Functional dissection and molecular characterization of calcium-sensitive actin-capping and actin-depolymerizing sites in villin.
All proteins of the villin superfamily, which includes the actin-capping and -severing proteins such as gelsolin, scinderin, and severin, are calcium-regulated actin-modifying proteins. Like some of these proteins, villin has morphologically distinct effects on actin assembly depending on the free calcium concentrations. At physiological calcium (Ca2+) villin nucleates and bundles actin, whereas at higher concentrations it caps (>50 microm) and severs (>200 microM) actin filaments. Although Ca(2+)-binding sites have been described in villin, the functional characterization of these sites has not been done previously. In the present study we functionally dissect the calcium-dependent actin-capping and -depolymerizing sites in villin. Our analysis reveals that villin binds Ca2+ with a Kd of 80.5 microM, a stoichiometry of 5.97, and a Hill's coefficient of 1.2. Using the NMR structure of villin 14T and the gelsolin-actin/Ca2+ crystal structure, six putative sites that result in Ca(2+)-induced conformational changes were identified in human villin and confirmed by mutational analysis. Molecular dynamics studies support the mutational analysis and provide a model for structural difference in the A93G mutant that prevents the calcium-induced conformational changes in the S1 domain of villin. Furthermore, we determined that villin expresses at least two types of Ca(2+)-sensitive sites that determine separate functional properties; site 1 (Glu-25, Asp-44, and Glu-74) regulates actin-capping, whereas sites 1 and 2 (Asp-86, Ala-93, and Asp-61), together with the intra-domain calcium-sensitive sites in villin, regulate actin depolymerization by villin. This is the first study that employs sequential mutagenesis to biochemically and functionally characterize the calcium-sensitive sites in villin. Such mutational analysis and functional characterization of the actin-capping and -depolymerizing sites are unknown for other proteins of the villin family.
Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
Narendra
N
Kumar
Alok
A
Tomar
Abby L
AL
Parrill
Seema
S
Khurana
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000409:Alanine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D018260:Gelsolin / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008840:Microfilament Proteins / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000494:pharmacology
D000199:Actins; D018260:Gelsolin; D008840:Microfilament Proteins; C026615:villin; D001224:Aspartic Acid; D014364:Tryptophan; D014508:Urea; D000409:Alanine; D002118:Calcium