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Research Article 481
The structure of the N-terminal actin-binding domain of human dystrophin and how mutations in this domain may cause Duchenne or Becker muscular dystrophy Fiona LM Norwood1†, Andrew J Sutherland-Smith1†*, Nicholas H Keep1,2 and John Kendrick-Jones1
Background: Dystrophin is an essential component of skeletal muscle cells. Its Addresses: 1Structural Studies Division, Medical N-terminal domain binds to F-actin and its C terminus binds to the dystrophin- Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK and associated glycoprotein (DAG) complex in the membrane. Dystrophin is 2Department of Crystallography, Birkbeck College, therefore thought to serve as a link from the actin-based cytoskeleton of the University of London, Malet Street, London WC1E muscle cell through the plasma membrane to the extracellular matrix. Pathogenic 7HX, UK. mutations in dystrophin result in Duchenne or Becker muscular dystrophy. *Corresponding author. E-mail: [email protected] Results: The crystal structure of the dystrophin actin-binding domain (ABD) has been determined at 2.6 Å resolution. The structure is an antiparallel dimer of †These authors contributed equally to this work. two ABDs each comprising two calponin homology domains (CH1 and CH2) that are linked by a central α helix. The CH domains are both α-helical globular Key words: actin binding, calponin homology domain, dystrophin, muscular dystrophy, utrophin folds. Comparisons with the structures of utrophin and fimbrin ABDs reveal that the conformations of the individual CH domains are very similar to those of Received: 1 December 1999 dystrophin but that the arrangement of the two CH domains within the ABD is Revisions requested: 10 February 2000 altered. The dystrophin dimer reveals a change of 72° in the orientation of one Revisions received: 23 February 2000 Accepted: 28 February 2000 pair of CH1 and CH2 domains (from different monomers) relative to the other pair when compared with the utrophin dimer. The dystrophin monomer is more Published: 25 April 2000 elongated than the fimbrin ABD. Structure 2000, 8:481–491
Conclusions: The dystrophin ABD structure reveals a previously 0969-2126/00/$ – see front matter uncharacterised arrangement of the CH domains within the ABD. This © 2000 Elsevier Science Ltd. All rights reserved. observation has implications for the mechanism of actin binding by dystrophin and related proteins. Examining the position of three pathogenic missense mutations within the structure suggests that they exert their effects through misfolding of the ABD, rather than through disruption of the binding to F-actin.
Introduction subsarcolemmal cytoskeleton and laminin in the extracel- Duchenne muscular dystrophy (DMD) is the most lular matrix via a glycoprotein complex in the sarcolemma common serious X-chromosome-linked genetic disorder, (plasma membrane; for a review see [11]). with an approximate incidence of one in 3500 live-born males. It is now more than ten years since the gene for The expression of the gene encoding dystrophin has been DMD was isolated [1,2] and shown to be allelic with that detected in many types of cell, including skeletal, cardiac for Becker muscular dystrophy. The protein encoded by and smooth muscle. Full-length dystrophin transcribed the gene is called dystrophin [3]. Initially, because of its from the muscle (M) promoter translates into a protein of size and inferred shape [4], dystrophin was thought to be a 3685 amino acids with a molecular mass of 427 kDa [3]. purely structural protein, providing mechanical strength On the basis of its primary sequence and homologies with for the muscle-cell membrane; localisation studies suggest other molecules, dystrophin has been divided into that this might be one of its roles although its precise cel- domains [4]. The 30kDa N-terminal domain is the focus lular function(s) remain to be established. In addition, dys- of this work. The large central domain comprises 24 spec- trophin has been shown to associate directly with a variety trin-like three-helix bundle motifs. The C-terminal region of intracellular components, such as the actin cytoskeleton interacts directly with the transmembrane protein β-dys- and the dystrophin-associated glycoprotein complex troglycan and hence indirectly with the other members of (DAG) in the plasma membrane [5–8], and indirectly with the dystrophin–glycoprotein complex. It contains several extracellular components such as laminin and merosin motifs, which are (from the N to C termini) the WW [9,10]. Thus dystrophin in a muscle cell acts as a linker domain (characterised by two highly conserved tryptophan molecule providing continuity between the F-actin-based residues), two putative Ca2+ binding EF hands, a ZZ 482 Structure 2000, Vol 8 No 5
