Biological Sciences - Biochemistry: Shalini Nag, Qing Ma, Hui Wang, Sakesit Chumnarnsilpa, Wei Lin Lee, Mårten Larsson, Balakrishnan Kannan, Maria Hernandez-Valladares, Leslie D. Burtnick, and Robert C. Robinson Ca2+ binding by domain 2 plays a critical role in the activation and stabilization of gelsolin PNAS 2009 106 (33) 13713-13718; published ahead of print August 4, 2009, doi:10.1073/pnas.0812374106
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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