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Solution Structure of the Calponin CH Domain and Fitting to the 3D-Helical Reconstruction of F-Actin:Calponin

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Solution Structure of the Calponin CH Domain and Fitting to the 3D-Helical Reconstruction of F-Actin:Calponin View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Structure, Vol. 10, 249–258, February, 2002, 2002 Elsevier Science Ltd. All rights reserved. PII S0969-2126(02)00703-7 Solution Structure of the Calponin CH Domain and Fitting to the 3D-Helical Reconstruction of F-Actin:Calponin Janice Bramham,1,6 Julie L. Hodgkinson,2 tropomyosin, caldesmon, and calponin. Basic calponin, Brian O. Smith,3 Dusan Uhrı´n,4 Paul N. Barlow,3,4 one of three genetic variants found almost exclusively in and Steven J. Winder5 smooth muscle, was first isolated from chicken gizzard 1 Department of Biochemistry smooth muscle as a 34 kDa protein that binds to filamen- University of Leicester tous actin (F-actin) [1]. It has been proposed that cal- Adrian Building ponin has a role both in muscle contraction and as a University Road structural component in the cytoskeleton of smooth Leicester LE1 7RH muscle cells. Calponin is associated with the contractile United Kingdom apparatus, along with actin, myosin, and caldesmon. It 2 Imperial College School of Science also occurs in the membrane skeleton that forms the Technology and Medicine interface between the contractile apparatus and the ex- National Heart and Lung Institute tracellular matrix, and it appears in the cytoskeleton, Dovehouse Street which surrounds and supports the contractile appara- London SW3 6LY tus, along with ␤-actin, filamin, and desmin [2]. United Kingdom In vitro studies have shown that calponin is an actin 3 Institute of Cell and Molecular Biology binding protein and a major regulator of muscle contrac- University of Edinburgh tion. The regulatory function arises from the ability of Mayfield Road calponin to modulate the interaction between myosin Edinburgh EH9 3JR and actin filaments by inhibition of the actomyosin Scotland ATPase [3]. In addition to its ability to bind to F-actin, 4 Department of Chemistry calponin is also reported to interact with myosin itself [4] Joseph Black Building and with a number of other cytoskeletal components, in- University of Edinburgh cluding tropomyosin [1], desmin intermediate filaments West Mains Road [5], tubulin [6], and phospholipids [7]. The reported inter- Edinburgh EH9 3JJ actions between calponin and several signal-transduc- Scotland ing proteins, such as the extracellular-regulated kinase 5 Institute of Biomedical and Life Sciences (ERK) and protein kinase C-⑀ (PKC⑀) [8,9], suggest fur- Division of Biochemistry and Molecular Biology ther potential functions for calponin as a signaling mole- Davidson Building cule or as an adaptor for the localization of these kinases. University of Glasgow The calponin molecule consists of four recognized Glasgow G12 8QQ domains: one at the N terminus, the calponin homology Scotland (CH) domain, and three repeating domains comprising the C terminus, the calponin-like repeats (CLR). The CH Summary domain has approximately 100 residues and was first identified in calponin from which its name is derived Calponin is involved in the regulation of contractility and [10]. Tandem pairs of CH domains are found in a number ␣ organization of the actin cytoskeleton in smooth muscle of actin binding proteins such as -actinin, dystrophin, cells. It is the archetypal member of the calponin homol- filamin, fimbrin and spectrin (reviewed by Stradal et al., ogy (CH) domain family of actin binding proteins that 1998 [11]). In these proteins, a role for the CH domain in includes cytoskeletal linkers such as ␣-actinin, spectrin, actin binding has been demonstrated: two CH domains and dystrophin, and regulatory proteins including VAV, positioned in tandem within a molecule are required to IQGAP, and calponin. We have determined the first form a high-affinity actin binding domain (ABD), thus structure of a CH domain from a single CH domain- potentially providing multiple sites for interaction with containing protein, that of calponin, and have fitted actin. The single CH domain of calponin is not part of the NMR-derived coordinates to the 3D-helical recon- an ABD and its precise role in actin binding has yet to struction of the F-actin:calponin complex using cryo- be determined [12]. Similarly, single copies of the CH electron microscopy. The tertiary fold of this single domain are found in other cytoskeletal and signaling CH domain is typical of, yet significantly different from, proteins where its function is unclear, including the Sac- those of the CH domains that occur in tandem pairs charomyces cerevisiae homolog Scp1, the smooth mus- to form high-affinity ABDs in other proteins. We thus cle protein SM22, the proto-oncogene Vav, and IQGAP provide a structural insight into the mode of interaction (reviewed by