Isolation and characterization of two mutants of human profilin I that do not bind poly(L-proline).
A simple procedure for the isolation of profilin mutants having a reduced capacity to bind poly(L-proline) is used to isolate two mutants of human profilin I, W3N and H133S. Binding of the mutants to poly(L-proline), actin, and phosphatidylinositol (4,5)-bisphosphate (PIP2) was studied. Both mutations abolished the poly(L-proline)-binding activity of profilin. This suggests that the arrangement of the N- and C-terminal helices forming the poly(L-proline)-binding site depends on the stabilizing interaction between W3 and W31 in the underlying beta-strand, and that the H133S mutation in the C-terminal helix also must have distorted the arrangement of the terminal helices. Both mutations caused a reduced affinity for actin, with the W3N replacement having the most pronounced effect. This shows that structural changes in the poly(L-proline)-binding region of profilin can affect the distantly located actin-binding site. Thus, ligands influencing the structure of the poly(L-proline)-binding site may regulate actin polymerization through profilin. This is consonant with the finding that PIP2, which changes the tryptophan fluorescence in wild-type profilin and dissociates the profilin:actin complex in vitro, binds more strongly to the W3N mutant profilin. Thus, the poly(L-proline)-binding surface represents a crucial regulatory site of profilin function.
Björkegren-Sjögren
C
C
Dept. of Cell Biology, WGI, Stockholm University, Sweden.
Korenbaum
E
E
Nordberg
P
P
Lindberg
U
U
Karlsson
R
R
D003285:Contractile Proteins; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D010455:Peptides / Q000378:metabolism*; D051304:Profilins; D011485:Protein Binding / Q000235:genetics
D003285:Contractile Proteins; D008840:Microfilament Proteins; C495738:PFN1 protein, human; D010455:Peptides; D051304:Profilins; C011083:polyproline