Structural determinants of the profilin/poly -L -proline interaction
Mahoney, Nicole Marie
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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.
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