U2 auxiliary factor (U2AF) is an essential splicing factor that recognizes the 3' splice site and recruits the U2 snRNP to the branch point. The X-ray structure of the human core U2AF heterodimer, consisting of the U2AF35 central domain and a proline-rich region of U2AF65, has been determined at 2.2 A resolution. The structure reveals a novel protein-protein recognition strategy, in which an atypical RNA recognition motif (RRM) of U2AF35 and the U2AF65 polyproline segment interact via reciprocal "tongue-in-groove" tryptophan residues. Complementary biochemical experiments demonstrate that the core U2AF heterodimer binds RNA, and that the interacting tryptophan side chains are essential for U2AF dimerization. Atypical RRMs in other splicing factors may serve as protein-protein interaction motifs elsewhere during spliceosome assembly.

Animals vaccinated with heat shock protein (HSP)-peptide complexes develop specific protective immunity against cancers from which the HSPs were originally isolated. This autologous specific immunity has been demonstrated using a number of HSP-peptide antigen complexes. A prototypical HSP-based cancer vaccine is the gp96-peptide antigen complex, which is currently undergoing human clinical trials. Here, we analyzed the structure of a recombinant wild-type and a mutant gp96 protein and their peptide complexes using a number of biophysical techniques. Gel filtration chromatography, dynamic light scattering, and equilibrium analytical ultracentrifugation demonstrated that both a wild-type gp96 and a gp96 mutant lacking a dimerization domain formed higher order structures. More detailed analysis using scanning transmission electron microscopy indicated that both the wild-type and dimerization deletion mutant gp96 protein were organized, unexpectedly, into large aggregates. Size distributions ranged from dimers to octamers and higher. Circular dichroism and intrinsic Trp fluorescence suggested that the gp96 dimerization domain deletion mutant protein was more compact than the wild-type gp96. A fluorescent peptide antigen was synthesized, and the peptide-binding properties of wild-type and the dimerization domain deletion mutant gp96 were studied. Fluorescence lifetime and anisotropy decay showed that the bound antigenic peptide was located in a hydrophobic pocket, with considerable free space for the rotation of the probe. Deletion of the dimerization domain affected the peptide-binding microenvironment, although peptide-binding affinity was reduced by only a small extent. Peptide-gp96 complexes were extremely stable, persisting for many days in the cold. The extraordinary stability of peptide-gp96 complexes and the plasticity of the peptide-binding pocket support the proposed relay of diverse peptides to MHC and/or other molecules via molecular recognition.Proper functioning of the immune system requires constant surveillance and evaluation of the peptide repertoire in cells and tissues. Peptide antigens of endogenous and exogenous origins are loaded on to major histocompatibility complex (MHC) 1 class I and class II molecules, respectively, and are displayed on the surfaces of antigen-presenting cells such as macrophages or dendritic cells (e.g., see ref 1). In turn, these antigen-presenting cells stimulate antigen-specific CD8 + (class I) and CD4 + (class II) lymphocytes (reviewed in (2-4)). A large body of experimental evidence stemming from tumor immunology suggests that peptide-binding heat shock/stress proteins (HSPs) of the endoplasmic reticulum (ER) and cytosol participate, in some cases, in the presentation of peptide antigens to the immune system (for reviews, see refs 5 and 6). The most compelling evidence for the interface of the heat shock/stress response with the immune defense system comes from studies of the ER glycoprotein gp96/GRP94. gp96/GRP94 is a member of the HSP90 family. The HSPs, a...