Importance of Stalk Segment S5 for Intramolecular Communication in the Sarcoplasmic Reticulum Ca2+-ATPase

Sørensen, Thomas; Andersen, Jens Peter

doi:10.1074/jbc.m004072200

Cited by 36 publications

(29 citation statements)

References 44 publications

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“…Upon dissociation of ADP the highly conserved TGES motif of domain A is inserted into the catalytic site of domain P, with the glutamate of TGES participating in the hydrolysis of the aspartyl phosphoryl bond as catalytic residue in the E2P form (10,20,21). The dephosphorylation of E2P has previously been found to be sensitive to amino acid substitutions or proteolytic cleavage in the linker segments connecting either domain A or domain P with the transmembrane segments M2, M3, and M5 (15,16,22,23). Hence, the linker segments appear to hold domain A and domain P in the optimal positions for catalysis of dephosphorylation.…”

Section: Resultsmentioning

confidence: 99%

“…Diffraction data were collected at the Protein Structure Factory beamline BL14.1 of Free University Berlin at BESSY and Deutsches Elektronen-Synchrotron (DESY/EMBL) beamline X11 and were processed and merged using the HKL package (14). Oligonucleotidedirected mutagenesis of cDNA encoding rabbit SERCA1a, expression in COS-1 cells, and functional analysis of the overall and partial reactions of expressed enzyme were carried out as described previously (15,16 …”

Section: Methodsmentioning

confidence: 99%

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Localization of a K+-binding Site Involved in Dephosphorylation of the Sarcoplasmic Reticulum Ca2+-ATPase

Sørensen

Clausen

Jensen

et al. 2004

Journal of Biological Chemistry

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Localization of a K+-binding Site Involved in Dephosphorylation of the Sarcoplasmic Reticulum Ca2+-ATPase

Sørensen

Clausen

Jensen

et al. 2004

Journal of Biological Chemistry

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“…Most of the other mutants showed wild type-like rates of H ϩ -ATPase-mediated proton transport. Targeted Proline Mutagenesis of S5-In the Ca 2ϩ -ATPase, M5 and S5 form a contiguous, long central helical element (3) that has been proposed to play a critical role in the coupling reaction (25). In contrast to the Ca 2ϩ -ATPase and related higher eukaryotic enzymes, the fungal H ϩ -ATPase has a naturally occurring proline, Pro 669 , as well as a Gly 670 , near the M5 interface, which should disrupt helical continuity in this region and create hinge-like flexibility.…”

Section: Resultsmentioning

confidence: 99%

Helical Stalk Segments S4 and S5 of the Plasma Membrane H+-ATPase from Saccharomyces cerevisiae Are Optimized to Impact Catalytic Site Environment

Soteropoulos¹,

Valiakhmetov²,

Kashiwazaki³

et al. 2001

Journal of Biological Chemistry

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The stalk segments of P-type ion-translocating enzymes are presumed to play important roles in energy coupling. In this work, stalk segments S4 and S5 of the yeast H ؉ -ATPase were examined for helical character, optimal length, and segment orientation by a combination of proline substitution, insertion/deletion mutagenesis, and second-site suppressor analyses. The substitution of various residues for helix-disrupting proline in both S4 (L353P,L353G; A354P; and G371P) and S5 (D676P and I684P) resulted in highly defective or inactive enzymes supporting the importance of helical character and/or the maintenance of essential interactions. The contiguous helical nature of transmembrane segment M5 and stalk element S5 was explored and found to be favorable, although not essential. The deletion or addition of one or more amino acids at positions Ala 354 in S4 and Asp 676 in S5, which were intended to either rotate helical faces or extend/reduce the length of helical segments, resulted in enzyme destabilization that abolished most enzyme assembly. Second-site suppressor mutations were obtained to primary site mutations G371A (S4) and D676G (S5) and were analyzed with a molecular structure model of the H ؉ -ATPase. Primary site mutations were predicted to alter the site of phosphorylation either directly or indirectly. The suppressor mutations either directly changed packing around the primary site or altered the environment of the site of phosphorylation. Overall, these data support the view that stalk segments S4 and S5 of the H ؉ -ATPase are helical elements that are optimized for length and interactions with other stalk elements and can influence the phosphorylation domain.The plasma membrane H ϩ -ATPase of yeast is an essential enzyme that actively pumps protons across the cellular membrane in order to maintain intracellular pH and the electrochemical proton gradient necessary for growth and development. It belongs to the superfamily of P-type ion-translocating ATPases for which there are more than 150 members known (1). The fungal H ϩ -ATPase is a class II, non-heavy metaltransporting enzyme that includes the plant H ϩ -ATPase and the animal Na ϩ ,K ϩ -ATPase, Ca 2ϩ

