title: Ca2+ occlusion and gating function of Glu309 in the ADP-fluoroaluminate analog of the Ca2+-ATPase phosphoenzyme intermediate.
abstract: In the absence of ATP the sarcoplasmic reticulum ATPase (SERCA) binds two Ca(2+) with high affinity. The two bound Ca(2+) rapidly undergo reverse dissociation upon addition of EGTA, but can be distinguished by isotopic exchange indicating fast exchange at a superficial site (site II), and retardation of exchange at a deeper site (site I) by occupancy of site II. Site II mutations that allow high affinity binding to site I, but only low affinity binding to site II, show that retardation of isotopic exchange requires higher Ca(2+) concentrations with the N796A mutant, and is not observed with the E309Q mutant even at millimolar Ca(2+). Fluoroaluminate forms a complex at the catalytic site yielding stable analogs of the phosphoenzyme intermediate, with properties similar to E2-P or E1-P.Ca(2). Mutational analysis indicates that Asp(351), Lys(352), Thr(353), Asp(703), Asn(706), Asp(707), Thr(625), and Lys(684) participate in stabilization of fluoroaluminate and Mg(2+) at the phosphorylation site. In the presence of fluoroaluminate and Ca(2+), ADP (or AMP-PCP) favors formation of a stable ADP.E1-P.Ca(2) analog. This produces strong occlusion of Ca(2+) bound to both sites (I and II), whereby dissociation occurs very slowly even following addition of EGTA. Occlusion by fluoraluminate and ADP is not observed with the E309Q mutant, suggesting a gating function of Glu(309) at the mouth of a binding cavity with a single path of entry. This phenomenon corresponds to the earliest step of the catalytic cycle following utilization of ATP. Experiments on limited proteolysis reveal that a long range conformational change, involving displacement of headpiece domains and transmembrane helices, plays a mechanistic role.
authors.affiliation: Department of Biochemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA. ginesi@umaryland.edu
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Inesi
authors.forename: Hailun
authors.initials: H
authors.lastname: Ma
authors.forename: David
authors.initials: D
authors.lastname: Lewis
authors.forename: Cheng
authors.initials: C
authors.lastname: Xu
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000535:Aluminum / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D005461:Fluorine / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; C053629:fluoroaluminum; D005461:Fluorine; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; D000535:Aluminum; D000252:Calcium-Transporting ATPases; D002118:Calcium