PDBsum entry 3dav

Protein binding

PDB id: 3dav

Contents

Protein chains

126 a.a.

Metals

_NA ×9

Waters ×231

References listed in PDB file

Key reference

Title

Incompatibility with formin cdc12p prevents human profilin from substituting for fission yeast profilin: insights from crystal structures of fission yeast profilin.

Authors

O.C.Ezezika, N.S.Younger, J.Lu, D.A.Kaiser, Z.A.Corbin, B.J.Nolen, D.R.Kovar, T.D.Pollard.

Ref.

J Biol Chem, 2009, 284, 2088-2097. [DOI no: 10.1074/jbc.M807073200]

PubMed id

19028693

Abstract

Expression of human profilin-I does not complement the temperature-sensitive cdc3-124 mutation of the single profilin gene in fission yeast Schizosaccharomyces pombe, resulting in death from cytokinesis defects. Human profilin-I and S. pombe profilin have similar affinities for actin monomers, the FH1 domain of fission yeast formin Cdc12p and poly-l-proline (Lu, J., and Pollard, T. D. (2001) Mol. Biol. Cell 12, 1161-1175), but human profilin-I does not stimulate actin filament elongation by formin Cdc12p like S. pombe profilin. Two crystal structures of S. pombe profilin and homology models of S. pombe profilin bound to actin show how the two profilins bind to identical surfaces on animal and yeast actins even though 75% of the residues on the profilin side of the interaction differ in the two profilins. Overexpression of human profilin-I in fission yeast expressing native profilin also causes cytokinesis defects incompatible with viability. Human profilin-I with the R88E mutation has no detectable affinity for actin and does not have this dominant overexpression phenotype. The Y6D mutation reduces the affinity of human profilin-I for poly-l-proline by 1000-fold, but overexpression of Y6D profilin in fission yeast is lethal. The most likely hypotheses to explain the incompatibility of human profilin-I with Cdc12p are differences in interactions with the proline-rich sequences in the FH1 domain of Cdc12p and wider "wings" that interact with actin.

Figure 2.
Fission yeast profilin (S. pombe PRF), but not human profilin (HPRF), allows Cdc12p-associated actin filaments to elongate their barbed ends. A, effects of profilins and formin Cdc12(FH1FH2)p on the time course of elongation of the barbed ends of preassembled filament seeds. Conditions: 10 mm imidazole, pH 7.0, 50 mm KCl, 1 mm MgCl[2], 1 mm EGTA, 0.5 mm DTT, 0.2 mm ATP, 90 μm CaCl[2]. Reactions were started by mixing 0.5 μm magnesium-ATP actin monomers (10% pyrene labeled) with 0.5 μm actin filaments: thick curve, actin alone; other samples contained 20 nm Cdc12(FH1FH2)p with: ×, no profilin; ⋄, 0.25 μm S. pombe profilin; □, 0.50 μm S. pombe profilin; ○, 2.5 μm S. pombe profilin; •, 2.5 μm Hs profilin-I. B, dependence of the initial rate of barbed end assembly (slope) with 20 nm Cdc12(FH1FH2)p on the concentrations of (○) S. pombe profilin or (•) Hs profilin-I. C, affinity of profilin for the Cdc12p proline-rich FH1 domain. Conditions: 20 mm Tris, pH 7.5, 150 mm KCl, 0.2 mm DTT. Either 0.5 μm S. pombe profilin (○) or Hs profilin (•) were incubated with a range of concentrations of Cdc12(FH1)p. The intrinsic tryptophan fluorescence of profilin was measured and plotted versus the concentration of Cdc12(FH1)p. Curve fits revealed the indicated equilibrium dissociation constants.

Figure 4.
Stereo diagrams of the homology model of S. pombe profilin bound to actin based on the bovine profilin-β actin crystal structure (PDB 1HLU). Color code for C-α traces: actin, dark blue; bovine profilin, cyan; S. pombe profilin, tan. ATP is yellow. Some residues discussed in the text are shown in stick representation. Subdomains 1–4 of actin are labeled in panel A. Important secondary structural elements are also labeled. The figure in panel B is oriented to highlight the β4/β5 and β5/β6 wings in bovine profilin. The figure was generated using Molscript and Raster3D.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 2088-2097) copyright 2009.