Solution Structure of the Calponin Homology (CH) Domain from the Smoothelin-like 1 Protein

The SMTNL1 protein contains a single type-2 calponin homology (CH) domain at its C terminus that shares sequence identity with the smoothelin family of smooth muscle-specific proteins. In contrast to the smoothelins, SMTNL1 does not associate with F-actin in vitro, and its specific role in smooth muscle remains unclear. In addition, the biological function of the C-terminal CH-domains found in the smoothelin proteins is also poorly understood. In this work, we have therefore determined the solution structure of the CH-domain of mouse SMTNL1 (SMTNL1-CH; residues 346-459). The secondary structure and the overall fold for the C-terminal type-2 CH-domain is very similar to that of other CH-domains. However, two clusters of basic residues form a unique surface structure that is characteristic of SMTNL1-CH. Moreover, the protein has an extended C-terminal α-helix, which contains a calmodulin (CaM)-binding IQ-motif, that is also a distinct feature of the smoothelins. We have characterized the binding of apo-CaM to SMTNL1-CH through its IQ-motif by isothermal titration calorimetry and NMR chemical shift perturbation studies. In addition, we have used the HADDOCK protein-protein docking approach to construct a model for the complex of apo-CaM and SMTNL1-CH. The model revealed a close interaction of SMTNL1-CH with the two Ca2+ binding loop regions of the C-terminal domain of apo-CaM; this mode of apo-CaM binding is distinct from previously reported interactions of apo-CaM with IQ-motifs. Finally, we comment on the putative role of the CH-domain in the biological function of SMTNL1. The SMTNL1 protein contains a single type-2 calponin homology (CH) domain at its C terminus that shares sequence identity with the smoothelin family of smooth muscle-specific proteins. In contrast to the smoothelins, SMTNL1 does not associate with F-actin in vitro, and its specific role in smooth muscle remains unclear. In addition, the biological function of the C-terminal CH-domains found in the smoothelin proteins is also poorly understood. In this work, we have therefore determined the solution structure of the CH-domain of mouse SMTNL1 (SMTNL1-CH; residues 346-459). The secondary structure and the overall fold for the C-terminal type-2 CH-domain is very similar to that of other CH-domains. However, two clusters of basic residues form a unique surface structure that is characteristic of SMTNL1-CH. Moreover, the protein has an extended C-terminal α-helix, which contains a calmodulin (CaM)-binding IQ-motif, that is also a distinct feature of the smoothelins. We have characterized the binding of apo-CaM to SMTNL1-CH through its IQ-motif by isothermal titration calorimetry and NMR chemical shift perturbation studies. In addition, we have used the HADDOCK protein-protein docking approach to construct a model for the complex of apo-CaM and SMTNL1-CH. The model revealed a close interaction of SMTNL1-CH with the two Ca2+ binding loop regions of the C-terminal domain of apo-CaM; this mode of apo-CaM binding is distinct from previously reported interactions of apo-CaM with IQ-motifs. Finally, we comment on the putative role of the CH-domain in the biological function of SMTNL1. Calponin is a key regulator of smooth muscle contraction (reviewed in Refs. 1Winder S.J. Walsh M.P. Cell. Signal. 1993; 5: 677-686Crossref PubMed Scopus (128) Google Scholar and 2Szymanski P.T. J. Muscle Res. Cell Motil. 2004; 25: 7-19Crossref PubMed Scopus (33) Google Scholar), and the calponin homology (CH) 5The abbreviations used are: CH, calponin homology; SMTNL1-CH, CH-domain of smoothelin-like 1; CaM, calmodulin; CaM-nt, the N-terminal domain of CaM; CaM-ct, the C-terminal domain of CaM; HSQC, heteronuclear single quantum correlation; RDC, residual dipolar coupling; NOE, nuclear Overhauser effect; ITC, isothermal titration calorimetry; CSP, chemical shift perturbation; r.m.s.d., root mean square deviation; TOCSY, total correlation spectroscopy; NOESY, NOE spectroscopy. domain was identified in the N-terminal portion of this protein as an ∼110-amino acid region that contributed to its actin-binding properties (3Castresana J. Saraste M. FEBS Lett. 1995; 