Calcium binding sequences in calmyrin regulates interaction with presenilin-2.
Calmyrin is a myristoylated calcium binding protein that contains four putative EF-hands. Calmyrin interacts with a number of proteins, including presenilin-2 (PS2). However, the biophysical properties of calmyrin, and the molecular mechanisms that regulate its binding to different partners, are not well understood. By site-directed mutagenesis and Ca2+ binding studies, we found that calmyrin binds two Ca2+ ions with a dissociation constant of approximately 53 microM, and that the two C-terminal EF-hands 3 and 4 bind calcium. Using ultraviolet spectroscopy, circular dichroism (CD), and NMR, we found that Ca(2+)-free and -bound calmyrin have substantially different protein conformations. By yeast two-hybrid assays, we found that both EF-hands 3 and 4 of calmyrin must be intact for calmyrin to interact with PS2-loop sequences. Pulse-chase studies of HeLa cells transfected with calmyrin expression constructs indicated that wild-type (Wt) calmyrin has a half-life of approximately 75 min, whereas a mutant defective in myristoylation turns over more rapidly (half-life of 35 min). By contrast, the half-lives of calmyrin mutants with a disrupted EF-hand 3 or EF-hand 4 were 52 and 170 min, respectively. Using immunofluorescence staining of HeLa cells transfected with Wt and mutant calmyrin cDNAs, we found that both calcium binding and myristoylation are important for dynamic intracellular targeting of calmyrin. Double immunofluorescence microscopy indicated that Wt and myristoylation-defective calmyrin proteins colocalize efficiently and to the same extent with PS2, whereas calmyrin mutants defective in calcium binding display less colocalization with PS2. Our results suggest that calmyrin functions as a calcium sensor and that calcium binding sequences in calmyrin are important for interaction with the PS2 loop.
Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD 21201, USA.
Jingsong
J
Zhu
Stacy M
SM
Stabler
James B
JB
Ames
Ilia
I
Baskakov
Mervyn J
MJ
Monteiro
D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation; D053766:Presenilin-2; D011485:Protein Binding; D011487:Protein Conformation
C109198:CIB1 protein, human; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; C508861:PSEN2 protein, human; D053766:Presenilin-2; D002118:Calcium