. However, the CYP1 target sites fail to display such a consensus sequence. One of the two CGG sites is poorly conserved and some experiments suggest a direct rather than an inverted repeat. Using 1H, 15N and 113Cd NMR spectroscopy, we have undertaken the analysis of the structural properties of the CYP1(56-126) fragment that consists of the zinc-cluster region, the linker peptide and a part of the dimerization helix. We have demonstrated that the six cysteine residues of the peptide chelate two cadmium ions as in GAL4 and PPR1. Fifteen structures of the zinc-cluster region (residues 60 to 100) were calculated, the linker peptide and the dimerization helix being unstructured under the conditions of our study. This region possesses the same overall fold as in GAL4 and PPR1, and most of the side-chains involved in the interaction with DNA are structurally conserved. This suggests that the CYP1 zinc-cluster region recognizes a CGG triplet in the same way as GAL4 and PPR1. In this case, the particular properties of CYP1 seem to be due to the structure of the linker peptide and/or of the dimerization helix.