CHARACTERIZATION OF THE 3-DIMENSIONAL SOLUTION STRUCTURE OF HUMAN PROFILIN - H-1, C-13, AND N-15 NMR ASSIGNMENTS AND GLOBAL FOLDING PATTERN

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