Stradal et al., 1998 [11]). between F-actin and CH domain-containing proteins. A number of in vitro studies have defined regions within the calponin molecule involved in its interactions Introduction with other proteins. One binding site for actin has been located between the CH domain and the first CLR in the The thin filaments of smooth muscle cells comprise actin C-terminal portion, in the region that is also required for filaments complexed with three other major proteins: Key words: actin binding, calponin-homology domain, NMR, struc- 6 Correspondence: [email protected] ture, calponin Structure 250 Figure 1. Analysis of the NOE and CSI Data for the Calponin CH Domain (A) Number of unambiguous NOE-derived distance restraints used in the structure calculations. Black, gray, and white bars correspond to sequential (i → i ϩ 1), medium range (i → i ϩ (2, 3, or 4)), and long range (i → i ϩ (Ͼ4)) NOEs, respectively. (B) NOEs between sequential backbone amide protons (Hn) and between Hn and H␣ (the heights of the bars are proportional to the NOE intensity). (C) Consensus CSI derived from the chemical shifts of C␣,C␤, CO, and H␣. (D) Cartoon representation of the secondary structure elements of the calponin CH domain. inhibition of the actomyosin ATPase (the so-called actin tion structure of any CH domain. The structure is com- binding site 1, ABS1) [13]. A second actin binding site pared to those of the crystal structures of the actin (ABS2) lies within the region spanning the three C-ter- binding CH domains and the implications for the ob- minal CLRs [14, 15]. Two binding sites for Ca2ϩ binding served differential actin binding properties are dis- proteins, such as calmodulin and S100, have been iden- cussed. The structure has also been fitted to the cryo- tified: one within an N-terminal region encompassing EM reconstruction of F-actin decorated with calponin; residues 7–45 and the second in the region between a comparison with the F-actin:CH domain reconstruc- residues 52 and 144; residues 142–227 contain a tropo- tions of other CH domains is discussed. myosin binding site [16]. Additionally, phosphorylation sites have been identified on all three CLRs [17]. The Results and Discussion relative significance of these reported in vitro interac- tions of calponin in vivo and of its distribution in smooth Structure Determination muscle has yet to be ascertained. Calponin may function A 1 mM sample of 15N,13C-labeled CH domain (residues separately both as a regulator of smooth muscle con- 27–134) yielded high-quality NMR spectra such that, traction and as a structural protein in smooth muscle. using a combination of complementary triple-resonance The distribution of the sites of interaction throughout experiments, nearly all of the 1H, 15N, and 13C nuclei in this modular molecule implies that the individual do- the protein could be unambiguously assigned [22]. The 1 13 13 mains may have separate functions in vivo. chemical shifts of all the assigned H␣, C␣, C␤, and The crystal structures of several CH domains from 13CO provided a consensus Chemical Shift Index (CSI) ABDs comprising tandem pairs of CH domains have that is consistent with a predominantly helical structure been reported [18–21]. In this report we describe the (Figure 1C). Five ␣ helices were predicted with no evi- three-dimensional solution structure of the archetypal dence of ␤ strands, concordant with the previously re- CH domain from chicken gizzard calponin, determined ported crystal structures of CH domains. Initial struc- by multinuclear NMR spectroscopy. This is the first tures were calculated using 3347 distance restraints structural investigation of a CH domain from a protein derived from the integrated crosspeaks in the 13C-edited containing only a single CH domain and is the first solu- NOESY-HSQC spectrum and the 15N-resolved cross- Solution Structure of the Calponin CH Domain 251 Table 1. Structural Statistics of the Final Ensemble of Twenty Refined Structures of the CH Domain of Chicken Calponin Ensemble Closest to Mean Number of NOE restraints used: Intraresidue (i → i) 1034 1034 Sequential (i → i ϩ 1) 320 320 Medium range (i → i ϩ (2 to 4)) 234 234 Long range (i → i ϩ (Ͼ4)) 315 315 Ambiguous 672 672 Total 2575 2575 Average NOE restraint violation (A˚ ) 0.031 Ϯ 0.007a 0.033 Lennard-Jones energy (kJmolϪ1)b Ϫ207 Ϯ 49 Ϫ222 Coordinate rmsd (A˚ ): All residues* (backbone heavy atoms) 0.580 0.493 (all heavy atoms) 0.990 0.873 Residues in helices† (backbone heavy atoms) 0.448 0.381 (all heavy atoms) 0.828 0.708 Parameter rmsd from idealized geometry: Bond lengths (A˚ ) 0.0018 Ϯ 6 ϫ 10Ϫ4c 0.0019 Angles (Њ) 0.331 Ϯ 0.007c 0.329 Improper dihedrals (Њ) 0.233 Ϯ 0.076c 0.232 Ramachandran assessment (%)d: Most favored region 84.5 87.2 Additionally allowed region 9.1 6.4 Generously allowed region 5.1 6.4 Disallowed region 1.3 0.0 a The sum of the NOE violations divided by the total number of restraints, averaged over the ensemble.
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