PMID- 18052080
OWN - NLM
STAT- MEDLINE
DCOM- 20080227
LR  - 20191210
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 46
IP  - 51
DP  - 2007 Dec 25
TI  - Inhibitors bound to Ca(2+)-free sarcoplasmic reticulum Ca(2+)-ATPase lock its 
      transmembrane region but not necessarily its cytosolic region, revealing the 
      flexibility of the loops connecting transmembrane and cytosolic domains.
PG  - 15162-74
AB  - Ca2+-free crystals of sarcoplasmic reticulum Ca2+-ATPase have, up until now, been 
      obtained in the presence of inhibitors such as thapsigargin (TG), bound to the 
      transmembrane region of this protein. Here, we examined the consequences of such 
      binding for the protein. We found that, after TG binding, an active site ligand 
      such as beryllium fluoride can still bind to the ATPase and change the 
      conformation or dynamics of the cytosolic domains (as revealed by the protection 
      afforded against proteolysis), but it becomes unable to induce any change in the 
      transmembrane domain (as revealed by the intrinsic fluorescence of the membranous 
      tryptophan residues). TG also obliterates the Trp fluorescence changes normally 
      induced by binding of MgATP or metal-free ATP, as well as those induced by 
      binding of Mg2+ alone. In the nucleotide binding domain, the environment of 
      Lys515 (as revealed by fluorescein isothiocyanate fluorescence after specific 
      labeling of this residue) is significantly different in the ATPase complex with 
      aluminum fluoride and in the ATPase complex with beryllium fluoride, and in the 
      latter case it is modified by TG. All these facts document the flexibility of the 
      loops connecting the transmembrane and cytosolic domains in the ATPase. In the 
      absence of active site ligands, TG protects the ATPase from cleavage by 
      proteinase K at Thr242-Glu243, suggesting TG-induced reduction in the mobility of 
      these loops. 2,5-Di-tert-butyl-1,4-dihydroxybenzene or cyclopiazonic acid, 
      inhibitors which also bind in or near the transmembrane region, also produce 
      similar overall effects on Ca2+-free ATPase.
FAU - Montigny, Cedric
AU  - Montigny C
AD  - CNRS, URA 2096 (Proteines Membranaires Transductrices d'Energie), F-91191 
      Gif-sur-Yvette, France.
FAU - Picard, Martin
AU  - Picard M
FAU - Lenoir, Guillaume
AU  - Lenoir G
FAU - Gauron, Carole
AU  - Gauron C
FAU - Toyoshima, Chikashi
AU  - Toyoshima C
FAU - Champeil, Philippe
AU  - Champeil P
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20071201
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Enzyme Inhibitors)
RN  - 67526-95-8 (Thapsigargin)
RN  - EC 3.4.21.64 (Endopeptidase K)
RN  - EC 7.2.2.10 (Calcium-Transporting ATPases)
RN  - K3Z4F929H6 (Lysine)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Calcium/metabolism
MH  - Calcium-Transporting ATPases/*antagonists & inhibitors/*metabolism
MH  - Cell Membrane/drug effects/*enzymology
MH  - Cytoplasm/drug effects/*enzymology
MH  - Endopeptidase K/metabolism
MH  - Enzyme Inhibitors/*pharmacology
MH  - Lysine/metabolism
MH  - Pliability
MH  - Sarcoplasmic Reticulum/*drug effects/*enzymology
MH  - Thapsigargin/pharmacology
EDAT- 2007/12/07 09:00
MHDA- 2008/02/28 09:00
CRDT- 2007/12/07 09:00
PHST- 2007/12/07 09:00 [pubmed]
PHST- 2008/02/28 09:00 [medline]
PHST- 2007/12/07 09:00 [entrez]
AID - 10.1021/bi701855r [doi]
PST - ppublish
SO  - Biochemistry. 2007 Dec 25;46(51):15162-74. doi: 10.1021/bi701855r. Epub 2007 Dec 
      1.