Mapping the functional surface of domain 2 in the gelsolin superfamily.
The crystal structure of the F-actin binding domain 2 of severin, the gelsolin homologue from Dictyostelium discoideum, has been determined by multiple isomorphous replacement and refined to 1.75 A resolution. The structure reveals an alpha-helix-beta-sheet sandwich similar to the domains of gelsolin and villin, and contains two cation-binding sites, as observed in other domain 1 and domain 2 homologues. Comparison of the structures of several gelsolin family domains has identified residues that may mediate F-actin binding in gelsolin domain 2 homologues. To assess the involvement of these residues in F-actin binding, three mutants of human gelsolin domain 2 were assayed for F-actin binding activity and thermodynamic stability. Two of the mutants, RRV168AAA and RLK210AAA, demonstrated a lowered affinity for F-actin, indicating a role for those residues in filament binding. Using both structural and biochemical data, we have constructed a model of the gelsolin domain 1-domain 2-F-actin complex. This model highlights a number of interactions that may serve as positive and negative determinants of filament end- and side-binding.
Puius
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YA
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Fedorov
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EV
Eichinger
L
L
Schleicher
M
M
Almo
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SC
D000199:Actins / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004023:Dictyostelium / Q000737:chemistry*; D018260:Gelsolin / Q000031:analogs & derivatives*; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics; D014508:Urea / Q000494:pharmacology
D000199:Actins; D018260:Gelsolin; D008840:Microfilament Proteins; D015800:Protozoan Proteins; C036571:severin protein, Dictyostelium; D014508:Urea