Autonomous folding of the recombinant large cytoplasmic loop of sarcoplasmic reticulum Ca2+-ATPase probed by affinity labeling and trypsin digestion.
Recombinant large cytoplasmic loop (LCL, residues 329-740) of sarcoplasmic reticulum Ca2+-ATPase, expressed in and purified from Escherichia coli, comprises most of the active site and binds ATP [Moutin, M.-J., Cuillel, M., Rapin, C., Miras, R., Anger, M., Lompré, A.-M. & Dupont, Y. (1994) J. Biol. Chem. 269, 11147-11154]. In this study, we show that fluorescein-5' isothiocyanate (FITC) specifically labels the same lysine residue as in the native Ca2+-ATPase (Lys515), with similar kinetics and pH dependence. ATP blocks the reaction with the lysine residue, but at higher concentrations compared with those for the native pump, in agreement with the lower ATP-binding affinity found previously. Graded tryptic digestion of LCL shows that favored cleavage is at the T1 site and that the N-terminal 75% of LCL are resistant to trypsin, as is native Ca2+-ATPase. Other experiments reveal differences to the native pump. (a) FITC derivatizes some -SH groups of LCL. (b) The C-terminal 25% of the polypeptide is susceptible to end-clipping by trypsin. (c) 2',3'-O-(2,4,6-trinitrophenyl)-ATP fails to specifically label the LCL (on the equivalent of Lys492), although it binds tightly (KD = 1.3 microM) and (d) Glutaraldehyde does not specifically cross-link LCL (between the equivalent of Lys492 and Arg678). These results could be explained by a flexible and loose structure of the hinge region of LCL (C-terminal 25%). Anchoring this region in the membrane and/or interaction with the missing beta-strand domain may be required for its compact folding and proper interaction with the rest of LCL. The results suggest that the N-terminal 75% of LCL expressed in E. coli folds autonomously to a fairly stable unit and native-like structure, encompassing the phosphorylation and central ATP binding sections. The hinge region does not appear to be part of the FITC-binding site but constitutes portions of the 2',3'-O-(2,4,6-trinitrophenyl)-ATP and, probably, ATP-binding site.
Moutin
M J
MJ
Laboratoire de Biophysique Moléculaire et Cellulaire, URA 520 CNRS, CEA, DBMS/CENG, Grenoble, France. moutin@dsvgre.cea.fr
Rapin
C
C
Miras
R
R
Vinçon
M
M
Dupont
Y
Y
McIntosh
D B
DB
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000345:Affinity Labels; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D003432:Cross-Linking Reagents; D003593:Cytoplasm; D005033:Ethylmaleimide / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005976:Glutaral; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014357:Trypsin
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011994:Recombinant Proteins; C058476:O(2',3')-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine; D005033:Ethylmaleimide; D005976:Glutaral