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.
Gombos
Zoltan
Z
Division of Molecular and Structural Biology, Ontario Cancer Institute, and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
Durussel
Isabelle
I
Ikura
Mitsuhiko
M
Rose
David R
DR
Cox
Jos A
JA
Chakrabartty
Avijit
A
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