Immunological tolerance has been demonstrated in double-transgenic mice expressing the genes for a neo-self antigen, hen egg lysozyme, and a high affinity anti-lysozyme antibody. The majority of anti-lysozyme B-cells did not undergo clonal deletion, but were no longer able to secrete anti-lysozyme antibody and displayed markedly reduced levels of surface IgM while continuing to express high levels of surface IgD. These findings indicate that self tolerance may result from mechanisms other than clonal deletion, and are consistent with the hypothesis that IgD may have a unique role in B-cell tolerance.

No abstract

Limited information is available on inherent stabilities of four-chain coiled-coils. We have developed a model system to study this folding motif using synthetic peptides derived from sequences contained in the tetramerization domain of Lac repressor. These peptides are tetrameric as judged by both gel filtration and sedimentation equilibrium and the tetramers are fully helical as determined by CD. The four-chain coiled-coils are well folded as judged by the cooperativity of thermal unfolding and by the extent of dispersion in aliphatic chemical shifts seen in NMR spectra. In addition, we measured the chain length dependence of this four-chain coiled-coil. To this end, we developed a general procedure for nonlinear curve fitting of denaturation data in oligomeric systems. The dissociation constants for bundles that contain a-helical chains 21, 28, and 35 amino acids in length are 3.1 x 6.7 X and 1.0 x M3, respectively. This corresponds to tetramer stabilities (in terms of the peptide monomer concentration) of 180 pM, 51 nM, and 280 f M , respectively. Finally, we discuss the rules governing coiledcoil formation in light of the work presented here.

Human profilin is a 15-kDa protein that plays a major role in the signaling pathway leading to cytoskeletal rearrangement. Essentially complete assignment of the 1H, 13C, and 15N resonances of human profilin have been made by analysis of multidimensional, double- and triple-resonance nuclear magnetic resonance (NMR) experiments. The deviation of the 13C alpha and 13C beta chemical shifts from their respective random coil values were analyzed and correlate well with the secondary structure determined from the NMR data. Twenty structures of human profilin were refined in the program X-PLOR using a total of 1186 experimentally derived conformational restraints. The structures converged to a root mean squared distance deviation of 1.5 A for the backbone atoms. The resultant conformational ensemble indicates that human profilin is an alpha/beta protein comprised of a seven-stranded, antiparallel beta-sheet and three helices. The secondary structure elements for human profilin are quite similar to those found in Acanthamoeba profilin I [Archer, S. J., Vinson, V. K., Pollard, T. D., & Torchia, D. A. (1993), Biochemistry 32, 6680-6687], suggesting that the three-dimensional structure of Acanthamoeba profilin I should be analogous to that determined here for human profilin. The structure determination of human profilin has facilitated the sequence alignment of lower eukaryotic and human profilins and provides a framework upon which the various functionalities of profilin can be explored. At least one element of the actin-binding region of human profilin is an alpha-helix. Two mechanisms by which phosphatidylinositol 4,5-bisphosphate can interfere with actin-binding by human profilin are proposed.

No abstract

The structure of human CTLA-4 reveals that residues Met 99, Tyr 100 and Tyr 104 of the M99YPPPY104 motif are adjacent to a patch of charged surface residues on the A'GFCC' face of the protein. Mutation of these residues, which are conserved in the CTLA-4/CD28 family, significantly reduces binding to CD80 and/or CD86, implicating this patch as a ligand binding site.

NMR spectroscopy has been used to characterize the protein-protein interactions between the mouse Grb2 (mGrb2) N-terminal SH3 domain complexed with a 15-residue peptide (SPLLPKLPP-KTYKRE) corresponding to residues 1264-1278 of the mouse Sos-2 (mSos-2) protein. Intermolecular interactions between the peptide and 13C-15N-labeled SH3 domain were identified in half-reverse-filtered 2D and 3D NOESY experiments. Assignments for the protons involved in interactions between the peptide and the SH3 domain were confirmed in a series of NOESY experiments using a set of peptides in which different leucine positions were fully deuterated. The peptide ligand-binding site of the mGrb2 N-terminal SH3 domain is defined by the side chains of specific aromatic residues (Tyr7, Phe9, Trp36, Tyr52) that form two hydrophobic subsites contacting the side chains of the peptide Leu4 and Leu7 residues. An adjacent negatively charged subsite on the SH3 surface is likely to interact with the side chain of a basic residue at peptide position 10 that we show to be involved in binding. The peptide-binding site of the SH3 is characterized by large perturbations of amide chemical shifts when the peptide is added to the SH3 domain. The mGrb2 N-terminal SH3 domain structure in the complex is well-defined (backbone RMSD of 0.56 +/- 0.21 calculated over the backbone N, C alpha, and C atoms of residues 1-54). The structure of the peptide in the complex is less well-defined but displays a distinct orientation.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrostatic interactions between charged amino acids often affect heterospecificity in coiled coils as evidenced by the interaction between the oncoproteins, fos and jun. Such interactions have been successfully exploited in the design of heteromeric coiled coils in a number of laboratories. It has been suggested that heterospecificity in these dimeric coiled-coil systems is driven not by specific electrostatic interactions in the heterodimers but rather by electrostatic repulsion acting to destabilize the homodimer state relative to the heterodimer state. We show that it is possible to design ion pair interactions that directly stabilize the heterotetrameric coiled-coil state. Synthetic peptides were used whose sequences are based on the C-terminal tetramerization domain of Lac repressor, as a model system for four-chain coiled coils (Fairman et al., 1995). These Lac-based peptides, containing either glutamic acid (Lac21E) or lysine (Lac21K) at all b and c heptad positions, only weakly self-associate but, when mixed, afford a highly stable heterotetramer. This study represents the first experimental evidence for the importance of the b and c heptad positions to the stability of coiled coils. Finally, pH dependence and NaCl dependence studies show that heterotetramer stability is driven by ion pair interactions between glutamate and lysine; these interactions contribute about 0.6 kcal/mol of stabilizing free energy for each potential glutamate-lysine pair.