The Crystal Structure of the Ca2+-ATPase 1 from Listeria monocytogenes reveals a Pump Primed for Dephosphorylation

Sara Basse Hansen,Mateusz Dyla,Caroline Neumann,Esben Meldgaard Hoegh Quistgaard,Jacob Lauwring Andersen,Magnus Kjaergaard,Poul Nissen

doi:10.1016/j.jmb.2021.167015

Abstract

Many bacteria export intracellular calcium using active transporters homologous to the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). Here we present three crystal structures of Ca2+-ATPase 1 from Listeria monocytogenes (LMCA1). Structures with BeF3- mimicking a phosphoenzyme state reveal a closed state, which is intermediate between the outward-open E2P and the proton-occluded E2-P* conformations known for SERCA. It suggests that LMCA1 in the E2P state is pre-organized for dephosphorylation upon Ca2+ release, consistent with the rapid dephosphorylation observed in single-molecule studies. An arginine side-chain occupies the position equivalent to calcium binding site I in SERCA, leaving a single Ca2+ binding site in LMCA1, corresponding to SERCA site II. Observing no putative transport pathways dedicated to protons, we infer a direct proton counter transport through the Ca2+ exchange pathways. The LMCA1 structures provide insight into the evolutionary divergence and conserved features of this important class of ion transporters.

Highlights

Ca2+ regulation is critical for all cells, and for bacterial cell biology and survival.[1]
The gram-positive bacterium Listeria monocytogenes expresses a Ca2+-ATPase (LMCA1), which is homologous to mammalian Ca2+-ATPases
The single-molecule FRET (smFRET) study of LMCA1 offers a rationale for this, as Ca2+ and ADP release was observed to be practically irreversible for LMCA1.11 despite the presence of high Ca2+ concentrations, LMCA1 apparently cannot enter this Ca2+-occluded E2P intermediate in the reverse direction, and the G4 mutant crystallized in a Ca2+-free form

Summary

Introduction

Ca2+ regulation is critical for all cells, and for bacterial cell biology and survival.[1] Active transporters pump Ca2+ across the membrane to maintain low intracellular Ca2+ concentrations.[2] Mechanistic details of the calcium transport mechanism have been derived mainly for the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and assumed to extrapolate to other calcium pumps. Ca2+-ATPases work in a range of different environments across the domains of life, and transport mechanisms must adapt to specific conditions. To understand how adaptive mechanisms translate sequence variations among Ca2+-ATPases to specific functions, detailed structural information of a more diverse pool of Ca2+ATPases is helpful. The gram-positive bacterium Listeria monocytogenes expresses a Ca2+-ATPase (LMCA1), which is homologous to mammalian Ca2+-ATPases. Soil is the natural habitat of Listeria, but they can develop into food borne pathogens, causing listeriosis through infection of

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