In Saccharomyces cerevisiae, efficient expression of glycolytic and translational component genes requires two DNA binding proteins, RAP1 (which binds to UASRPG) and GCR1 (which binds to the CT box). We generated deletions in GCR1 to test the validity of several different models for GCR1 function. We report here that the C‐terminal half of GCR1, which includes the domain required for DNA binding to the CT box in vitro, can be removed without affecting GCR1‐dependent transcription of either the glycolytic gene ADH1 or the translational component genes TEF1 and TEF2. We have also identified an activation domain within a segment of the GCR1 protein (the N‐terminal third) that is essential for in vivo function. RAP1 and GCR1 can be co‐immunoprecipitated from whole cell extracts, suggesting that they form a complex in vivo. The data are most consistent with a model in which GCR1 is attracted to DNA through contact with RAP1.

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Helper T-cell activation generally requires the coreceptor CD4, which binds MHC class II molecules. A remarkable feature of the CD4-MHC class II interaction is its exceptionally low affinity, which ranges from K D = w200 μM to >2 mM. Investigating the biological role of the much lower affinity of this interaction than those of other cell-cell recognition molecules will require CD4 mutants with enhanced binding to MHC class II for testing in models of T-cell development. To this end, we used in vitro-directed evolution to increase the affinity of human CD4 for HLA-DR1. A mutant CD4 library was displayed on the surface of yeast and selected using HLA-DR1 tetramers or monomers, resulting in isolation of a CD4 clone containing 11 mutations. Reversion mutagenesis showed that most of the affinity increase derived from just two substitutions, Gln40Tyr and Thr45Trp. A CD4 variant bearing these mutations bound HLA-DR1 with K D = 8.8 μM, compared with >400 μM for wild-type CD4. To understand the basis for improved affinity, we determined the structure of this CD4 variant in complex with HLA-DR1 to 2.4 Å resolution. The structure provides an atomiclevel description of the CD4-binding site on MHC class II and reveals how CD4 recognizes highly polymorphic HLA-DR, -DP, and -DQ molecules by targeting invariant residues in their α2 and β2 domains. In addition, the CD4 mutants reported here constitute unique tools for probing the influence of CD4 affinity on T-cell activation and development.T he analysis of leukocyte surface molecules involved in cellcell recognition has been critical to advancing our understanding of immunological phenomena, such as T-cell activation. From the wide range of interactions examined to date, it has emerged that leukocyte surface molecules interact with remarkably low affinities, with dissociation constants (K D 's) generally between 1 and 100 μM, mainly due to fast off-rates (k off >1 s −1 ) (1-3). For example, T-cell receptors (TCRs) bind cognate peptide-MHC (pMHC) complexes with K D 's between 1 and 100 μM, compared with w10 nM for typical antibody-antigen interactions. The low affinities and rapid kinetics (both on-and offrates) of TCR binding to pMHC are believed to allow T cells to scan with high speed and sensitivity the numerous self-pMHC complexes on antigen-presenting cells (APCs) to detect and respond to antigens expressed at low numbers (4).Of all leukocyte cell-cell recognition molecules characterized so far, the T-cell coreceptor CD4 is the most enigmatic in terms of its binding properties (1). The interaction of CD4 with MHC class II molecules greatly augments cytokine production by helper T cells (5) and substantially reduces the number of antigenic peptides on APCs required for T-cell triggering (6). Surprisingly, however, CD4 binds MHC class II with exceptionally low affinity compared with other cell-cell recognition molecules (K D = 1-100 μM) (1-3), including the T-cell coreceptor CD8, which binds MHC class I molecules. For the CD4-MHC class II interaction, K D 's have been vario...