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Ca²⁺ Binding by Domain 2 Plays a Critical Role in the Activation and Stabilization of Gelsolin

Shalini Nag, Qing Ma, Hui Wang, Sakesit Chumnarnsilpa, Wei Lin Lee, Mårten Larsson, Balakrishnan Kannan, Maria Hernandez-Valladares, Leslie D. Burtnick, Robert C. Robinson and Thomas D. Pollard
Proceedings of the National Academy of Sciences of the United States of America
Vol. 106, No. 33 (Aug. 18, 2009), pp. 13713-13718
Stable URL: http://www.jstor.org/stable/40484306
Page Count: 6
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Ca²⁺ Binding by Domain 2 Plays a Critical Role in the Activation and Stabilization of Gelsolin
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Abstract

Gelsolin consists of six homologous domains (G1-G6), each containing a conserved Ca-binding site. Occupation of a subset of these sites enables gelsolin to sever and cap actin filaments in a Ca-dependent manner. Here, we present the structures of Ca-free human gelsolin and of Ca-bound human G1-G3 in a complex with actin. These structures closely resemble those determined previously for equine gelsolin. However, the G2 Ca-binding site is occupied in the human G1-G3/actin structure, whereas it is vacant in the equine version. In-depth comparison of the Ca-free and Ca-activated, actin-bound human gelsolin structures suggests G2 and G6 to be cooperative in binding Ca²⁺ and responsible for opening the G2-G6 latch to expose the F-actin-binding site on G2. Mutational analysis of the G2 and G6 Ca-binding sites demonstrates their interdependence in maintaining the compact structure in the absence of calcium. Examination of Ca binding by G2 in human G1-G3/actin reveals that the Ca²⁺ locks the G2-G3 interface. Thermal denaturation studies of G2-G3 indicate that Ca binding stabilizes this fragment, driving it into the active conformation. The G2 Ca-binding site is mutated in gelsolin from familial amyloidosis (Finnish-type) patients. This disease initially proceeds through protease cleavage of G2, ultimately to produce a fragment that forms amyloid fibrils. The data presented here support a mechanism whereby the loss of Ca binding by G2 prolongs the lifetime of partially activated, intermediate conformations in which the protease cleavage site is exposed.

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