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A DISCRETE SERCA N‐DOMAIN LOOP PLAYS A ROLE IN PUMP STRUCTURAL DYNAMICS AND FUNCTIONAL REGULATION
Author(s) -
Raguimova Olga N,
Smolin Nikolai,
Bovo Elisa,
Zima Aleksey V,
Robia Seth L
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.762.1
Subject(s) - serca , phospholamban , endoplasmic reticulum , calcium pump , biophysics , chemistry , calcium , atpase , phosphorylation , microbiology and biotechnology , biochemistry , biology , organic chemistry , enzyme
The sarco/endoplasmic reticulum calcium ATPase (SERCA) and its inhibitor phospholamban (PLB) are major regulators of calcium handling in cardiomyocytes. We have previously hypothesized that a compact structure of SERCA is the signature of a high efficiency catalytic pathway. Previous molecular dynamics (MD) simulations of SERCA spontaneous movements showed that a SERCA headpiece open‐to‐closed transition was initiated by an interaction between the A‐domain and the beta 5,6‐loop of the N‐domain. Here, we used additional MD simulations to identify specific residues of the loop that facilitate SERCA headpiece disorder‐to‐order transition. Fluorescence resonance energy transfer (FRET) measurements between fluorescently tagged A‐ and N‐domains of SERCA confirmed that mutation of key loop residues reduced the rate of SERCA headpiece closure during calcium cycling. As a functional consequence of loop mutations, SERCA ATPase activity was impaired by 60% without a reduction in calcium sensitivity. Calcium transport by SERCA was also reduced by loop mutations, as measured by a low affinity calcium sensor (R‐CEPIA1er) in the ER of HEK cells. Interestingly, MD simulations of a SERCA‐PLB complex model showed that PLB phosphorylation stabilizes the SERCA headpiece in an ordered conformation via an interaction with N‐loop. Thus our data suggest that the beta 5,6‐loop of SERCA N‐domain is important for stabilization of the SERCA compact conformation during calcium transport and plays a role in functional regulation by PLB phosphorylation. This small discrete structure represents a potential target for rational design of novel therapeutics to modulate cardiac function.