The role of the calponin homology domain of smoothelin-like 1 (SMTNL1) in myosin phosphatase inhibition and smooth muscle contraction

Arginine-specific ADP-ribosyltransferase (Art) catalyzes the mono-ADP-ribosylation, in which it transfers a single ADP-ribose moiety of NAD to the arginine residue(s) of target proteins, and may regulate the function of the proteins or peptides in cellular processes. In vertebrates, Art family is consisted of seven members (Arts1-7), and these Arts are distributed among various tissues except B lymphocytes. Previously, we described molecular cloning, characterization and distribution of glycosylphosphatidylinositol (GPI)-anchored Arts, Art7.1 and Art7.2 (formerly, we referred as cgArt1 and cgArt2, respectively) in chicken tissues (Terashima et al (2005) Biochem J 389:853-861). Here, we demonstrate for the first time that Art7.1 was predominantly expressed on the surface of B cells from the bursa of Fabricius as a GPI-anchored form, as well as on T cells from the thymocytes. Furthermore, we show that the expression of Art7.1 molecules on B cells could modulate the B cell receptor (BCR) signalling and direct the B cell fate to maturation. Thus, our present observation sheds light on the Art molecule expressed on B cells and its possible functional role in BCR signalling.     

Intrinsically disordered N-terminus of calponin homology-associated smooth muscle protein (CHASM) interacts with the calponin homology domain to enable tropomyosin binding

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.     

Solution structure of the calponin CH domain and fitting to the 3D-helical reconstruction of F-actin:calponin

Calponin is involved in the regulation of contractility and organization of the actin cytoskeleton in smooth muscle cells. It is the archetypal member of the calponin homology (CH) domain family of actin binding proteins that includes cytoskeletal linkers such as alpha-actinin, spectrin, and dystrophin, and regulatory proteins including VAV, IQGAP, and calponin. We have determined the first structure of a CH domain from a single CH domain-containing protein, that of calponin, and have fitted the NMR-derived coordinates to the 3D-helical reconstruction of the F-actin:calponin complex using cryo-electron microscopy. The tertiary fold of this single CH domain is typical of, yet significantly different from, those of the CH domains that occur in tandem pairs to form high-affinity ABDs in other proteins. We thus provide a structural insight into the mode of interaction between F-actin and CH domain-containing proteins.     

Solution structure of a C-terminal coiled-coil domain from bovine IF(1): the inhibitor protein of F(1) ATPase

Bovine IF(1) is a basic, 84 amino acid residue protein that inhibits the hydrolytic action of the F(1)F(0) ATP synthase in mitochondria under anaerobic conditions. Its oligomerization state is dependent on pH. At a pH value below 6.5 it forms an active dimer. At higher pH values, two dimers associate to form an inactive tetramer. Here, we present the solution structure of a C-terminal fragment of IF(1) (44-84) containing all five of the histidine residues present in the sequence. Most unusually, the molecule forms an anti-parallel coiled-coil in which three of the five histidine residues occupy key positions at the dimer interface.     

Formin homology domain protein (FHOD1) is a cyclic GMP-dependent protein kinase I-binding protein and substrate in vascular smooth muscle cells

Cyclic GMP-dependent protein kinase I (PKGI) mediates vascular relaxation by nitric oxide and related nitrovasodilators and inhibits vascular smooth muscle cell (VSMC) migration. To identify VSMC proteins that interact with PKGI, the N-terminal protein interaction domain of PKGIalpha was used to screen a yeast two-hybrid human aortic cDNA library. The formin homology (FH) domain-containing protein, FHOD1, was found to interact with PKGIalpha in this screen. FH domain-containing proteins bind Rho-family GTPases and regulate actin cytoskeletal dynamics, cell migration, and gene expression. Antisera to FHOD1 were raised and used to characterize FHOD1 expression and distribution in vascular cells. FHOD1 is highly expressed in human coronary artery, aortic smooth muscle cells, and in human arterial and venous endothelial cells. In glutathione S-transferase pull-down experiments, the FHOD1 C terminus (amino acids 964-1165) binds full-length PKGI. Both in vitro and intact cell studies demonstrate that the interaction between FHOD1 and PKGI is decreased 3- to 5-fold in the presence of the PKG activator, 8Br-cGMP. Immunofluorescence studies of human VSMC show that FHOD1 is cytoplasmic and is concentrated in the perinuclear region. PKGI also directly phosphorylates FHOD1, and studies with wild-type and mutant FHOD1-derived peptides identify Ser-1131 in the FHOD1 C terminus as the unique PKGI phosphorylation site in FHOD1. These studies demonstrate that FHOD1 is a PKGI-interacting protein and substrate in VSMCs and show that cyclic GMP negatively regulates the FHOD1-PKGI interaction. Based on the known functions of FHOD1, the data are consistent with a role for FHOD1 in cyclic GMP-dependent inhibition of VSMC stress fiber formation and/or migration.     

