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J. Biol. Chem., Vol. 277, Issue 31, 28319-28329, August 2, 2002
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From the Krannert Institute of Cardiology and the Department of
Medicine, Indiana University School of Medicine,
Indianapolis, Indiana 46202
Phospholamban (PLB) is a 52-amino acid inhibitor
of the Ca2+-ATPase in cardiac sarcoplasmic reticulum
(SERCA2a), which acts by decreasing the apparent affinity of the enzyme
for Ca2+. To localize binding sites of SERCA2a for PLB, we
performed Cys-scanning mutagenesis of PLB, co-expressed the PLB mutants
with SERCA2a in insect cell microsomes, and tested for cross-linking of
the mutated PLB molecules to SERCA2a using 1,6-bismaleimidohexane, a
10-Å-long, homobifunctional thiol cross-linking agent. Of several mutants tested, only PLB with a Cys replacement at position 30 (N30C-PLB) cross-linked to SERCA2a. Cross-linking occurred specifically and with high efficiency. The process was abolished by micromolar Ca2+ or by an anti-PLB monoclonal antibody and was
inhibited 50% by phosphorylation of PLB by cAMP-dependent
protein kinase. The SERCA2a inhibitors thapsigargin and cyclopiazonic
acid also completely prevented cross-linking. The two essential
requirements for cross-linking of N30C-PLB to SERCA2a were a
Ca2+-free enzyme and, unexpectedly, a micromolar
concentration of ATP or ADP, demonstrating that N30C-PLB cross-links
preferentially to the nucleotide-bound, E2 state of
SERCA2a. Sequencing of a purified proteolytic fragment in combination
with SERCA2a mutagenesis identified Cys318 of SERCA2a as
the sole amino acid cross-linked to N30C-PLB. The proximity of residue
30 of PLB to Cys318 of SERCA2a suggests that PLB may
interfere with Ca2+ activation of SERCA2a by a protein
interaction occurring near transmembrane helix M4.
Close Proximity between Residue 30 of Phospholamban and Cysteine
318 of the Cardiac Ca2+ Pump Revealed by Intermolecular
Thiol Cross-linking*
,
*
This work was supported by National Institutes of Health
Grant HL49428.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Krannert Institute of
Cardiology, 1800 N. Capitol Ave., Indianapolis, IN 46202. Tel.:
317-962-0095; Fax: 317-962-0113; E-mail: lrjones@iupui.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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