title: Nucleotide activation of the Ca-ATPase.
abstract: We have used fluorescence spectroscopy, molecular modeling, and limited proteolysis to examine structural dynamics of the sarcoplasmic reticulum Ca-ATPase (SERCA). The Ca-ATPase in sarcoplasmic reticulum vesicles from fast twitch muscle (SERCA1a isoform) was selectively labeled with fluorescein isothiocyanate (FITC), a probe that specifically reacts with Lys-515 in the nucleotide-binding site. Conformation-specific proteolysis demonstrated that FITC labeling does not induce closure of the cytoplasmic headpiece, thereby assigning FITC-SERCA as a nucleotide-free enzyme. We used enzyme reverse mode to synthesize FITC monophosphate (FMP) on SERCA, producing a phosphorylated pseudosubstrate tethered to the nucleotide-binding site of a Ca(2+)-free enzyme (E2 state to prevent FMP hydrolysis). Conformation-specific proteolysis demonstrated that FMP formation induces SERCA headpiece closure similar to ATP binding, presumably due to the high energy phosphoryl group on the fluorescent probe (ATP·E2 analog). Subnanosecond-resolved detection of fluorescence lifetime, anisotropy, and quenching was used to characterize FMP-SERCA (ATP·E2 state) versus FITC-SERCA in Ca(2+)-free, Ca(2+)-bound, and actively cycling phosphoenzyme states (E2, E1, and EP). Time-resolved spectroscopy revealed that FMP-SERCA exhibits increased probe dynamics but decreased probe accessibility compared with FITC-SERCA, indicating that ATP exhibits enhanced dynamics within a closed cytoplasmic headpiece. Molecular modeling was used to calculate the solvent-accessible surface area of FITC and FMP bound to SERCA crystal structures, revealing a positive correlation of solvent-accessible surface area with quenching but not anisotropy. Thus, headpiece closure is coupled to substrate binding but not active site dynamics. We propose that dynamics in the nucleotide-binding site of SERCA is important for Ca(2+) binding (distal allostery) and phosphoenzyme formation (direct activation).
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Autry
authors.forename: John E
authors.initials: JE
authors.lastname: Rubin
authors.forename: Bengt
authors.initials: B
authors.lastname: Svensson
authors.forename: Ji
authors.initials: J
authors.lastname: Li
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D016880:Anisotropy; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D019277:Entropy; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009132:Muscles / Q000378:metabolism; D009711:Nucleotides / Q000235:genetics*; D010710:Phosphates / Q000737:chemistry; D011487:Protein Conformation; D011817:Rabbits; D012997:Solvents / Q000737:chemistry; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D008024:Ligands; D009711:Nucleotides; D010710:Phosphates; D012997:Solvents; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium