VISUALIZING THE NUCLEATING AND CAPPED STATES OF F-ACTIN BY Ca ²⁺ -GELSOLIN: SAXS DATA BASED STRUCTURES OF BINARY AND TERNARY COMPLEXES

Structural insight eludes on how full-length gelsolin depolymerizes and caps F-actin, while the same entity can nucleate polymerization of G-actins. Employing small angle X-ray scattering (SAXS) data analysis, we deciphered these two contrasting assemblies. Mixing Ca ²⁺ -gelsolin with F-actin in high salt F-buffer resulted in depolymerization of ordered F-actin rods to smaller sized species which became monodisperse upon dialysis with low salt G-buffer. These entities were the ternary (GA ₂ ) and binary (GA) complexes of gelsolin and actin with radius of gyration and maximum linear dimension of 4.55 and 4.68 nm, and 15 and 16 nm, respectively. In contrast, upon mixing G-actin with Ca ²⁺ -gelsolin in G-buffer, rapid association of higher order species started. Using size exclusion chromatography in-line with SAXS, we confirmed that initially GA and GA ₂ species are formed as seen upon depolymerization of F-actin, followed by dialysis. Interestingly, while GA ₂ could seed formation of native-like F-actin in both G- and F-buffer, GA failed in G-buffer. Thus, GA ₂ and GA are the central species formed via depolymerization or towards nucleation. SAXS profile referenced modeling revealed that: 1) in GA, actin is bound to the C-terminal half of gelsolin, and 2) in GA ₂ , second actin binds to the open N-terminal half accompanied by dramatic rearrangements across gelsolin’s g1-g2 and g3-g4 linkers. Importantly, first structural insight is provided into the two probable models for GA ₂ with two actins in parallel, but differentially stacked: one in polymerization competent, and other in incompetent manner, suggesting latter to represent capped state along with the inert GA.

TOC Abstract

Highlights

• Orderly decrement in the length of F-actin by Ca ²⁺ -gelsolin was tracked by SAXS.

• Residual re-association in 1:2 ratio in F-buffer was quenched by dialysis in G-buffer.

• Identical GA and GA ₂ entities formed upon mixing F- or G-actin with Ca ²⁺ -gelsolin.

• Models of nucleation competent, GA ₂ showed differential stacking of two G-actins.

• N-terminal half of gelsolin reposition as GA ₂ changes to or from GA, the capped state.