Intrinsically Disordered N-Terminus of Calponin Homology-Associated Smooth Muscle Protein (CHASM) Interacts with the Calponin Homology Domain to Enable Tropomyosin Binding

Abstract

The calponin homology-associated smooth muscle (CHASM) protein plays an important adaptive role in smooth and skeletal muscle contraction. CHASM is associated with increased muscle contractility and can be localized to the contractile thin filament via its binding interaction with tropomyosin. We sought to define the structural basis for the interaction of CHASM with smooth muscle tropomyosin as a first step to understanding the contribution of CHASM to the contractile capacity of smooth muscle. Herein, we provide a structure-based model for the tropomyosin-binding domain of CHASM using a combination of hydrogen/deuterium exchange mass spectrometry (HDX-MS) and NMR analyses. Our studies provide evidence that a portion of the N-terminal intrinsically disordered region forms intramolecular contacts with the globular C-terminal calponin homology (CH) domain. Ultimately, cooperativeness between these structurally dissimilar regions is required for CHASM binding to smooth muscle tropomyosin. Furthermore, it appears that the type-2 CH domain of CHASM is required for tropomyosin binding and presents a novel function for this protein domain.

The C Terminus of the Catalytic Domain of Type A Botulinum Neurotoxin May Facilitate Product Release from the Active Site

“…Recent years have witnessed a tremendous proliferation of three-dimensional structure determinations by the advent of high throughput x-ray crystallography soon after the sequence and adequate expression of a protein became available (1,2). In some proteins, no electron density can be observed for stretches of the amino acid sequence especially at the N or C terminus (3)(4)(5)(6)(7)(8). Thus, a functional role for such regions is not always discernible from their three-dimensional structures.…”

Binding of smoothelin-like 1 to tropomyosin and calmodulin is mutually exclusive and regulated by phosphorylation

“…A clone comprising the Tpm-binding region of SMTNL1 (SMTNL1-TMB, amino acids 195–459; NP_077192) was described previously [18, 19]. SMTNL1-TMB was expressed as a glutathione S -transferase (GST)-fusion protein in E. coli and purified with glutathione-Sepharose.…”

“…In smooth muscle, two Tpm isoforms (Tpm1.4 and Tpm2.1; previously identified as Tmsm-α/Tm6 and Tmsm-β/Tm1β, respectively [16]) are predominantly expressed [17]. SMTNL1 was shown to associate with smooth muscle Tpm α/β dimers with a binding surface comprising of its N-terminal intrinsically-disordered region and the C-terminal calponin homology domain [18]. …”

“…Previous in vitro studies have defined SMTNL1 as a Tpm- and CaM-binding protein [10–12, 18, 19]. Moreover, the phosphorylation of SMTNL1 was identified during calcium desensitization of smooth muscle [1, 5].…”

“…Intriguingly, the binding domains for CaM (CBD1 and CBD2) are localized in close vicinity to the Tpm-binding surface on the SMTNL1 protein. In addition, the Ser301 phosphorylation site targeted by cyclic nucleotide-dependent protein kinases (i.e., PKA and PKG) is located near CBD1 and within the Tpm-binding region [11, 18]. This led us to inquire about the interplay of SMTNL1 phosphorylation and calcium levels on the multi-functionality of Tpm- and CaM-binding.…”