Modeling of the inhibitory interaction of phospholamban with the Ca ²⁺ ATPase

Abstract

The inhibitory interaction of phospholamban (PLN) with the sarco(endo)plasmic reticulum Ca 2؉ ATPase isoform 1 (SERCA1a) was modeled on the basis of several constraints which included (i) spontaneous formation of SS-bridges between mutants L321C in transmembrane helix 4 (M4) of SERCA1a and N27C in PLN and between V89C (M4) and V49C (PLN); (ii) definition of the face of the PLN transmembrane helix that interacts with SERCA; (iii) crosslinking between Lys-3 of PLN and Lys-397 and Lys-400 of SERCA2a. The crystal structure of SERCA1a in the absence of Ca 2؉ , which binds PLN, was used as the structure into which an atomic model of PLN was built. PLN can fit into a transmembrane groove formed by the juxtaposition of M2, the upper part of M4, M6, and M9. In the SERCA1a structure with bound Ca 2؉ , this groove is closed, accounting for the ability of Ca 2؉ to disrupt PLN-SERCA interactions. Near the cytoplasmic surface of the bilayer, the PLN helix is disrupted to prevent its collision with M4. The model can be extended into the cytoplasmic domain so that Lys-3 in PLN can be cross-linked with Lys-397 and Lys-400 in SERCA1a with little unwinding of the N-terminal helix of PLN. S arco(endo)plasmic reticulum Ca 2ϩ ATPases (SERCAs) have proven to be exceptionally useful models for the investigation of structure-function relationships and the mechanism of action of P-type cation pumps (1-4). SERCA1a is also the first P-type ATPase for which high-resolution crystal structures have been presented in both E1 (5) and E2 (6) conformations. The high-resolution structures have not only defined the gross movements of the major actuator (A), nucleotide binding (N), phosphorylation (P), and Ca 2ϩ binding domains, but have provided precise information on the relationship among individual amino acids and helices.The activity of SERCA2a is regulated by phospholamban (PLN) (7,8), a member of a family of low-molecular-mass transmembrane proteins that includes sarcolipin (SLN) (9-11). Because PLN has been demonstrated to be a major regulator of the kinetics of cardiac contractility through its effects on SERCA2a in the heart (12, 13), it is a potential drug target for management of cardiac function. Accordingly, it has been of great interest to determine the sites of interaction between PLN and SERCA2a or other SERCA isoforms.James et al. (14) cross-linked PLN to SERCA2a by using a cross-linking agent (the Denny-Jaffe reagent) to show that Lys-3 of PLN lies within 15 Å of both Lys-397 and Lys-400 in the sequence Lys-Asp-Asp-Lys-400 in what is now recognized as the cytosolic N domain of SERCA2a. Studies using chimera formation and mutagenesis confirmed that the sequence, LysAsp-Asp-Lys-Pro-Val-402 is involved in PLN-SERCA2a interactions (15). Further studies then showed that a series of charged and hydrophobic amino acids between Glu-2 and Ile-18 in PLN were also mutation-sensitive in the assay used for measurement of Ca 2ϩ affinity of SERCA2a (16). The C-terminal region of PLN, involving either Leu-28-Leu-52 or Asn-30-Leu-52 fused to a var...

Molecular mechanisms in therapy of acid-related diseases

“…The development of methods to produce crystal structures of the SERCA ATPase at high resolution by Toyoshima and colleagues revolutionized understanding of the conformational changes during the catalytic cycle of these enzymes [44][45][46]. Using the crystal structure of the SERCA Ca ATPase in different conformations as a template, computer-generated homology modeling and site-directed mutagenesis resulted in not only an expanded picture of the ion transport mechanism of the gastric ATPase but also gave insight as to how the covalently binding PPIs and the K + -competitive APAs inhibit the pump.…”

Sarcolipin regulates sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) by binding to transmembrane helices alone or in association with phospholamban

“…In previous papers (10,21), we showed that the diminished Ca 2ϩ affinity that is associated with PLN inhibition of SERCA activity was reversed partially for the SERCA1a mutants V49C, L321A, V795A, L802A, T805A, and F809A. We also showed that coimmunoprecipitation with PLN was diminished for mutants V795A and L802A.…”

“…The results, presented in Table 1, show that the apparent Ca 2ϩ affinity (K Ca ) of wt SERCA1a was reduced by 0.22 pCa (Ϫlog 10 [Ca]) unit through coexpression with NF-SLN; by 0.36 pCa unit through coexpression with PLN; and by 0.93 pCa unit through coexpression with NF-SLN and PLN. The reduction in apparent Ca 2ϩ affinity caused by interaction with NF-SLN or PLN was reversed significantly with SERCA1a mutants V89C, L321A, V795A, L802A, T805A, and F809A.…”

“…Modeling from high-resolution crystal and NMR structures has identified additional amino acids that interact between PLN domain Ia and the cytosolic domains of SERCA1a and between PLN domains Ib and II and transmembrane helices M2, M4, M6, and M9 in SERCA1a (10,23).…”

“…SLN is a 31-aa membrane protein that resembles PLN in these essential inhibitory features (5,8). The two proteins have similar transmembrane sequences (4, 9) but differ at their C termini, where PLN ends with the sequence Met-Leu-Leu-52 (10), whereas SLN ends with the more hydrophilic sequence, ArgSer-Tyr-Gln-Tyr-31. They also differ at their N termini: phosphorylation of PLN in a well conserved 30-aa cytosolic domain disrupts inhibitory interactions, accounting, in part, for the inotropic response of the heart to adrenergic stimulation (2,7).…”