The Plant Plasma Membrane Ca2+ Pump ACA8 Contains Overlapping as Well as Physically Separated Autoinhibitory and Calmodulin-binding Domains

Michael G Palmgren,Laura Luoni,Lone Bækgaard,Maria Ida De Michelis

doi:10.1074/jbc.m508299200

Michael G Palmgren, Laura Luoni + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m508299200

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Abstract

In plant Ca(2+) pumps belonging to the P(2B) subfamily of P-type ATPases, the N-terminal cytoplasmic domain is responsible for pump autoinhibition. Binding of calmodulin (CaM) to this region results in pump activation but the structural basis for CaM activation is still not clear. All residues in a putative CaM-binding domain (Arg(43) to Lys(68)) were mutagenized and the resulting recombinant proteins were studied with respect to CaM binding and the activation state. The results demonstrate that (i) the binding site for CaM is overlapping with the autoinhibitory region and (ii) the autoinhibitory region comprises significantly fewer residues than the CaM-binding region. In a helical wheel projection of the CaM-binding domain, residues involved in autoinhibition cluster on one side of the helix, which is proposed to interact with an intramolecular receptor site in the pump. Residues influencing CaM negatively are situated on the other face of the helix, likely to face the cytosol, whereas residues controlling CaM binding positively are scattered throughout. We propose that early CaM recognition is mediated by the cytosolic face and that CaM subsequently competes with the intramolecular autoinhibitor in binding to the other face of the helix.

Highlights

The P2B Ca2ϩ-ATPases including PMCAs from animals and ACAs from plants are autoinhibitory proteins regulated by CaM
To investigate the relationship between autoinhibitory and CaM binding residues in a CaM-activated Ca2ϩ pump we focused on ACA8, an Arabidopsis plasma membrane Ca2ϩ-ATPase with a CaMbinding site at its N terminus [18]
Mutations of Trp-47 and Phe-60 to alanines both resulted in severely reduced affinity for CaM supporting the hypothesis that these residues function as anchor points in the CaM-binding domain (CaMBD) of ACA8

Summary

Introduction

The P2B Ca2ϩ-ATPases including PMCAs (plasma membrane Ca2ϩATPases) from animals and ACAs (autoinhibited Ca2ϩ-ATPases) from plants are autoinhibitory proteins regulated by CaM. The regulatory region consisting of a CaMBD and an autoinhibitory domain is located in the C terminus in animals and N terminus in plants [9, 10] These two domains are likely to be at least partly overlapping in both plant and mammalian Ca2ϩ-ATPases, as has been shown for the human PMCA4b (isoform 4, splice variant b) [11], Arabidopsis ACA2 (isoform 2) [12, 13], and ACA8 [14]. The mechanism behind CaM activation of an autoinhibited enzyme has been studied using the P2B plasma membrane Ca2ϩ-ATPase ACA8 from Arabidopsis [18]. Both the autoinhibitory domain and the CaMBD have recently been localized to the N terminus of ACA8 [14, 18, 19].

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