The Crystal Structure of the Actin Binding Domain from α-Actinin in its Closed Conformation: Structural Insight into Phospholipid Regulation of α-Actinin

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α-Actinin is the major F-actin crosslinking protein in both muscle and non-muscle cells. We report the crystal structure of the actin binding domain of human muscle α-actinin-3, which is formed by two consecutive calponin homology domains arranged in a “closed” conformation. Structural studies and available biochemical data on actin binding domains suggest that two calponin homology domains come in a closed conformation in the native apo-form, and that conformational changes involving the relative orientation of the two calponin homology domains are required for efficient binding to actin filaments. The actin binding activity of muscle isoforms is supposed to be regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which binds to the second calponin homology domain. On the basis of structural analysis we propose a distinct binding site for PtdIns(4,5)P2, where the fatty acid moiety would be oriented in a direction that allows it to interact with the linker sequence between the actin binding domain and the first spectrin-like repeat, regulating thereby the binding of the C-terminal calmodulin-like domain to this linker.

Introduction

The cytoskeleton consists of a number of filamentous systems composed of polymers of actin, tubulin and intermediate filament proteins. These proteins form the filaments of actin stress fibers, microtubules and intermediate filaments. Their filamentous state provides the cell with networks of structures that are highly dynamic as well as highly stable. These networks give the cell internal scaffolds for maintaining and modelling cell shape and provide routes for inter- and intracellular traffic and signalling. An important family of cytoskeleton proteins are those that crosslink or bundle actin filaments (α-actinin, filamin, and fimbrin) or link the actin filaments to the cell membrane (β-spectrin, dystrophin and utrophin) or other filamentous systems (plectin). The common functional domain of these proteins is the actin binding domain (ABD).

α-Actinin is the major F-actin crosslinking protein in both muscle and non-muscle cells.1 α-Actinin is a functional antiparallel homodimer and is composed of an N-terminal ABD, followed by a rod domain consisting of four spectrin-like (SR) repeats and a C-terminal calmodulin-like (CaM) domain. The actin binding domain (ABD) consists of two consecutive calponin homology (CH) domains.2 Several α-actinin isoforms exist,3 which are regulated differently: actin binding in the non-muscle isoforms is Ca2+-sensitive, whereas in muscle α-actinin it is not.4 The CaM domain consists of two EF-hand motifs. The CaM domain of the non-muscular isoforms can bind Ca2+ and thereby regulate the actin binding activity. The CaM domain of the muscle isoforms does not bind Ca2+ and their activity is Ca2+-insensitive. The actin binding activity of muscle isoforms might instead be regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and the binding site was mapped to a region of the second CH domain in ABD.5 This region is also involved in the regulated interaction of the CaM domain with titin.6

Apart from its interaction with actin, α-actinin has emerged as a major multivalent platform mediating interactions with many cytoskeletal or regulatory proteins.7, 8 At the membrane, in focal adhesions and cell–cell contacts, α-actinin interacts with capZ, vinculin, zyxin and cell surface receptors such as the NMDA or integrin receptors. Along the actin cytoskeleton, α-actinin interacts with signalling molecules such as the protein kinases MEKK1 and PKN, zinc-finger proteins like ALP, and PDZ domain proteins like ZASP/cypher.7, 9 In muscle, in addition to some of these proteins, the interactions with sarcomeric proteins are functionally possibly most important: α-actinin binds to the giant actin filament ruler titin and to the novel Z-disk proteins myotilin and ZASP.9, 10

Striated muscle α-actinin, predominantly isoforms 2,11 interacts with two classes of binding sites for the Z-disk portion of titin;12 the highly homologous skeletal muscle-specific α-actinin-3 shows similar binding (Figure 1(a)). The titin Z-repeats provide multiple binding sites for the C-terminal CaM domain of α-actinin,6 whose differential splicing appears to determine Z-disk thickness by determining the number of cross-links in this structure.13, 14 At the Z-disk periphery, a unique site interacts with the two central spectrin-like repeats of the α-actinin rod.12 Controlled activation of protein–protein interactions is a major mechanism for the sequential integration of protein components into cytoskeletal structures also in muscle, and muscle α-actinin is accordingly regulated in a specific way. The interaction of muscle α-actinin with titin is regulated intrasterically by a phospholipid-dependent mechanism that controls its interaction with the titin,6 and requires the presence of both CH domains for full regulation. However, no structural information on α-actinin ABDs is available so far that would allow us to analyse this regulation mechanism in atomic detail.

