Ca2+ binding by domain 2 plays a critical role in the activation and stabilization of gelsolin.
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 Ca(2+) 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 Ca(2+) 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.
Nag
Shalini
S
Institute of Molecular and Cell Biology, ASTAR, 61 Biopolis Drive, Proteos, Singapore 138673.
Ma
Qing
Q
Wang
Hui
H
Chumnarnsilpa
Sakesit
S
Lee
Wei Lin
WL
Larsson
Mårten
M
Kannan
Balakrishnan
B
Hernandez-Valladares
Maria
M
Burtnick
Leslie D
LD
Robinson
Robert C
RC
D000199:Actins / Q000737:chemistry; D000682:Amyloid / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D018260:Gelsolin / Q000737:chemistry*; D006736:Horses; D006801:Humans; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D013045:Species Specificity
D000199:Actins; D000682:Amyloid; D018260:Gelsolin; D010447:Peptide Hydrolases; D002118:Calcium