374: 149-151Crossref PubMed Scopus (124) Google Scholar). CH-domains have since been identified in a number of cytoskeletal and signaling proteins (4Stradal T. Kranewitter W. Winder S.J. Gimona M. FEBS Lett. 1998; 431: 134-137Crossref PubMed Scopus (113) Google Scholar, 5Gimona M. Djinovic-Carugo K. Kranewitter W.J. Winder S.J. FEBS Lett. 2002; 513: 98-106Crossref PubMed Scopus (267) Google Scholar, 6Korenbaum E. Rivero F. J. Cell Sci. 2002; 115: 3543-3545Crossref PubMed Scopus (113) Google Scholar). The CH-domain has a highly conserved structure that is associated with diverse biological functions. Although the various CH-domains share relatively little amino acid sequence identity, a number of strictly conserved hydrophobic residues give rise to an almost invariant hydrophobic core (see Fig. 1). Thus, all of the CH-domain structures that have been determined to date are very similar. Despite a common overall fold, different CH-domains serve to interface with a wide variety of proteins involved in cytoskeletal dynamics and/or signal transduction. Therefore, the divergence in CH-domain function is thought to result from discrete sequence elements that are exposed on the protein surface. CH-domains have been classified into several families (summarized in Ref. 5Gimona M. Djinovic-Carugo K. Kranewitter W.J. Winder S.J. FEBS Lett. 2002; 513: 98-106Crossref PubMed Scopus (267) Google Scholar). The type-1 and type-2 CH-domains are normally arranged in tandem and are found in many actin-binding proteins, including members of the spectrin, α-actinin, dystrophin, and fimbrin protein families. Single CH-domains are found in several proteins, such as calponin and IQGAP, and are usually classified as type-3 CH-domains. The type-2 CH-domain can also exist as an isolated CH-domain, and it is found in a few proteins, including smoothelins, MICALs, and RP/EBs (6Korenbaum E. Rivero F. J. Cell Sci. 2002; 115: 3543-3545Crossref PubMed Scopus (113) Google Scholar). Calponin and other CH-domain proteins may regulate smooth muscle contractility via the thin filament regulatory system. In a previous report, Borman et al. (7Borman M.A. MacDonald J.A. Haystead T.A. FEBS Lett. 2004; 573: 207-213Crossref PubMed Scopus (31) Google Scholar) identified a novel ∼60 kDa protein that was phosphorylated by cGMP-dependent protein kinase during cGMP-induced Ca2+ densensitization in ileal smooth muscle. This protein was shown to contain a single type-2 CH-domain at its C terminus, which shared sequence similarity with the smoothelin family of smooth muscle-specific proteins (reviewed in Ref. 8van Eys G.J. Niessen P.M. Rensen S.S. Trends Cardiovasc. Med. 2007; 17: 26-30Crossref PubMed Scopus (80) Google Scholar). The 459-residue protein, initially called CHASM (calponin homology-associated smooth muscle), is termed SMTNL1 (smoothelin-like 1). However, unlike the smoothelins, it did not associate with actin filaments in vitro, and hence the specific role of SMTNL1 in smooth muscle relaxation remains undefined. Also, the interaction of smoothelin proteins with actin is mediated by additional N-terminal actin-binding domains such that their CH-domain was neither necessary nor sufficient for actin binding (9Quensel C. Kramer J. Cardoso M.C. Leonhardt H. J. Cell. Biochem. 2002; 85: 403-409Crossref PubMed Scopus (16) Google Scholar). Therefore, the biological role of the C-terminal type-2 CH-domain in the smoothelin protein family remains unclear. Close inspection of their amino acid sequences reveals that the CH-domains of the smoothelins and SMTNL1 contain a putative calmodulin (CaM)-binding IQ-motif sequence (Fig. 1). It has been reported that CaM can bind to many other CH-domain proteins, even though these generally do not posses IQ-motifs. These proteins include calponin (10Takahashi K. Hiwada K. Kokubu T. Biochem. Biophys. Res. Commun. 1986; 141: 20-26Crossref PubMed Scopus (261) Google Scholar, 11Winder S.J. Walsh M.P. Vasulka C. Johnson J.D. Biochemistry. 