domain (a putative zinc-binding motif) and two predicted We present the crystal structure of the human dystrophin α helices (reviewed in [12]). ABD. This study was undertaken for several reasons. Dys- trophin deficiency is the molecular defect underlying Improved detection methods have produced extensive DMD and this is the first report describing the tertiary lists of individual disease-causing mutations within the structure of part of this protein. A possible therapeutic dystrophin gene. The distribution of large deletions and strategy to alleviate muscle-cell damage in DMD patients duplications is non-random, with clustering around is to replace the function of the missing protein with a regions of exons 2–20 and 45–53 (N-terminal domain and molecule that has an equivalent function. The current the beginning and end of the helical repeat region, e.g., interest in utrophin centres on its potential for replacing [13]). Point mutations, accounting for the remaining third dystrophin, which is absent in DMD patients [26], and of the mutations, are distributed more evenly along the therefore the demonstration that their actin-binding gene [14], a few of them have been detected in the N-ter- regions are equivalent is important. The position of three minal domain. Thus, although defects in dystrophin are regions of primary sequence involved in actin binding have the underlying cause of Duchenne and Becker muscular been proposed through peptide-binding, sequence compar- dystrophies, the exact pathogenic mechanism involved ison and deletion analysis experiments [27–29], although remains unclear. the tertiary arrangement of these sequences remained unknown. Disease-causing mutations affecting the N-ter- The possibility that the dystrophin N-terminal region minal region [14] might be assumed to produce their dele- might bind to F-actin was first noticed through its primary terious effect through modulating this region’s binding to sequence similarity to the corresponding actin-binding F-actin in some way. In order to resolve these issues, the regions of chicken α-actinin [15,16]. Members of this structure of the N-terminal actin-binding region of human ‘spectrin superfamily’, which now also contains utrophin, dystrophin has been determined at 2.6 Å resolution. are proposed to derive from a common ancestor [17–19] and to contain regions that are structurally and function- Results and discussion ally analogous, leading to suggestions that members of the The final model comprises four dystrophin ABDs assem- superfamily might be able to complement each other [16]. bled into two dimers within the asymmetric unit. The The sequence from which the actin-binding regions are N terminus is disordered resulting in a dystrophin ABD proposed to derive is homologous with the smooth muscle model consisting of residues 9–246. A Ramachandran plot protein calponin and hence has been designated as a of mainchain torsion angles (φ,ψ) calculated using the calponin homology (CH) domain [20]. Proteins containing program MOLEMAN2 [30] reveals that 97% of residues lie CH domains include spectrin, α-actinin, dystrophin, within the favoured region. The average temperature utrophin and fimbrin. Apart from calponin itself and in factors for the protein mainchain, protein sidechain and SM22 (transgelin), the signalling protein IQGAP and the solvent atoms are 37.5 Å2, 37.7 Å2, and 38.4 Å2, respectively. proto-oncoprotein Vav with a single CH domain [17], these domains occur in tandem, each pair represented Two dystrophin ABDs assemble into an antiparallel dimer once in each actin-binding domain (ABD); the exception (approximate dimensions 80 × 50 × 30Å) with the N-termi- is fimbrin, which has two pairs of CH domains organised nal CH1 domain of one monomer in close association with into two ABDs. The N-terminal (CH1) domain from one the C-terminal CH2 domain of the other (Figure 1a). Each protein bears closer resemblance to the equivalent CH1 ABD monomer adopts an extended dumbell shape com- domain of a homologous protein than to the C-terminal prising the two CH domains connected by an α helix (helix (CH2) domain of its own molecule. I, Figure 1b). The CH2 domain is formed from a scaffold of seven helices (which are shown mapped to the sequence in The crystal structures of the CH2 domain from spectrin Figure 2 and are labelled A–G, as in [21]), with the major [21,22] and from utrophin [23] revealed that the CH helices A, C, E, and G comprising the core of the domain. domain is mainly α-helical, with a core of four helices. Helices C, E and G are approximately parallel and form a Three of the helices are roughly parallel to each other and triple helical bundle; the N-terminal A helix lies perpendic- the fourth is approximately perpendicular to the other ular to this bundle, packing against the C and G helices. three. In addition, the structures of the N-terminal Helices B, D and F are short two and three turn helices that fimbrin ABD [24] and the ABD from utrophin [25] have connect