Study the Structural Topology, Dynamic Properties and Functional Model of Phage 21 Holin Protein using EPR Spectroscopy

Abstract

Calcium (Ca2þ) dysregulation is a hallmark of heart failure and is characterized by impaired Ca 2þ sequestration into the sarcoplasmic reticulum (SR) by the SR-Ca 2þ -ATPase (SERCA). Regulins, single-pass membrane proteins, bind and inhibit SERCA by allosterically modulating the affinity of its Ca 2þ binding sites. DWarf Open Reading Frame (DWORF) is a 35 amino acid regulin that enhances SERCA2a activity in cardiomyocytes. As a first step to understanding DWORF regulation of SERCA, we used Oriented-Sample Solid-State NMR spectroscopy (OS-ssNMR) and computational methods to determine the structure and topology of DWORF in liquid crystalline lipid bilayers. We found that DWORF adopts a helical structure, with a pronounced kink at the N-terminus. Restrained molecular dynamics calculations using backbone dipolar couplings and chemical shift anisotropy show the dynamic topology of DWORF, with the N-terminal helix spanning tilt angles ranging from 50-60 degrees and C-terminal helix ranging from 30-40 degrees. The two helical domains are anchored to both membrane leaflets by polar residues. This topology differs from that of phospholamban, a main regulator of SERCA, and might explain the opposite effects on the ATPase's apparent affinity for Ca 2þ ions of these two regulins.