The Length of the A-M3 Linker Is a Crucial Determinant of the Rate of the Ca2+ Transport Cycle of Sarcoplasmic Reticulum Ca2+-ATPase

Abstract

Ion translocation by the sarcoplasmic reticulum Ca 2؉ -ATPase depends on large movements of the A-domain, but the driving forces have yet to be defined. The A-domain is connected to the ion-binding membranous part of the protein through linker regions. We have determined the functional consequences of changing the length of the linker between the A-domain and transmembrane helix M3 ("A-M3 linker") by insertion and deletion mutagenesis at two sites. It was feasible to insert as many as 41 residues (polyglycine and glycine-proline loops) in the flexible region of the linker without loss of the ability to react with Ca 2؉ and ATP and to form the phosphorylated Ca 2 E1P intermediate, but the rate of the energy-transducing conformational transition to E2P was reduced by >80%. Insertion of a smaller number of residues gave effects gradually increasing with the length of the insertion. Deletion of two residues at the same site, but not replacement with glycine, gave a similar reduction as the longest insertion. Insertion of one or three residues in another part of the A-M3 linker that forms an ␣-helix ("A3 helix") in E2/E2P conformations had even more profound effects on the ability of the enzyme to form E2P. These results demonstrate the importance of the length of the A-M3 linker and of the position and integrity of the A3 helix for stabilization of E2P and suggest that, during the normal enzyme cycle, strain of the A-M3 linker could contribute to destabilize the Ca 2 E1P state and thereby to drive the transition to E2P.The sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA) 2 is a membrane-bound ion pump that transports Ca 2ϩ against a steep concentration gradient, utilizing the energy derived from ATP hydrolysis (1-3). It belongs to the family of P-type ATPases, in which the ␥-phosphoryl group of ATP is transferred to a conserved aspartic acid residue during the reaction cycle. Both phospho and dephospho forms of the enzyme undergo transitions between so-called E1 and E2 conformations (Scheme 1). The E1 and E1P states display specificity for reaction with ATP and ADP, respectively ("kinase activity"), whereas E2P and E2 react with water and P i instead of nucleotide ("phosphatase activity"). The E1 dephosphoenzyme of the Ca 2ϩ

P-type ATPases at a glance

“…Several studies that have probed shortening, lengthening or proteolytic cleavage of these linkers in SERCA demonstrate a severe effect on the communication between the phosphorylation site and the ion-binding site, which causes impairment of pump function (Daiho et al, 2003;Daiho et al, 2007;Holdensen and Andersen, 2009;Lenoir et al, 2004;Moller et al, 2002). When phosphoryl transfer releases the tight connection between the N-and Pdomains, and when the A-domain rotates into the emerging gap of the phosphoenzyme, this extensive (almost 120°) rotation exerts a pull on the three linkers.…”

Coarse‐Grained Simulations of Transitions in the E2‐to‐E1 Conformations for Ca ATPase (SERCA) Show Entropy–Enthalpy Compensation

“…In an experimental work, 16 the insertions, deletions, and substitutions in the A-M3 region resulted in a dramatic change in the reaction rates of this transition. In order to combine information from the sampled intermediates with an estimate for the changes in kinetics, we constructed an approximation of the total free-energy changes along the computed DIMS transitions.…”

“…All inserts were modeled as coil regions due to the nature of the residues that comprised it, which were then minimized keeping the rest of the protein fixed so that the inserts could adopt a favorable conformation. Following the experimental study, 16 we used two sites to insert residues: site 1, between Glu243-Gln244; and site 2, Gly233-Lys234 (Fig. 2).…”

“…2) on kinetics between the E2 and E1 states of the pump cycle. A set of deletion and insertion mutants was found to have a large and consistent change on the measured kinetics 16 ( Table 1). The questions that were raised in the experimental study, and partially answered by the experiments, were as follows: Why did those particular mutations had the measured effects?…”

“…5,9,12 The rotation of the A domain is critical in the E1P → E2P conversion. 9,[13][14][15][16] From the X-ray structures, the A domain seems to rotate about 90°a nd comes in close proximity to the P domain. 2,[7][8][9] The Asp351 is phosphorylated in the E1P state and is dephosphorylated by the TGE 183 S motif in the A domain to form the E2P state.…”