Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volume 1844, Issue 9, Pages 1580-1590Publisher
ELSEVIER
DOI: 10.1016/j.bbapap.2014.05.011
Keywords
SMTNL1; CaM; IQ-motif; CHASM; Calmodulin-binding domain; Intrinsically-disordered protein
Categories
Funding
- Canadian Institutes of Health Research [MOP-97931]
- Natural Sciences and Engineering Research Council
- Heart & Stroke Foundation of Canada Fellowship
- Alberta Innovates-Health Solutions (AIHS) Senior Scholar award
- Canada Research Chair (Tier II) in Smooth Muscle Pathophysiology
- AIHS
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The smoothelin-like 1 protein (SMTNL1) is a modulator of smooth and skeletal muscle contractility and can bind to calmodulin and tropomyosin. Calmodulin is the major calcium sensor of eukaryotic cells and it can cycle between calcium-free (apo-CaM) and calcium-bound (Ca-CaM) forms. Bioinformatic screening of the SMTNL1 sequence predicted a second CaM-binding region (CBD1) that is located N-terminal to the previously defined apo-CaM-binding site (CBD2). Pull-down assays, surface plasmon resonance, isothermal calorimetry and NMR techniques were used to determine that CBD1 associated preferentially to Ca-CaM while CBD2 bound preferentially to apo-CaM. Mutation of hydrophobic residues abolished Ca-CaM-binding to CBD1 while acidic residues in CBD2 were necessary for apo-CaM-binding to CBD2. The dissociation constant (K-d) for Ca-CaM-binding to a CBD1 peptide was 26 (*) 10(-6)M while the value for binding to a longer protein construct was 0.5 (*) 10(-6)M. The binding of SMTNL1 to both apo-CaM and Ca-CaM suggests that endogenous CaM is continuously associated with SMTNL1 to allow for quick response to changes in intracellular calcium levels. We also found that the intrinsically disordered N-terminus of SMTNL1 can reduce binding to apo-CaM and increase binding to Ca-CaM. This finding suggests that an additional CaM-binding region may exist and/or that intramolecular interactions between the N-terminus and the folded C-terminus reduce apo-CaM-binding to CBD2. Intriguingly, CBD1 is located close to the SMTNL1 phosphorylation site and tropomyosin-binding region. We discuss the possibility that all three signals are integrated at the region surrounding CBD1. (C) 2014 Elsevier B.V. All rights reserved.
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