Structure-dynamic determinants governing a mode of regulatory response and propagation of allosteric signal in splice variants of Na+/Ca2+ exchange (NCX) proteins.
The Ca(2+)-dependent allosteric regulation of Na(+)/Ca(2+) exchanger (NCX) proteins represents Ca(2+) interaction with the cytosolic domains, CBD1 (calcium-binding domain 1) and CBD2, which is associated either with activation, inhibition or no response to regulatory Ca(2+) in a given splice variant. CBD1 contains a high affinity Ca(2+)-sensor (which is highly conserved among splice variants), whereas primary information upon Ca(2+) binding to CBD1 is modified by alternative splicing of CBD2, yielding the diverse regulatory responses to Ca(2+). To resolve the structure-dynamic determinants of splicing-dependent regulation, we tested two-domain tandem (CBD12) constructs possessing either positive, negative or no response to Ca(2+) using hydrogen-deuterium exchange MS (HDX-MS), SAXS, equilibrium 45Ca(2+) binding and stopped-flow kinetics. Taken together with previously resolved crystallographic structures of CBD12, the data revealed that Ca(2+) binding to CBD1 rigidifies the main-chain flexibility of CBD2 (but not of CBD1), whereas CBD2 stabilizes the apo-CBD1. Strikingly, the extent and strength of Ca(2+)-dependent rigidification of CBD2 is splice-variant dependent, where the main-chain rigidification spans from the Ca(2+)-binding sites of CBD1, through a helix of CBD2 (positioned at the domains' interface) up to the tip of CBD2 [>50 Å (1 Å = 0.1 nm)] or alternatively, it stops at the CBD2 helix in the splice variant exhibiting an inhibitory response to regulatory Ca(2+). These results provide a structure-dynamic basis by which alternative splicing diversifies the regulatory responses to Ca(2+) as well as controls the extent and strength of allosteric signal propagation over long distance.
*Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel.
Moshe
M
Giladi
†School of Pharmacy, Sungkyunkwan University, Jangan-gu, Suwon 440-746, South Korea.
Su Youn
SY
Lee
*Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel.
Reuben
R
Hiller
†School of Pharmacy, Sungkyunkwan University, Jangan-gu, Suwon 440-746, South Korea.
Ka Young
KY
Chung
*Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel.
Daniel
D
Khananshvili
D000494:Allosteric Regulation / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D004285:Dogs; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D015398:Signal Transduction / Q000502:physiology*; D019831:Sodium-Calcium Exchanger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014961:X-Ray Diffraction
D020033:Protein Isoforms; D019831:Sodium-Calcium Exchanger; D002118:Calcium