A CapG gain-of-function mutant reveals critical structural and functional determinants for actin filament severing.
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.
Zhang
Y
Y
Department of Medicine, University of Florida, Gainesville, 32610, USA.
Vorobiev
Sergey M
SM
Gibson
Bruce G
BG
Hao
Binghua
B
Sidhu
Gurjit S
GS
Mishra
Vishnu S
VS
Yarmola
Elena G
EG
Bubb
Michael R
MR
Almo
Steven C
SC
Southwick
Frederick S
FS
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
D000199:Actins; D018260:Gelsolin; D050505:Mutant Proteins