Solution structure of the C-terminal domain from poly(A)-binding protein in Trypanosoma cruzi: a vegetal PABC domain

PABC is a phylogenetically conserved peptide-binding domain primarily found within the C terminus of poly(A)-binding proteins (PABPs). This domain recruits a series of translation factors including poly(A)-interacting proteins (Paip1 and Paip2) and release factor 3 (RF3/GSPT) to the initiation complex on mRNA. Here, we determine the solution structure of the Trypanosoma cruzi PABC domain (TcPABC), a representative of the vegetal class of PABP proteins. TcPABC is similar to human PABC (hPABC) and consists of five alpha-helices, in contrast to the four helices observed in PABC domains from yeast (yPABC) and hyper plastic disk proteins (hHYD). A mobile N-terminal helix is observed in TcPABC that does not pack against the core of the protein, as found in hPABC. Characteristic to all PABC domains, the last four helices of TcPABC fold into a right-handed super coil. TcPABC demonstrates high-affinity binding to PABP interacting motif-2 (PAM-2) and reveals a peptide-binding surface homologous to that of hPABC. Our results demonstrate the last four helices in TcPABC are sufficient for peptide recognition and we predict a similar binding mode in PABC domains. Furthermore, these results point to the presence of putative PAM-2 site-containing proteins in trypanosomes.     

Solution structure of the Eps15 homology domain of a human POB1 (partner of RalBP1)

The solution structure of the Eps15 homology (EH) domain of a human POB1 (partner of RalBP1) has been determined by uniform ¹³C/¹⁵N labeling and heteronuclear multidimensional nuclear magnetic resonance spectroscopy. The POB1 EH domain consists of two EF-hand structures, and the second one binds a calcium ion. In the calcium-bound state, the orientation of the fourth α-helix relative to the other helices of the POB1 EH domain is slightly different from that of calbindin, and much more different from those of calmodulin and troponin C, on the basis of their atomic coordinates.     

Solution structure of the RecQ C-terminal domain of human Bloom syndrome protein

RecQ C-terminal (RQC) domain is known as the main DNA binding module of RecQ helicases such as Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) that recognizes various DNA structures. Even though BLM is able to resolve various DNA structures similarly to WRN, BLM has different binding preferences for DNA substrates from WRN. In this study, we determined the solution structure of the RQC domain of human BLM. The structure shares the common winged-helix motif with other RQC domains. However, half of the N-terminal has unstructured regions (α1–α2 loop and α3 region), and the aromatic side chain on the top of the β-hairpin, which is important for DNA duplex strand separation in other RQC domains, is substituted with a negatively charged residue (D1165) followed by the polar residue (Q1166). The structurally distinctive features of the RQC domain of human BLM suggest that the DNA binding modes of the BLM RQC domain may be different from those of other RQC domains.     

Functional role of the C-terminal domain of smooth muscle myosin light chain kinase on the phosphorylation of smooth muscle myosin

Smooth muscle myosin light chain kinase (MLCK) is known to bind to thin filaments and myosin filaments. Telokin, an independently expressed protein with an identical amino acid sequence to that of the C-terminal domain of MLCK, has been shown to bind to unphosphorylated smooth muscle myosin. Thus, the functional significance of the C-terminal domain and the molecular morphology of MLCK were examined in detail. The C-terminal domain was removed from MLCK by alpha-chymotryptic digestion, and the activity of the digested MLCK was measured using myosin or the isolated 20-kDa light chain (LC20) as a substrate. The results showed that the digestion increased K(m) for myosin 3-fold whereas it did not change the value for LC20. In addition, telokin inhibited the phosphorylation of myosin by MLCK by increasing K(m) but only slightly increased K(m) for LC20. Electron microscopy indicated that MLCK was an elongated molecule but was flexible so as to form folded conformations. MLCK was crosslinked to unphosphorylated heavy meromyosin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the absence of Ca(2+)/calmodulin (CaM), and electron microscopic observation of the products revealed that the MLCK molecule bound to the head-tail junction of heavy meromyosin. These results suggest that MLCK binds to the head-tail junction of unphosphorylated myosin through its C-terminal domain, where LC20 can be promptly phosphorylated through its catalytic domain following the Ca(2+)/CaM-dependent activation.     

