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Two domains of the smoothelin‐like 1 protein bind apo‐and calcium‐calmodulin independently
Author(s) -
UlkeLemee Annegret,
Ishida Hiroaki,
Vogel Hans J,
MacDonald Justin A
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1036.2
Subject(s) - calmodulin , chemistry , calcium binding protein , calcium , binding site , calcium in biology , biophysics , plasma protein binding , binding domain , calponin , biochemistry , intracellular , microbiology and biotechnology , biology , actin , organic chemistry
The smoothelin‐like 1 protein (SMTNL1; also known as calponin homology (CH) domain‐associated smooth muscle, CHASM) is a modulator of vascular smooth muscle contractility and can bind to calmodulin (CaM) and tropomyosin. The intracellular calcium receptor CaM switches between calcium‐free (apo‐CaM) and calcium‐bound (Ca‐CaM) forms. Bioinformatic analysis of the SMTNL1 sequence predicted two CaM‐binding regions: CBD1, N‐terminal to the CH domain and CBD2, a previously described IQ motif within the CH domain. We sought to describe the contribution of SMTNL1's two CBDs to CaM binding in the presence or absence of calcium. Using a variety of assays (biochemical pull‐downs, surface plasmon resonance, NMR and intracellular co‐localization by microscopy), we determined that CBD1 binds preferentially to Ca‐CaM while CBD2 binds preferentially to apo‐CaM. Mutation of key acidic residues abolished apo‐CaM binding to CBD2, while hydrophobic residues in CBD1 were required for Ca‐CaM binding. The binding affinities (~4 ×10 −6 M) were similar for both. The association of SMTNL1 with apo‐CaM and Ca‐CaM suggest that endogenous SMTNL1 is always associated with CaM. This lead us to the conclusion that changes in the CaM binding to CBD1 or CBD2 may allow for downsteam effects, such as disruption of SMTNL1 binding to tropomyosin.