1993; 32: 13327-13333Crossref PubMed Scopus (49) Google Scholar), spectrin (12Sobel J.S. Goldstein E.G. Venuti J.M. Welsh M.J. Dev. Biol. 1988; 126: 47-56Crossref PubMed Scopus (20) Google Scholar), dystrophin (13Jarrett H.W. Foster J.L. J. Biol. Chem. 1995; 270: 5578-5586Abstract Full Text PDF PubMed Scopus (70) Google Scholar), and filamin A (14Nakamura F. Hartwig J.H. Stossel T.P. Szymanski P.T. J. Biol. Chem. 2005; 280: 32426-32433Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), in which CaM modulates the Ca2+ dependence of actin binding (13Jarrett H.W. Foster J.L. J. Biol. Chem. 1995; 270: 5578-5586Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 14Nakamura F. Hartwig J.H. Stossel T.P. Szymanski P.T. J. Biol. Chem. 2005; 280: 32426-32433Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 15Kolakowski J. Makuch R. Stepkowski D. Dabrowska R. Biochem. J. 1995; 306: 199-204Crossref PubMed Scopus (57) Google Scholar). Some CH-domain proteins, such as IQGAP, also contain IQ-motifs, though these are located outside of the CH-domain region of the protein (16Briggs M.W. Sacks D.B. EMBO Rep. 2003; 4: 571-574Crossref PubMed Scopus (250) Google Scholar). In this study, we provide the first insight into the structure of the C-terminal type-2 CH-domain from SMTNL1 (SMTNL1-CH) and discuss its structural characteristics with respect to previously reported CH-domains. We also demonstrate the binding of apo-CaM to SMTNL1-CH via the IQ-motif sequence and calculate a docking model for the SMTNL1-CH·apo-CaM complex using the HADDOCK protein-protein docking program (17Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2217) Google Scholar). Expression and Purification of SMTNL1-CH—The full-length mouse SMTNL1 cDNA was generated from IMAGE clone 3593616 as described previously (7Borman M.A. MacDonald J.A. Haystead T.A. FEBS Lett. 2004; 573: 207-213Crossref PubMed Scopus (31) Google Scholar). A fragment of SMTNL1 encoding the CH-domain of SMTNL1 (residues 346-459) was then amplified by standard PCR techniques and subcloned into the pGEX-6P1 vector (GE Healthcare) using BamHI/NotI sites. The construct was verified by DNA sequencing. The GST-SMTNL1-CH fusion protein was produced in Escherichia coli, strain BL21(DE3) in LB medium. Uniformly 15N-labeled and15N,13C-labeledGST-SMTNL1-CH proteins were prepared in M9 medium containing 0.5 g/liter 15NH4Cl and 1 g/liter [13C6] glucose (or unlabeled glucose). The fusion proteins were isolated using glutathione-Sepharose 4B resin and cleaved “on-column” by treatment with PreScission Protease (GE Healthcare). The eluted protein contained the cloning artifact “GPLGS” at its N terminus. The SMTNL1-CH construct was concentrated and exchanged into 1 mm sodium phosphate buffer with an Amicon centrifugal filter (Millipore). Expression and Purification of Calmodulin (CaM)—Chicken CaM was expressed from the pET30b(+) vector in E. coli strain BL21(DE3) grown in LB medium as described previously (18Nakashima K. Maekawa H. Yazawa M. Biochemistry. PubMed Scopus Google Scholar). Uniformly 15N-labeled CaM was prepared in M9 medium containing 0.5 g/liter CaM the N-terminal domain of CaM residues and the C-terminal domain of CaM residues were produced as described previously M. T. J. Biochem. PubMed Scopus Google Scholar). NMR NMR contained mm 15N-labeled 1 mm sodium phosphate buffer mm and 0.5 mm acid in SMTNL1-CH was also prepared in The used for residual dipolar also contained mm mm and to the interaction SMTNL1-CH and apo-CaM also contain an additional 1 mm were on a of mm CaM, CaM-nt, with mm and 1 mm mm were into a containing SMTNL1-CH in the contained 1 mm to in SMTNL1-CH. The of protein was determined using their CaM, CaM-ct, CaM-nt, and SMTNL1-CH, were at and the were to a binding model to NMR NMR were at on NMR with with a single of and of SMTNL1-CH were using and a of including and were using TOCSY, and and were also for the T. J.D. J. Am. Chem. Soc. 1993; 115: Scopus Google Scholar). including the and were with a of heteronuclear NOE were on a with a of R. T. J.D. Biochemistry. PubMed Scopus Google Scholar). The were and were using the M. F. J. 1998; PubMed Scopus Google Scholar). The chemical shift perturbation were by the of 15N-labeled SMTNL1-CH and 15N-labeled apo-CaM by unlabeled apo-CaM and unlabeled SMTNL1-CH, The was then as a chemical shift of and using the A.M. J.S. J. S.J. P.T. Biol. PubMed Scopus Google Scholar). in all were using acid to and chemical J. F. E. J.L. J. 1995; PubMed Scopus Google Scholar). were using F. J. J. 1995; PubMed Scopus Google Scholar) and using the J. 4: PubMed Scopus Google Scholar). SMTNL1-CH structure was with J. J. 2003; 4: PubMed Scopus Google Scholar) using from the NOE were with F. J. PubMed Scopus Google Scholar), and were on secondary structure from a chemical shift for the and structural with the of were by J. 2003; PubMed Scopus Google Scholar). for the of the and the were on the structure by using M. J. Am. Chem. Soc. Scopus Google Scholar). Finally, the structures from a total of were and for the on the a docking model for the SMTNL1-CH·apo-CaM complex was with the program in with J.S. J. M. T. Biol. 1998; PubMed Scopus Google Scholar). The HADDOCK program is for the of complex structures on (17Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2217) Google Scholar). to the of the and the C-terminal domain of apo-CaM was in this The for SMTNL1-CH in this and the for the C-terminal domain of apo-CaM (residues J. PubMed Scopus Google Scholar) were used as the The in the structures were by The residues that a for SMTNL1-CH and for the C-terminal domain of apo-CaM were as residues (see The residues residues with a were as The residues in SMTNL1-CH were and The associated residues were and the C-terminal domain of the residues were and and the residues were and The of the residues for proteins are on their structure (see In the first of the an of docking was The complex were then and for a of with The in the were in and using a Finally, the complex were from the with the HADDOCK and used for the were with R. M. K. J. Scopus Google Scholar). of the and all for and were in the (Fig. The were from the TOCSY, TOCSY, and SMTNL1-CH contains a total of the and were also to of the of the total were and used in the structure The structures were using the dynamics and the NOE with the first a total of NOE were identified on the and as as the in NOE were by and this generated The of the was determined as a function of number (Fig. The for the region (residues of the structures with the and was not we during the of the structure with the program The were for of the residues and and with a of used for the of the structure are in The number of was The of the structures and the of the structure of SMTNL1-CH are shown in Fig. a and The for the and the in the region (residues were and The determined with the from the structures of SMTNL1-CH with correlation and of and The structures were with the program M.W. E.G. J.M. PubMed Scopus Google Scholar), and of the residues were found in regions of the the residues were all found in the regions 1). The SMTNL1-CH is a containing and with structure (Fig. The are located from residues and which with the chemical shift from the and (Fig. were also identified for residues and structural for SMTNL1-CH. The number of for the structure of the structures and the NOE are shown as a function of In the and the number of medium P.T. J. Muscle Res. Cell Motil. 2004; 25: 7-19Crossref PubMed Scopus (33) Google Scholar, J. Saraste M. FEBS Lett. 1995; 374: 149-151Crossref PubMed Scopus (124) Google Scholar, T. Kranewitter W. Winder S.J. Gimona M. FEBS Lett. 1998; 431: 134-137Crossref PubMed Scopus (113) Google Scholar), and In the the for the and all the are shown with a and chemical shift for and The secondary structures from the structures are also The with the of the are also found in the by the with a and structural of the structures of of from from from in in additional in in of regions (residues in a the of structures of SMTNL1-CH are for the region (residues The are from to the of the structure is The conserved hydrophobic residues the CH-domains are also and the surface properties of SMTNL1-CH are from different of SMTNL1-CH with of the structure of SMTNL1-CH was with the CH-domain structures of smoothelin structure was determined as of a structural the are in the for this and was (Fig. 1 (Fig. E. F. F. R. J. Biol. PubMed Scopus Google Scholar), spectrin (Fig. Saraste M. