Substrate-induced conformational fit and headpiece closure in the Ca2+ATPase (SERCA).
Protection of the Ca2+ATPase (SERCA) from proteinase K digestion has been observed following the addition of Ca2+, Mg2+, and nucleotide and interpreted as a substrate-dependent conformational change (1). The protected digestion site is located on the loop connecting the A domain and the M3 transmembrane helix. We studied by mutational analysis the protective effect of AMP-PCP, an ATP analog that is not utilized for enzyme phosphorylation. We found that the nucleotide protective effect is interfered with by single mutations of Arg-560 and Glu-439 in the N domain and Lys-352, Lys-684, Thr-353, Asp-703, and Asp-707 in the P domain. This is consistent with a transition from the open to the compact configuration of the ATPase headpiece and approximation of the N and P domains by interactions with the nucleotide adenosine and phosphate moieties, respectively. The A domain-M3 loop is consequently involved. Protection by nucleotide substrate increased following the mutations of Asp-351 (the residue undergoing phosphorylation by ATP) and neighboring Asn-706 to Ala, underlying the importance of side chain specificity in positioning the nucleotide terminal phosphate and limiting the stability of the substrate-enzyme complex. Protection is not observed when AMP-PCP is added in the absence of Ca2+ or following mutations (E771Q or N796A) that interfere with Ca2+ binding. Therefore, nucleotide binds to the Ca2+-activated enzyme in the open headpiece conformation and the consequent approximation of the N and P domains occurs while the transmembrane domain is still in the Ca2+-bound conformation. Mg2+ is not required for the protective effect of nucleotide, even though it is specifically required for the subsequent catalytic reactions.
Ma
Hailun
H
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Inesi
Giuseppe
G
Toyoshima
Chikashi
C
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002413:Cations, Divalent; D002645:Chickens; D019286:Endopeptidase K / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D011994:Recombinant Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013329:Structure-Activity Relationship
D002413:Cations, Divalent; D010446:Peptide Fragments; D011994:Recombinant Proteins; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000255:Adenosine Triphosphate; D019286:Endopeptidase K; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium