title: Two domains of the smoothelin-like 1 protein bind apo- and calcium-calmodulin independently.
abstract: 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 (Kd) 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, 3280 Hospital Drive NW, Calgary, AB, T2N 4Z6, Canada. Electronic address: aulke@ucalgary.ca.
authors.forename: Annegret
authors.initials: A
authors.lastname: Ulke-Lemée
authors.affiliation: Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, T2N 1N4, Canada. Electronic address: hishida@ucalgary.ca.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Ishida
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, 3280 Hospital Drive NW, Calgary, AB, T2N 4Z6, Canada. Electronic address: mchappel@ucalgary.ca.
authors.forename: Mona
authors.initials: M
authors.lastname: Chappellaz
authors.affiliation: Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, T2N 1N4, Canada. Electronic address: vogel@ucalgary.ca.
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Vogel
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, 3280 Hospital Drive NW, Calgary, AB, T2N 4Z6, Canada. Electronic address: jmacdo@ucalgary.ca.
authors.forename: Justin A
authors.initials: JA
authors.lastname: MacDonald
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000096985:Calponins
chemical_list: D001059:Apoproteins; D002118:Calcium; D002147:Calmodulin; D009124:Muscle Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D014335:Tropomyosin; C491838:SMTNL1 protein, mouse; D000096985:Calponins
keywords: CHASM; CaM; Calmodulin-binding domain; IQ-motif; Intrinsically-disordered protein; SMTNL1