title: A CapG gain-of-function mutant reveals critical structural and functional determinants for actin filament severing.
abstract: CapG is the only member of the gelsolin family unable to sever actin filaments. Changing amino acids 84-91 (severing domain) and 124-137 (WH2-containing segment) simultaneously to the sequences of gelsolin results in a mutant, CapG-sev, capable of severing actin filaments. The gain of severing function does not alter actin filament capping, but is accompanied by a higher affinity for monomeric actin, and the capacity to bind and sequester two actin monomers. Analysis of CapG-sev crystal structure suggests a more loosely folded inactive conformation than gelsolin, with a shorter S1-S2 latch. Calcium binding to S1 opens this latch and S1 becomes separated from a closely interfaced S2-S3 complex by an extended arm consisting of amino acids 118-137. Modeling with F-actin predicts that the length of this WH2-containing arm is critical for severing function, and the addition of a single amino acid (alanine or histidine) eliminates CapG-sev severing activity, confirming this prediction. We conclude that efficient severing utilizes two actin monomer-binding sites, and that the length of the WH2-containing segment is a critical functional determinant for severing.
authors.affiliation: Department of Medicine, University of Florida, Gainesville, 32610, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.forename: Sergey M
authors.initials: SM
authors.lastname: Vorobiev
authors.forename: Bruce G
authors.initials: BG
authors.lastname: Gibson
authors.forename: Binghua
authors.initials: B
authors.lastname: Hao
authors.forename: Gurjit S
authors.initials: GS
authors.lastname: Sidhu
authors.forename: Vishnu S
authors.initials: VS
authors.lastname: Mishra
authors.forename: Elena G
authors.initials: EG
authors.lastname: Yarmola
authors.forename: Michael R
authors.initials: MR
authors.lastname: Bubb
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.forename: Frederick S
authors.initials: FS
authors.lastname: Southwick
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D018360:Crystallography, X-Ray; D018260:Gelsolin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011817:Rabbits; D013329:Structure-Activity Relationship
chemical_list: D000199:Actins; D018260:Gelsolin; D050505:Mutant Proteins