title: Conformational changes produced by ATP binding to the plasma membrane calcium pump.
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.
authors.affiliation: From the Instituto de Química y Fisicoquímica Biologicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Junín 956 (1113) Buenos Aires, Argentina.
authors.forename: Irene C
authors.initials: IC
authors.lastname: Mangialavori
authors.forename: Mariela S
authors.initials: MS
authors.lastname: Ferreira-Gomes
authors.forename: Nicolás A
authors.initials: NA
authors.lastname: Saffioti
authors.forename: Rodolfo M
authors.initials: RM
authors.lastname: González-Lebrero
authors.forename: Rolando C
authors.initials: RC
authors.lastname: Rossi
authors.forename: Juan Pablo F C
authors.initials: JP
authors.lastname: Rossi
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000201:enzymology*; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D008956:Models, Chemical; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: D010713:Phosphatidylcholines; D000255:Adenosine Triphosphate; D053499:Plasma Membrane Calcium-Transporting ATPases
keywords: ATP; Calcium; Calcium ATPase; Calcium Transport; Protein Conformation; Reaction Cycle; Vanadate
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