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(Received for publication, March 31, 1997, and in revised form, August 25, 1997)
From the Nitric oxide synthases (NOSs) are classified
functionally, based on whether calmodulin binding is
Ca2+-dependent (cNOS) or
Ca2+-independent (iNOS). This key dichotomy has not been
defined at the molecular level. Here we show that cNOS isoforms contain
a unique polypeptide insert in their FMN binding domains which is not
shared with iNOS or other related flavoproteins. Previously identified
autoinhibitory domains in calmodulin-regulated enzymes raise the
possibility that the polypeptide insert is the autoinhibitory domain of
cNOSs. Consistent with this possibility, three-dimensional molecular
modeling suggested that the insert originates from a site immediately
adjacent to the calmodulin binding sequence. Synthetic peptides derived
from the 45-amino acid insert of endothelial NOS were found to potently
inhibit binding of calmodulin and activation of cNOS isoforms. This
inhibition was associated with peptide binding to NOS, rather than free
calmodulin, and inhibition could be reversed by increasing calmodulin
concentration. In contrast, insert-derived peptides did not interfere
with the arginine site of cNOS, as assessed from
[3H]NG-nitro-L-arginine
binding, nor did they potently effect iNOS activity. Limited
proteolysis studies showed that calmodulin's ability to gate electron
flow through cNOSs is associated with displacement of the insert
polypeptide; this is the first specific calmodulin-induced change in
NOS conformation to be identified. Together, our findings strongly
suggest that the insert is an autoinhibitory control element, docking
with a site on cNOSs which impedes calmodulin binding and enzymatic
activation. The autoinhibitory control element molecularly defines
cNOSs and offers a unique target for developing novel NOS activators
and inhibitors.
Volume 272, Number 47,
Issue of November 21, 1997
pp. 29769-29777
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
An Autoinhibitory Control Element Defines Calcium-regulated
Isoforms of Nitric Oxide Synthase
,
,
,
,
,
,
,
,
and
Department of Biology,
Department of Biochemistry, The University of Texas
Health Science Center, San Antonio, Texas 78284-7760, ** Department of
Pharmacology, Cornell University Medical College, New York, New York
10021, and Program in Biochemistry and
Structural Biology, The Cornell University Graduate School of Medical
Sciences, New York, New York 10021
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