Intrinsically Disordered HAX-1 Regulates SERCA in a Calcium-Dependent Manner

Abstract

Hematopoietic-subtrate-1 associated protein X-1 (HAX-1) is a 279 aa protein expressed ubiquitously. In the cardiac muscle, HAX-1 was found to inhibit the sarco-endoplasmic reticulum calcium ATPase (SERCA) by shifting the pCa to higher Ca2+ concentrations. Co-immunoprecipitation assays suggest that HAX-1 binds to phospholamban (PLN), enhancing its inhibitory action. HAX-1 function is reversed upon PLN phosphorylation by cAMP-dependent protein kinase A. Our activity assays, show that HAX-1 shifts SERCA's pCa to lower values, inhibiting the ATPase activity even in the absence of PLN. Using a combination of NMR and CD spectroscopy, we found that HAX-1 is intrinsically disordered in the absence of a binding partner. Chemical denaturation experiments show only a few portions of the protein might adopt a defined secondary structure conformation, while the remainder is essentially unfolded. In agreement with previous reports, we found that HAX-1 binds Ca2+ ions and PLN's cytoplasmic domain. We propose that HAX-1 regulates the SERCA/PLN complex in a Ca2+-dependent manner. This would add another layer of control in Ca2+ homeostatic balance in the heart muscle.