High-resolution structural analysis of mammalian profilin 2a complex formation with two physiological ligands: the formin homology 1 domain of mDia1 and the proline-rich domain of VASP.

Profilins are small proteins capable of binding actin, poly-l-proline and other proline-rich sequences, and phosphatidylinositol (4,5)-bisphosphate. A number of proline-rich ligands for profilin have been characterised, including proteins of the Ena/VASP and formin families. We have determined the high-resolution crystal structures of mouse profilin 2a in complex with peptides from two functionally important ligands from different families, VASP and mDia1. The structures show that the binding mode of the peptide ligand is strongly affected by the non-proline residues in the sequence, and the peptides from VASP and mDia1 bind to profilin 2a in distinct modes. The high resolution of the crystallographic data allowed us to detect conserved CH-pi hydrogen bonds between the peptide and profilin in both complexes. Furthermore, both peptides, which are shown to have micromolar affinity, induced the dimerisation of profilin, potentially leading to functionally different ligand-profilin-actin complexes. The peptides did not significantly affect actin polymerisation kinetics in the presence or in the absence of profilin 2a. Mutant profilins were tested for binding to poly-L-proline and the VASP and mDia1 peptides, and the F139A mutant bound proline-rich ligands with near-native affinity. Peptide blotting using a series of designed peptides with profilins 1 and 2a indicates differences between the two profilins towards proline-rich peptides from mDia1 and VASP. Our data provide structural insights into the mechanisms of mDia1 and VASP regulated actin polymerisation.

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