Substrate-induced Conformational Fit and Headpiece Closure in the Ca2+ATPase (SERCA)

Abstract

Protection of the Ca2؉ ATPase (SERCA) from proteinase K digestion has been observed following the addition of Ca 2؉ , Mg 2؉ , 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 Ca 2؉ or following mutations (E771Q or N796A) that interfere with Ca 2؉ binding. Therefore, nucleotide binds to the Ca 2؉ -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 Ca 2؉ -bound conformation. Mg 2؉ is not required for the protective effect of nucleotide, even though it is specifically required for the subsequent catalytic reactions. The sarcoplasmic reticulum (SR)1 ATPase is a 110-kDa enzyme that utilizes 1 mol of ATP for active transport of two calcium ions in exchange for 2 mol of protons. The catalytic cycle begins with cooperative binding of two calcium ions from the cytosolic medium followed by ATP utilization and formation of a phosphorylated enzyme intermediate. The bound calcium is then released onto the luminal side of the SR, and the cycle is completed by hydrolytic cleavage of acylphosphate.The ATPase molecular structure includes a transmembrane region made of 10 clustered helical segments including the Ca 2ϩ binding domain and a cytosolic headpiece including the nucleotide binding (N), phosphorylation (P), and actuator (A) domains. Structural studies have shown that if crystallization is performed in the presence of Ca 2ϩ the three headpiece domains are distinctly separate in an open configuration (2). On the other hand, in the absence of Ca 2ϩ , those domains gather to form a compact headpiece (3, 4). This finding suggests that in solution the three domains undergo fluctuations, yielding different enzyme conformations depending on the presence of specific ligands. Taking advantage of a selective ATPase cleavage (5), it was then shown that the addition of nucleotides in the presence of Mg 2ϩ and Ca 2ϩ ...

Deletions of Any Single Residues in Glu40-Ser48 Loop Connecting A Domain and the First Transmembrane Helix of Sarcoplasmic Reticulum Ca2+-ATPase Result in Almost Complete Inhibition of Conformational Transition and Hydrolysis of Phosphoenzyme Intermediate

“…Single deletions or substitutions of the residues in the adjacent N-terminal (His 32 -Asn 39 ) and C-terminal (Leu 49 -Ile 54 ) regions of the Glu 40 -Ser 48 loop had only slight or moderate effect on the activity, except that the specific substitutions of Asn 39 (N39D and N39T, but not N39A) had a significantly reduced activity. Quadruple alanine substitutions of Asn 39 , Glu 40 , Glu 44 , and Glu 45 were previously shown to cause no loss of function (22), and the present results are in essential agreement.…”

“…These facts, together with the observed blocking of the E1P to E2P transition by the specific substitutions, strongly suggest that the above residues (Asp 601 , Pro 603 , Asp 627 , and Asp 703 ) or at least some of them are involved in the A-P domain interaction together with the TGES 184 loop for the loss of the ADP sensitivity, although information on the threedimensional structure of E2PCa 2 formed in step 4 (the ADPinsensitive EP with bound or occluded (39) Ca 2ϩ ) is yet unavailable. Importantly, Asp 627 , Asp 703 , Lys 684 , Asp 601 , and Pro 603 were all found previously (30,31,40) to be crucial also for the conformation of catalytic site and for the formation and hydrolysis of EP as their substitutions (other than the specific one stated above) diminished the phosphorylation from ATP and from P i . Therefore, they seem to play multiple essential roles and the roles likely alter as the steps proceed in the Ca 2ϩ transport cycle, i.e.…”

“…3). Deletions of any single residues in the Glu 40 -Ser 48 loop resulted in almost complete loss of the activity, while their nonconservative substitutions caused only partial decrease or rather slight increase in the activity. Single deletions or substitutions of the residues in the adjacent N-terminal (His 32 -Asn 39 ) and C-terminal (Leu 49 -Ile 54 ) regions of the Glu 40 -Ser 48 loop had only slight or moderate effect on the activity, except that the specific substitutions of Asn 39 (N39D and N39T, but not N39A) had a significantly reduced activity.…”

“…the ATP binding and phosphorylation, the isomeric transition of EP (the domain association), and its hydrolysis. Lys 684 seems not to be involved in the domain interaction in E2P (E2V), but likely functions in coordination of the covalently bound phosphate in EP (38,40) and thus in the possible change in the immediate vicinity of the phosphate for the loss of ADP sensitivity.…”

“…1). The single deletions in the Glu 40 Table I. The fraction of E2P determined at each time point (as described in the legend to Fig.…”