Conformational coupling of Mg2+ and Ca2+ on the three-state folding of calexcitin B.
Calexcitin (CE) is a calcium sensor protein that has been implicated in associative learning through the Ca(2+)-dependent inhibition of K(+) channels and activation of ryanodine receptors. CE(B), the major CE variant, was identified as a member of the sarcoplasmic Ca(2+) binding protein family: proteins that can bind both Ca(2+) and Mg(2+). We have now determined the intrinsic Ca(2+) and Mg(2+) binding affinities of CE(B) and investigated their interplay on the folding and structure of CE(B). We find that urea denaturation of CE(B) displays a three-state unfolding transition consistent with the presence of two structural domains. Through a combination of spectroscopic and denaturation studies we find that one domain likely possesses molten globule structure and contains a mixed Ca(2+)/Mg(2+) binding site and a Ca(2+) binding site with weak Mg(2+) antagonism. Furthermore, ion binding to the putative molten globule domain induces native structure formation. The other domain contains a single Ca(2+)-specific binding site and has native structure, even in the absence of ion binding. Ca(2+) binding to CE(B) induces the formation of a recessed hydrophobic pocket. On the basis of measured ion binding affinities and intracellular ion concentrations, it appears that Mg(2+)-CE(B) represents the resting state and Ca(2+)-CE(B) corresponds to the active state, under physiological conditions.
Division of Molecular and Structural Biology, Ontario Cancer Institute, and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
Zoltan
Z
Gombos
Isabelle
I
Durussel
Mitsuhiko
M
Ikura
David R
DR
Rose
Jos A
JA
Cox
Avijit
A
Chakrabartty
D001665:Binding Sites; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000494:pharmacology
D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins; D015221:Potassium Channels; D013438:Sulfhydryl Compounds; D014364:Tryptophan; D014508:Urea; D019204:GTP-Binding Proteins; C102751:cex-2 protein, C elegans; D008274:Magnesium; D003545:Cysteine; D002118:Calcium