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), and (Fig. H. H. J. J. J. J. PubMed Scopus (16) Google Scholar). The were using the regions of SMTNL1-CH and the regions of other CH-domain structures that were by sequence (Fig. 1). The was and with spectrin, and of the with SMTNL1-CH—The binding of apo-CaM with SMTNL1-CH was first characterized by an interaction with SMTNL1-CH in the of of binding was in the of Ca2+ (Fig. We also with the and N-terminal of an interaction with a similar to that with However, did not bind to SMTNL1-CH (Fig. determined from the of SMTNL1-CH the of unlabeled CaM, were as a function of number (Fig. that to residues located on the IQ-motif sequence of SMTNL1-CH to regions that were in close (Fig. the were in the of apo-CaM by unlabeled SMTNL1-CH into the NMR (Fig. with were found in the C-terminal domain of CaM and were located its two Ca2+ binding (Fig. The structure of complex and a of the model for the interaction of SMTNL1-CH with the C-terminal domain of apo-CaM were generated (Fig. The for the complex was In Fig. the that form at the interface of the docking model are also were as to and on SMTNL1-CH that form a to the of and on structure of SMTNL1-CH with the C-terminal domain of of of the complex of SMTNL1-CH with the C-terminal domain of apo-CaM of the The that to the of are also The and basic are in and is shown in We have a structural of the C-terminal type-2 CH-domain of the smoothelin-like 1 protein to in of the biological function of this novel of the smoothelin family of smooth muscle-specific proteins. we that the CH-domain of SMTNL1 a structure that is of that found for the CH-domains of many other proteins (Fig. A number of conserved hydrophobic including the two residues in SMTNL1-CH, to a hydrophobic core that is conserved in the structure of other CH-domains 1 and The NOE in the region (residues is a very protein (Fig. This is similar to the reported NOE for the type-3 CH-domain of calponin J. J.L. D. Winder S.J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). SMTNL1-CH shares sequence identity with the CH-domain of and not the is the SMTNL1-CH structure was on the CH-domain structure of smoothelin (Fig. Despite the relatively of sequence identity and to the CH-domains of spectrin, and the structures were very similar to (Fig. and The in the structure of SMTNL1-CH as with the other CH-domains is the extended C-terminal (Fig. The unique basic at the of a highly basic surface to the SMTNL1-CH 1 and basic surface is by residues and 1 and and is also unique to the CH-domains of the smoothelin family proteins. the of the which are generally located at the N terminus of proteins, of SMTNL1-CH is located at the C terminus of the SMTNL1 protein and is exposed to the The reported solution structure of the type-2 CH-domain H. H. J. J. J. J. PubMed Scopus (16) Google Scholar) also a at its C terminus. However, this CH-domain is located in the of the protein, and the basic is T. T. T. K. C. H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). A from this is the of a IQ-motif on of SMTNL1-CH that is also conserved in the other smoothelin family members (Fig. The IQ-motif, with sequence was first characterized in the of many proteins as tandem Cell Biol. 4: PubMed Scopus Google Scholar) CaM T. J.S. Cell Motil. PubMed Google Scholar, M. FEBS Lett. 2002; 513: PubMed Scopus Google Scholar). The IQ-motif sequence with has also been identified in many proteins such as and proteins M. FEBS Lett. 2002; 513: PubMed Scopus Google Scholar, H.W. PubMed Scopus Google Scholar, 2004; PubMed Google Scholar). We CaM was of binding to SMTNL1-CH, and the by isothermal titration calorimetry was for apo-CaM (Fig. This is in with many previously apo-CaM protein the reported are for J. K. D. D. M. Biochem. 2003; PubMed Scopus Google Scholar) and M.A. J. Res. PubMed Scopus Google Scholar) and for Walsh J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar) and Biochem. Biophys. Res. Commun. 2004; PubMed Scopus Google Scholar), from to for the in P.M. FEBS Lett. 2004; PubMed Scopus (31) Google Scholar). These apo-CaM are distinct from the that a binding that to The of apo-CaM CaM from in the The of CaM at specific in the is to a to an of Ca2+ J. Biol. Chem. Full Text PDF PubMed Google Scholar, R. D. J. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The titration the of a complex with on the NMR not The from was (Fig. and this was by the of the binding of the relatively interaction by the of protein Although are to bind to proteins and containing are also of binding M. FEBS Lett. 2002; 513: PubMed Scopus Google Scholar). binding of with SMTNL1-CH (Fig. with the of these calorimetry in titration with we a few that to the N terminus of SMTNL1-CH that in the on the NMR which by in the NMR not We therefore that apo-CaM to SMTNL1-CH. This that of apo-CaM binding to and in which poorly with these proteins M. FEBS Lett. 2002; 513: PubMed Scopus Google Scholar, H.W. PubMed Scopus Google Scholar, Biochem. Biophys. Res. Commun. 2004; PubMed Scopus Google Scholar). in the of we on the novel interaction identified apo-CaM and SMTNL1-CH. The and the that the interface of apo-CaM binding was located the IQ-motif sequence of SMTNL1-CH (Fig. of the structure of with the protein, with that CaM to and using its C-terminal the N-terminal domain in solution M. R. Sci. 2005; PubMed Scopus Google Scholar). In SMTNL1-CH, the a similar binding to that with CaM, the did not binding (Fig. However, the of generated by apo-CaM binding to SMTNL1-CH was almost that by (Fig. the other the in the of apo-CaM that were by binding to SMTNL1-CH to the C-terminal domain of apo-CaM (Fig. and which is with the and are in the N-terminal These that apo-CaM to SMTNL1-CH through its C-terminal the N-terminal domain the of the in is relatively is of the structures of proteins that is by the on these we can therefore construct a docking model for the SMTNL1-CH complex with the C-terminal domain of apo-CaM using the HADDOCK The interaction to several residues and of the Ca2+ binding of the C-terminal domain of apo-CaM form to basic residues and of SMTNL1-CH. In this the and that are located on the C-terminal region of SMTNL1-CH structure were not However, their relatively as as their close to the CaM protein, and/or are also to form additional interactions to The structure of apo-CaM with from that the C-terminal domain of CaM a to the first of the IQ-motif through a hydrophobic interaction E. C. Sci. PubMed Scopus Google Scholar). In this the at the first and a hydrophobic at the of the IQ-motif sequence hydrophobic to the hydrophobic of the C-terminal domain of apo-CaM (Fig. 1). However, in SMTNL1-CH, the first is in the protein structure and is therefore not to such a hydrophobic In addition, the hydrophobic at of the IQ-motif is with in SMTNL1-CH (Fig. 1). of the C-terminal domain of apo-CaM that form to SMTNL1-CH, including residues and also serve as the Ca2+ binding that this interaction CaM Ca2+ This is in with and NMR in which apo-CaM of binding to SMTNL1-CH Fig. The of SMTNL1 can relaxation of ileal smooth muscle (7Borman M.A. MacDonald J.A. Haystead T.A. FEBS Lett. 2004; 573: 207-213Crossref PubMed Scopus (31) Google Scholar), and of muscle using SMTNL1 that the of SMTNL1-CH is for the of the the other the SMTNL1 can muscle relaxation is not understood. Although at this the role of the interaction has not been we that the of apo-CaM with SMTNL1 has are several a interaction the contractility of smooth muscle. The of CaM with the CH-domain of SMTNL1 for the of with other proteins of SMTNL1. CaM binding the of SMTNL1 may CaM binding of Ca2+ The cGMP-dependent protein kinase (7Borman M.A. MacDonald J.A. Haystead T.A. FEBS Lett. 2004; 573: 207-213Crossref PubMed Scopus (31) Google Scholar) is located outside of the CH-domain in close to the CaM binding The C-terminal basic of SMTNL1 that also in smoothelin with other and of the with the CaM binding CaM with SMTNL1 with common residues on CaM for Ca2+ binding J.L. Walsh M.P. Biochem. J. 2007; PubMed Scopus Google Scholar), it the Ca2+ and/or of CaM, as has also been in the of J.A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Finally, as we binding of apo-CaM to proteins, by of has been described as a for of CaM J. Biol. Chem. Full Text PDF PubMed Google Scholar). the for in CaM binding is of the of the CH-domain of SMTNL1-CH also a we have described in this the C-terminal type-2 CH-domain has distinct structural properties from the other of CH-domains. Therefore, of additional proteins that with the CH-domains of smoothelin and SMTNL1 in smooth muscle is necessary for a of the function of the C-terminal type-2

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