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“…The kinetic simulation software SimZyme (43,44) was used to simulate the amount of phosphoenzyme formed as a function of time.…”

Section: Methodsmentioning

confidence: 99%

Involvement of the L6–7 Loop in SERCA1a Ca2+-ATPase Activation by Ca2+ (or Sr2+) and ATP

Lenoir

Picard

Møller

et al. 2004

Journal of Biological Chemistry

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Wild-type (WT) and the double mutant D813A,D818A (ADA) of the L6 -7 loop of SERCA1a were expressed in yeast, purified, and reconstituted into lipids. This allowed us to functionally study these ATPases by both kinetic and spectroscopic means, and to solve previous discrepancies in the published literature about both experimental facts and interpretation concerning the role of this loop in P-type ATPases. We show that in a solubilized state, the ADA mutant experiences a dramatic decrease of its calcium-dependent ATPase activity. On the contrary, reconstituted in a lipid environment, it displays an almost unaltered maximal calcium-dependent ATPase activity at high (millimolar) ATP, with an apparent affinity for Ca 2؉ altered only moderately (3-fold). In the absence of ATP, the true affinity of ADA for Ca 2؉ is, however, more significantly reduced (20 -30-fold) compared with WT, as judged from intrinsic (Trp) or extrinsic (fluorescence isothiocyanate) fluorescence experiments. At low ATP, transient kinetics experiments reveal an overshoot in the ADA phosphorylation level primarily arising from the slowing down of the transition between the nonphosphorylated "E2" and "Ca 2 E1" forms of ADA. At high ATP, this slowing down is only partially compensated for, as ADA turnover remains more sensitive to orthovanadate than WT turnover. ADA ATPase also proved to have a reduced affinity for ATP in studies performed under equilibrium conditions in the absence of Ca 2؉ , highlighting the long range interactions between L6 -7 and the nucleotide-binding site. We propose that these mutations in L6 -7 could affect protonation-dependent winding and unwinding events in the nearby M6 transmembrane segment.Muscle relaxation occurs by of the removal of calcium from the cytosol and its accumulation into sarcoplasmic reticulum (SR) 1 through an active transport that consumes ATP (1, 2). In fast twitch muscle this transport is achieved by the SERCA1a isoform of Ca 2ϩ -ATPase (3), with a stoichiometry of two calcium ions transported per ATP molecule hydrolyzed (see reviews in Refs. 4 and 5). SR Ca 2ϩ -ATPase belongs to the family of P-type ATPases, which transport cations such as H ϩ , Na ϩ , K ϩ , or Ca 2ϩ across the membrane and share a common mechanism that involves autophosphorylation of the protein (see review in Ref. 6): transport is achieved through a reversible cycle, during which the ATPase is thought to sequentially adopt two main conformations (7, 8), E1 and E2, with a high and low affinity for the transported ion, and the overall cycle corresponds to completion of the following scheme: E2 3 Ca 2 E1 3 Ca 2 E1 ϳ P 3 E2P 3 E2 (for review, see Refs. 9 and 10). Much effort has gone into determining the structural organization of SR Ca 2ϩ -ATPase from primary structure predictions (11), electron microscopy (12), and three-dimensional crystallogenesis (13-15) (for a recent review see Ref. 16). Finally, the structure has been solved at the atomic level, first in a Ca 2 E1 state (14) and then in a thapsigargin-stabilized E2 state (15). T...

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Importance of Stalk Segment S5 for Intramolecular Communication in the Sarcoplasmic Reticulum Ca2+-ATPase

Abstract: Sixteen residues in stalk segment S5 of the Ca 2؉ -ATPase of sarcoplasmic reticulum were studied by sitedirected mutagenesis.

Cited by 36 publications

References 44 publications

Localization of a K+-binding Site Involved in Dephosphorylation of the Sarcoplasmic Reticulum Ca2+-ATPase

Localization of a K+-binding Site Involved in Dephosphorylation of the Sarcoplasmic Reticulum Ca2+-ATPase

Helical Stalk Segments S4 and S5 of the Plasma Membrane H+-ATPase from Saccharomyces cerevisiae Are Optimized to Impact Catalytic Site Environment

Involvement of the L6–7 Loop in SERCA1a Ca2+-ATPase Activation by Ca2+ (or Sr2+) and ATP

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