Structural determinants of the profilin/poly -L -proline interaction

Date

1999

Authors

Mahoney, Nicole Marie

Journal Title

Journal ISSN

Volume Title

Publisher

ProQuest Dissertations & Theses

YU Faculty Profile

Abstract

Motility, cytokinesis, mRNA localization, and cell signaling depend on the site specific assembly of multi-protein complexes which may serve to link extracellular cues with cytoskeletal rearrangements. Profilin, a 13--15 kDa actin monomer binding protein that regulates actin structures in vivo, is incorporated into specific multi-protein assemblies via proline-rich ligands. All cellular profilins bind poly-L-proline and several proline-rich proteins interact with profilin in vitro and co-localize with profilin in vivo. Profilin's ligands include the focal adhesion proteins VASP (vasodilator s&barbelow;timulated p&barbelow;hosphoprotein) and Mena (m&barbelow;ammalian homologue of Drosophila enabled) and the remains. These proteins are molecular scaffolds that recruit signaling and actin regulatory proteins to cellular regions undergoing cytoskeletal rearrangements during morphogenesis and development.;We examined the structure of human platelet profilin (HPP) bound to proline-rich peptides by x-ray crystallography, fluorescence and NMR spectroscopy to determine the structural basis for, and the biochemical consequences of, complex formation between profilin and proline-rich proteins. The 2.2A crystal structure of a complex between HPP and a proline decamer (L-Pro)10 was solved by multiple isomorphous replacement. This structure identified the poly-L-proline binding site as a conserve patch of surface exposed aromatic residues where the (LPro)10 peptide binds in a left-handed poly-L-proline type II (PPII) helical conformation. The HPP/(L-Pro)10 Interaction has been analyzed in the context of other known proline-rich binding modules, such as SH3 and WW domains, which are not structurally homologous to profilin but utilize a similar ligand binding strategy.;SH3 domains bind proline-rich ligands; in either of two amide backbone orientations, leading to speculation that the specific orientations will have different effects on macromolecular complex organization and function. A similar mechanism may be operating for profilin, as the (L-Pro)10 directionality was initially unclear due to the pseudo-two-fold symmetry inherent in proline oligomers. This ambiguity was overcome by examining the interaction between HPP and terminally tagged or spin labeled proline-rich peptides using x-ray crystallography and a novel NMR technique, respectively. Together, the results of these experiments revealed an extended poly-L-proline recognition site on profilin and most importantly indicate that profilin, like SH3 domains, can bind peptide ligands in either of two distinct amide backbone orientations.

Description

Keywords

Biochemistry., Cellular biology.

Citation

Source: Dissertation Abstracts International, Volume: 61-02, Section: B, page: 8420.;Advisors: Steven C. Almo.