Conformational Changes Produced by ATP Binding to the Plasma Membrane Calcium Pump

Irene C Mangialavori,Mariela S Ferreira-Gomes,Nicolás A Saffioti,Rodolfo M González-Lebrero,Rolando C Rossi,Juan Pablo F.C Rossi

doi:10.1074/jbc.m113.494633

Abstract

The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca(2+) with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. To assess the conformational behavior of the Ca(2+) binding domain, we also studied the occlusion of Ca(2+), both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca(2+) and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only.

Highlights

The plasma membrane calcium ATPase (PMCA) reaction cycle is associated with conformational changes
Effects of Vanadate and Lanthanum on the PMCA Transmembrane Domain Conformation—To investigate the structure-function relationship of intermediates of the reaction cycle of PMCA, we studied the effects of (VO4)3Ϫ and LaIII on the conformation of the transmembrane domain of the pump under conditions similar to those used in the inhibition experiments of Fig. 1, except for the fact that all experiments were performed in equilibrium
There are several crystal structures of the Ca2ϩ pump of sarcoplasmic reticulum corresponding to different conformations reached during the reaction cycle [2, 3], and crystal structures of the Hϩ-ATPase [59] and of the Naϩ/Kϩ-ATPase were reported as well [60, 61]

Summary

Introduction

The plasma membrane calcium ATPase (PMCA) reaction cycle is associated with conformational changes. The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3Hdiazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3phosphocholine to the protein. This allowed the development of a model that describes the transport of Ca2؉ and its relation with ATP hydrolysis This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only

Objectives

Results

Conclusion