The Molecular Basis for Cyclopiazonic Acid Inhibition of the Sarcoplasmic Reticulum Calcium Pump

Karine Moncoq,Catharine A Trieber,Howard S Young

doi:10.1074/jbc.m611653200

Abstract

The sarcoplasmic reticulum Ca(2+)-ATPase is essential for calcium reuptake in the muscle contraction-relaxation cycle. Here we present structures of a calcium-free state with bound cyclopiazonic acid (CPA) and magnesium fluoride at 2.65 A resolution and a calcium-free state with bound CPA and ADP at 3.4A resolution. In both structures, CPA occupies the calcium access channel delimited by transmembrane segments M1-M4. Inhibition of Ca(2+)-ATPase is stabilized by a polar pocket that surrounds the tetramic acid of CPA and a hydrophobic platform that cradles the inhibitor. The calcium pump residues involved include Gln(56), Leu(61), Val(62), and Asn(101). We conclude that CPA inhibits the calcium pump by blocking the calcium access channel and immobilizing a subset of transmembrane helices. In the E2(CPA) structure, ADP is bound in a distinct orientation within the nucleotide binding pocket. The adenine ring is sandwiched between Arg(489) of the nucleotide-binding domain and Arg(678) of the phosphorylation domain. This mode of binding conforms to an adenine recognition motif commonly found in ATP-dependent proteins.

Highlights

IntroductionA luminal exit channel has not been observed in the available crystal structures [4, 6]
Cyclopiazonic acid (CPA) is a toxic indole tetramic acid produced by certain fungi (Penicillium or Aspergillus) [20] that are found as contaminants in foods [21]
We describe crystal structures of calcium-free conformations of Ca2ϩ-ATPase solved to 2.65 Å resolution with bound CPA and magnesium fluoride and solved to 3.4 Å resolution with bound CPA and ADP

Summary

Introduction

A luminal exit channel has not been observed in the available crystal structures [4, 6] This has recently been addressed in a biochemical study [15], which demonstrated that luminal access to the calcium binding sites is dependent on the inhibitors and/or phosphate analogues used to stabilize the complex. Despite these complications, the inhibitors and substrate analogs are necessary to stabilize calcium-free conformations for crystallization. We describe crystal structures of calcium-free conformations of Ca2ϩ-ATPase solved to 2.65 Å resolution with bound CPA and magnesium fluoride and solved to 3.4 Å resolution with bound CPA and ADP. A distinct mode of adenine recognition is identified in the E2 calcium-free conformation with bound ADP

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