Three-dimensional structures of actin binding domains have been reported for the following actin binding proteins: plectin,15, 16, 17 human fimbrin,18 and fimbrins from Arabidopsis thaliana and Schizosaccharomyces pombe.19 The structures of plectin and fimbrin ABD revealed two CH domains to be in intimate contact, and are therefore referred to as “closed” conformations. Interestingly, structural flexibility within ABD domains from two crystallographically independent fimbrin core molecules was observed: the CH1 domain undergoes a rotation of about 50° in these two molecules.19 ABDs of utrophin and dystrophin assemble into antiparallel dimers, with the N-terminal CH1 of one monomer in close association with the C-terminal CH2 of the other. Each ABD monomer adopts an “open” conformation comprising the two CH domains connected by an α-helix.

In order to gain further insight into the molecular details of filamentous actin cross-linking structures in the muscle Z-disk, and the structural basis of phospholipid regulation via the actin binding domain, we determined the molecular structure of the actin binding domain of human skeletal muscle α-actinin (isoform 3) in two crystal forms.

Section snippets

Titin binding

Similar to α-actinin-2, the highly homologous α-actinin-3 was also found to show phospholipid-regulated binding to the titin Z-repeats. Binding could be stimulated by negatively charged phospholipids like phosphatidyl-inositol 4,5-bisphosphate (PtdIns(4,5)P2) (Figure 1(a)) as well as by lysophosphatidic acid (not shown). No activation was observed by the phospholipid head groups alone, in agreement with earlier observations for α-actinin-2.6

This interaction is in agreement with the high level

Conclusions

The cytoskeleton consists of a number of filamentous systems composed of polymers of actin, tubulin and intermediate filament proteins. An important family of cytoskeletal proteins crosslink or bundle actin filaments. The major F-actin crosslinking protein in both muscle and non-muscle cells is α-actinin. The actin binding domain of human muscle α-actinin-3 is formed by two consecutive calponin homology domains that are arranged in the closed conformation in the native apo-form.

The conservation

Protein preparation, crystallisation and titin binding

The actin binding domain of α-actinin-3 (residues 26–273, Swissprot entry Q08043) was cloned into a modified pET vector with an N-terminal His6-tag and with a TEV-protease cleavage site. Protein expression was carried out in BL21[DE3] cells at 30° C for six hours after induction at A600 0.5 with 0.1 mM IPTG and gave a yield of ca 15 mg/l of culture. After affinity purification on a Ni-NTA matrix (Qiagen), the protein was cleaved with TEV protease at a ratio of 1:100 (w/w) overnight, and purified

Accession numbers

The coordinates and structure factors have been deposited in the Brookhaven PDB as entries 1TJT and 1WKU for crystal forms L and H, respectively.

Acknowledgements

B.S. is a recipient of a post-doctoral fellowship funded by Research Training Network CYTONET (contract no. HPRN-CT-2000-00096), which also supported the work of M.G., who is a recipient of an MRC International Appointment Initiative Award. Oliviero Carugo (University of Pavia, Italy) is gratefully acknowledged for help with structural analyses of CH domains. The excellent technical assistance of Nathalie Bleimling is gratefully acknowledged.

Competing interest statement. The authors declare

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    Present addresses: B. Sjöblom & K. Djinović Carugo, Max F. Perutz Laboratories, University Departments at Vienna Biocenter, Department for Biomolecular Structural Chemistry, University of Vienna, Campus Vienna Biocenter 6/1, Rennweg 95b, A-1030 Vienna, Austria.

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