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

Structural analysis of human profilin has revealed two tryptophan residues, W3 and W31, which interact with polyproline. The codons for these residues were mutated to encode phenylalanine and the mutant proteins overexpressed in Eschericia coli. The isolated proteins were diminished in their ability to bind polyproline, whereas phosphatidylinositol 4,5-bisphosphate (PIP 2 ) binding remained unchanged. In many strains of Saccharomyces cerevisiae, disruption of the gene encoding profilin, PFY1, is lethal. It was found that expression of the gene for human profilin is capable of suppressing this lethality. The polyproline-binding mutant alleles of the human gene were cloned into various yeast expression vectors. Each of the mutant genes resulted in suppression of the lethality of pfy1D. It was observed that the mutant protein expression levels paralleled the growth rates of the strains. The severity of various morphological abnormalities of the strains was also attenuated with increased protein levels, suggesting that profilin polyproline-binding mutations are deleterious to cell growth unless overexpressed. Both tryptophan mutations were combined to give a third mutant allele that was found both unable to bind polyproline and to suppress the lethality of a pfy1 deletion. Immunoprecipitation experiments suggested that the mutants were unaltered in their affinity for actin and PIP 2 . These data strongly suggest that polyproline binding is an essential function of profilin.Profilin is a ubiquitous eukaryotic protein that binds actin, phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and poly-l-proline [1]. In resting mammalian cells, binding of profilin to PIP 2 inhibits phospholipid hydrolysis by phospholipase Cg (PLCg) [2]. When mammalian cells are stimulated to divide through the activation of receptor tyrosine kinases, PLCg becomes phosphorylated and hydrolyzes PIP 2 in the presence of profilin. It has been hypothesized that this hydrolysis not only releases the second messenger inositol trisphosphate from the plasma membrane but also releases profilin, allowing it to interact with actin in the cytosol [3]. When bound to actin, profilin has been shown to catalyze the release of ADP from monomeric (G-) actin [4], which increases the exchange of ADP for ATP. ATP/G-actin is activated for polymerization into microfilaments and facilitates the local reorganization of the cytoskeleton [5]. This competition between PIP 2 and actin for profilin binding is therefore an important regulatory component of the eukaryotic cell cycle. In the yeast Saccharomyces cerevisiae, the gene for profilin, PFY1, has been cloned and characterized [18]. Deletion of the gene is a lethal event in many strains [18,19], while in others, the disruption produces strains with highly abnormal morphologies and severely retarded growth rates [20]. The effects of yeast profilin on actin binding and polymerization have been studied and found to be very similar to those of multicellular organisms [21]. The PIP 2 -dependent translocation of profilin from t...

No abstract

No abstract