The [alpha]-Helical Propensity of the Cytoplasmic Domain of Phospholamban: A Molecular Dynamics Simulation of the Effect of Phosphorylation and Mutation

INTRODUCTION

The 52-mer protein phospholamban (PLB) regulates calcium stores by inhibiting the sarcoplasmic reticulum Ca-ATPase (SERCA), a membrane-bound enzymatic calcium pump that triggers cardiac relaxation by lowering cytosolic Ca^sup 2+^ concentrations. Phosphorylation of PLB relieves the inhibition, resulting in a return of Ca^sup 2+^ into the sarcoplasmic reticulum and the start of a new cardiac cycle (MacLennan and Kranias, 2003).

Phospholamban (shown schematically in Fig. 1) regulates SERCA by binding to both membrane and cytosolic regions of the enzyme. The transmembrane domain inhibits SERCA at submicromolar [Ca^sup 2+^], whereas phosphorylation of the cytoplasmic domain at Ser-16 relieves this inhibition. The interaction mode has recently been modeled by docking the NMR-derived structure of PLB (Mortishire-Smith et al., 1995; Pollesello et al., 1999; Lamberth et al., 2000; Zamoon et al., 2003) to the x-ray structure of SERCA-1A in its thapsigargin-bound, calcium-free state (Toyoshima et al., 2003). According to this model, the transmembrane helix of PLB fits into a groove formed by helices M2, M4, M6, and M9 of SERCA. In the cytosol, residue K3 of PLB reaches near residue K400 of SERCA, whereas the loop domain (residues 17-21) interacts with residues in loop 67 of the pump (Asahi et al., 2000). The model could not clearly define the interaction of domain IA with SERCA because of a poor fit between SERCA and the NMR-derived structure of PLB in this region. Satisfactory docking was achieved after unfolding the domain I helix observed by NMR. It was proposed that unfolding was a necessary step before phosphorylation at Ser16 by protein kinase A (PKA) (Toyoshima et al., 2003).

Recent studies have suggested that domain IA maintains a helical structure when bound to SERCA-1A, whereas the loop domain undergoes conformational changes responsible for PKA recognition (Chen et al., 2003). The importance of structural and/or chemical integrity of domain IA for recognition by the pump is further shown by the effect of mutations in this region. Mutation of Arg-9 to Cys severely diminishes PLB inhibitory activity in vitro and blocks wildtype PLB phosphorylation, resulting in dilated cardiomyopathy in dominant phenotypes (Schmitt et al., 2003). An extensive site-directed mutagenesis study has shown that region IA is highly sensitive to side-chain replacement, as single point mutations of 13 out of 20 residues either reduce or abolish PLB's inhibitory potency (Toyofuku et al., 1994).

Domain IA of PLB experiences environments of very different polarities. As an isolated monomer, it lies on the membrane surface (Mascioni et al., 2002; Zamoon et al., 2003; Karim et al., 2004), but it is in the cytosol when interacting with SERCA (Toyoshima et al., 2003; Kirby et al., 2004). Experimental studies have shown that the helical order of domain IA of PLB is highly sensitive to the chemical environment (Karim et al., 2004), suggesting that the helix/coil equilibrium of this region is the key for understanding both the interaction with the pump and its recognition by PKA. Studies of PLB obtained in low-polarity media generally agree on the helical structure of membrane-spanning domain. However, the helical content of domain IA decreases in polar solvents and upon phosphorylation. Circular dichroism (CD) spectra of PLB^sub 1-25^ show 60% helical content in 30% TFE (Mortishire-Smith et al., 1998) but only 17% in water (Lockwood et al., 2003). NMR spectra of PLB^sub 1-25^ in TFE have shown a shortening of the domain IA helix down to residues 2-12 upon phosphorylation (Mortishire-Smith et al., 1995; Pollesello and Annila, 2002). The structural details of domain IA also vary with solvent. In TFE, the domain IA helix spans residues 3-18, followed by a ß-III turn from residue 19 to 21 (Pollesello et al., 1999). The domain IA helix is shorter in a CH^sub 4^/CHC1^sub 3^ mixture, spanning residues 4-16, whereas residues 17-20 form a short hinge before the start of the domain II helix at Pro-21 (Lamberth et al., 2000). In DPC micelles, domain IA is helical from residue 2-16, whereas residues 17-21 form a ß-IV turn of five amino acids, (Zamoon et al., 2003).

Molecular dynamics (MD) simulations have successfully studied the relative stability of the helical conformation in short peptides (Levy et al., 2001; Bystroff and Garde, 2003). The effect of phosphorylation as a function of sequence position has been studied using Monte Carlo/stochastic dynamics simulations (Smart and McCammon, 1999) and applications of a modified Lifson-Roig helix-coil model (Andrew et al., 2002). In this study, we have used simulations to obtain atomic-level insight into the mechanism of peptide dynamics of domain I of PLB and the effect of phosphorylation and mutation on the helix. The peptides used in the study were PLB^sub 1-25^, PLB^sub 1-25^ phosphorylated at S16 (P^sub 16^-PLB^sub 1-25^), and a mutant in which ARG-9 is replaced with cysteine (R9C-PLB^sub 1-25^). Peptides PLB^sub 1-25^ and P^sub 16^-PLB^sub 1-25^ were chosen for comparison with experimental results (Mortishire-Smith et al., 1995, 1998; Lockwood et al., 2003). A shorter peptide capped at Pro-21, PLB^sub 1-21^ was used to study unfolding from an ideal a-helical conformation and to rationalize the differences in helical content observed in various solvents. Comparison of the helical propensity of wild-type versus the R9C mutant provided a mechanistic explanation for the observed inactivity of the mutant (Schmitt et al., 2003).

METHODS

Molecular dynamics simulations