Solution structure of the epsin N-terminal homology (ENTH) domain of human epsin

Epsin is a protein that binds to the Eps15 homology (EH) domains, and is involved in clathrin-mediated endocytosis. The epsin N-terminal homology (ENTH) domain (about 140 amino acid residues) is well conserved in eukaryotes and is considered to be important for actin cytoskeleton organization in endocytosis. In this study, we have determined the solution structure of the ENTH domain (residues 1–144) of human epsin by multidimensional nuclear magnetic resonance spectroscopy. In the ENTH-domain structure, seven -helices form a superhelical fold, consisting of two antiparallel two-helix HEAT motifs and one three-helix ARM motif, with a continuous hydrophobic core in the center. We conclude that the seven-helix superhelical fold defines the ENTH domain, and that the previously-reported eight-helix fold of a longer fragment of rat epsin 1 is divided into the authentic ENTH domain and a C-terminal flanking -helix.     

NMR-based homology model for the solution structure of the C-terminal globular domain of EMILIN1

EMILIN1 is a glycoprotein of elastic tissues that has been recently linked to the pathogenesis of hypertension. The protein is formed by different independently folded structural domains whose role has been partially elucidated. In this paper the solution structure, inferred from NMR-based homology modelling of the C-terminal trimeric globular C1q domain (gC1q) of EMILIN1, is reported. The high molecular weight and the homotrimeric structure of the protein required the combined use of highly deuterated ¹⁵N, ¹³C-labelled samples and TROSY experiments. Starting from a homology model, the protein structure was refined using heteronuclear residual dipolar couplings, chemical shift patterns, NOEs and H-exchange data. Analysis of the gC1q domain structure of EMILIN1 shows that each protomer of the trimer adopts a nine-stranded β sandwich folding topology which is related to the conformation observed for other proteins of the family. Distinguishing features, however, include a missing edge-strand and an unstructured 19-residue loop. Although the current data do not allow this loop to be precisely defined, the available evidence is consistent with a flexible segment that protrudes from each subunit of the globular trimeric assembly and plays a key role in inter-molecular interactions between the EMILIN1 gC1q homotrimer and its integrin receptor α4β1. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Crystal structure of the C-terminal domain of a flagellar hook-capping protein from Xanthomonas campestris

The crystal structure of the C-terminal domain of a hook-capping protein FlgD from the plant pathogen Xanthomonas campestris (Xc) has been determined to a resolution of ca 2.5 Å using X-ray crystallography. The monomer of whole FlgD comprises 221 amino acids with a molecular mass of 22.7 kDa, but the flexible N-terminus is cleaved for up to 75 residues during crystallization. The final structure of the C-terminal domain reveals a novel hybrid comprising a tudor-like domain interdigitated with a fibronectin type III domain. The C-terminal domain of XcFlgD forms three types of dimers in the crystal. In agreement with this, analytical ultracentrifugation and gel filtration experiments reveal that they form a stable dimer in solution. From these results, we propose that the Xc flagellar hook cap protein FlgD comprises two individual domains, a flexible N-terminal domain that cannot be detected in the current study and a stable C-terminal domain that forms a stable dimer.     

Solution structure of the 162 residue C-terminal domain of human elongation factor 1Bgamma

The multisubunit elongation factor 1 (eEF1) is required for the elongation step of eukaryotic protein synthesis. The eEF1 complex consists of four subunits: eEF1A, a G-protein that shuttles aminoacylated tRNAs to the ribosome; eEF1Balpha and eEF1Bbeta, two guanine nucleotide exchange factors, and eEF1Bgamma. Although its exact function remains unknown, this latter subunit is present in all eukaryotes. Recombinant human eEF1Bgamma has been purified and shown to consist of two independent domains. We have utilized high resolution NMR to determine the three-dimensional structure of the 19 kDa C-terminal fragment (domain 2). The structure consists of a five-stranded anti-parallel beta-sheet surrounded by alpha-helices and resembles a contact lens. Highly conserved residues are mainly located on the concave face, suggesting thereby that this side of the molecule might be involved in some biologically relevant interface(s). Although the isolated domain 2 appears to be mostly monomeric in solution, biochemical and structural data indicate a potential homodimer. The proposed dimer model can be further positioned within the quaternary arrangement of the whole eEF1 assembly.