Correlating Structure and Function in Human Genetic Variants of Phospholamban and Sarcolipin

Abstract

Calcium is an essential signal carrier that enables cellular processes such as muscle contraction, secretion, and metabolism. Consequently, calcium dysregulation is associated with many human diseases, including heart failure, cancer, diabetes, and many others. Considering heart failure alone, 26 million people currently suffer worldwide. The principal site for cellular calcium homeostasis is the endo- and sarcoplasmic reticulum, where resident calcium pumps (SERCA) actively maintain cytosolic calcium. SERCA is so important for cellular function that it is controlled by a number of tissue-specific master regulators – phospholamban (PLN), sarcolipin (SLN) and myoregulin (MLN). These SERCA regulators control cardiac contractility, thermogenesis, and skeletal muscle performance. Herein we present mutagenesis and structure-function studies of PLN and SLN, with an emphasis on clinically relevant mutations. As genome sequencing projects rapidly expand the spectrum of human genetic variants in PLN and SLN, this information provides a basis for targeted structure-function analyses. Focusing on missense variants and chimeras of these peptides, we use membrane reconstitution, functional assays and biophysical approaches – CD spectroscopy of PLN and SLN variants and electron cryo-microscopy (cryoEM) of two-dimensional crystals of SERCA-PLN and SERCA-SLN. Some of the mutants investigated include Ala15-Thr, Pro21-Thr, and Leu51-Pro. The Ala15-Thr mutant has comparable inhibitory activity to wild-type PLN, but the interaction with protein kinase A is altered. The Pro21-Thr mutant has comparable inhibitory activity to wild-type PLN, but it has a more helical conformation reminiscent of early models for the PLN pentamer. Finally, the Leu51-Pro mutant is a less helical form of PLN with complete loss of inhibitory function. Since Pro21-Thr and Leu51-Pro alter PLN structure, they are ideal candidates for two-dimensional crystallization with SERCA and cryoEM. These studies are underway, and our current progress on PLN, SLN and MLN will be presented.