Thomas B. Lavoie

Solution structure of human CTLA-4 and delineation of a CD80/CD86 binding site conserved in CD28.

The structure of human CTLA-4 reveals that residues Met 99, Tyr 100 and Tyr 104 of the M99 YPPPY104 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.

Characterization of the three-dimensional solution structure of human profilin: proton, carbon-13, and nitrogen-15 NMR assignments and global folding pattern

Abstract 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.

Orientation of peptide fragments from Sos proteins bound to the N-terminal SH3 domain of Grb2 determined by NMR spectroscopy.

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)

Relaxation study of the backbone dynamics of human profilin by two‐dimensional 1H‐15N NMR

The dynamic properties of 111 backbone HN sites in uncomplexed human profilin, a protein of 139 residues, have been characterized by two‐dimensional inverse‐detected 1H‐15N NMR spectroscopy. Heteronuclear {1H}‐15N nuclear Overhauser effects and 15N longitudinal and transverse relaxation rates have been analyzed in terms of model‐free spectral density functions and exchange contributions to transverse relaxation rates. Relatively high mobilities on the nanosecond timescale are observed for Asp26 and Ser27, which form part of a loop connecting β‐strands A and B, and for Thr92 through Ala95, which are in a loop connecting β‐strands E and F. Significant exchange contributions, indicative of motions on the microsecond to millisecond timescale, have been obtained for 30 residues. These include Leu77, Asp80 and Gly81 of a loop between β‐strands D and E, Ser84 and Met85 of β‐strand E, Gly121 of a loop connecting β‐strand G and the C‐terminal helix, and Gln138, which is next to the C‐terminal residue Tyr139. Some of the regions showing high flexibility in profilin are known to be involved in poly‐L‐proline binding.

Crystallization and preliminary X-ray analysis of CTLA-4 (CD152) membrane-external domain

CTLA-4 (CD152) is involved in T-lymphocyte co-stimulatory pathways modulating both humoral and cellular immune response. The membrane-external domain has been prepared and crystallized. The unit-cell parameters are a = b = 43, c = 143 A with the symmetry of space group P3(1)21 or its enantiomer and the crystals diffract to 2. 7 A resolution at synchrotron beamlines.