the major structural elements. The CH1 domain also been determined recently, and again the core CH exhibits the same overall fold as the CH2 region. Superpo- domain structure is preserved. The manner in which the sition of the CH1 and CH2 domains on the core A, C, E two domains are organised within the ABD, however, is and G helices (55 Cα atoms) results in a root mean square strikingly different. The fimbrin ABD crystallises as a deviation (rmsd) of 1.4 Å, whereas an overlay using all Cα monomer with a compact globular structure, whereas atoms within the CH domains (104 Cα atoms) results in a utrophin forms an antiparallel dimer of two ABDs, each superposition with an rmsd of 3.7 Å. These comparisons adopting a more elongated dumbell shape. show that substantial differences exist between the loop Research Article Crystal structure of the dystrophin actin-binding domain Norwood et al. 483
Figure 1
(a) (b) F ACH1 ACH1 BCH2 BCH2 E A100 A100 CH2 D B N N B200 A50 B200 A50 G C
B150 B150 A C C C C C A150 A150 I
C A200 A200 G N N N A B50 B50 E CH1 ACH2 ACH2 B100 B100
BCH1 BCH1 F Structure
Structure of the dystrophin actin-binding domain. (a) Stereoview Cα trace of the dystrophin dimer. Every tenth residue is highlighted and every fiftieth numbered. (b) Ribbon diagram of the dystrophin ABD. The ribbon representation is colour ramped from the N terminus (blue) to the C terminus (red); the helices are labelled. regions of the CH1 and CH2 domains even though the core both sets of comparisons the superpositions were calcu- structures of the two domains are very similar. The short B lated on the A, C, E and G helices (46 Cα atoms only). and D helices are not present in the CH1 domain and are replaced with loop regions and an extra turn at the N termi- The antiparallel quaternary structure of the dystrophin nus of the E helix. In addition, the CH1 domain has two dimer brings the CH1 domain of one monomer into residues deleted in the FG loop (between helices F and G) contact with the CH2 domain from the other, forming two when compared with the CH2 domain. The regions C-ter- domain-swapped CH1–CH2 interfaces. These dimer con- minal to the core CH fold exhibit quite different conforma- tacts are supplemented by extensive contacts between tions in the two domains. The polypeptide chain extends helix I and the corresponding region in the other from the G helix in CH1 forming helix I connecting the monomer, in addition to interactions with the C termini. two CH domains. The C terminus of CH2 contains an Hydrophobic patches formed by residues Val123, Met124, extended region that interacts with the noncrystallographic Ile127, Met128, Ala129, Gly130 and Leu131 on a face of symmetry (NCS) C terminus from the other monomer and helix I, and residues Val243 and Ile245 at the C terminus, packs so that it is perpendicular to the connecting I helices are buried upon dimer formation (Figure 3). A total of at the dimer interface. 2447 Å2 accessible surface area of one monomer is excluded from solvent at the dimer interface, correspond- The dystrophin and utrophin ABDs share 72% sequence ing to 19% of its total surface area. The shape complemen- identity (Figure 2). This high degree of conservation is tarity of the dimer interface can be described by reflected in a comparison of the structures of the individ- calculation of the gap index value, defined as the ratio of ual CH domains. Superposition of the CH1 domains from the gap volume between the monomers and the interface- dystrophin and utrophin results in an rmsd of 0.87 Å (cal- accessible surface area [31]. The gap index for the dys- culated on 106 Cα atoms), whereas comparison of the trophin dimer is 1.5 Å, a value that is indicative of a high CH2 domains yields an rmsd of 1.06 Å (calculated on 101 degree of complementarity for the interface compared Cα atoms). The structural core of CH domains from the with that of many other oligomeric structures analysed by ABDs of other actin-binding proteins is also highly con- this algorithm. The shape complementarity and total area served. Overlays of the CH1 domains from dystrophin and of the interface imply a relatively stable dimer [31]. the N-terminal ABD of fimbrin result in a superposition with an rmsd of 1.48 Å. The dystrophin CH2 domain A comparison of the dystrophin and utrophin dimers superimposes on the CH2 structures of fimbrin and spec- reveals a large conformational shift (Figure 4). The trans- trin with an rmsd of 1.12 Å and 0.58 Å, respectively. In formation is a 72° rigid-body rotation of a CH1–CH2 pair 484 Structure 2000, Vol 8 No 5
Figure 2
Sequence alignment of ABDs from dystrophin, 10 20 ABS1 30 Dystrophin ------MLWWEEVEDCYEREDVQKKTF TKW V N AQFS α β Utrophin ------MAKYGEHEASPDNGQNEFSDI I KSRSDEHNDVQKKTF TKW I N ARFS utrophin, -actinin, -spectrin and T-fimbrin A-Actinin ------MDHYDSQQTNDYMQPEEDWDRDLLLDPAWEKQQRKTF TAW C N SHLR B-Spectrin MTSATEFENVGNQPPYSRI NARWDAPDDELDNDNSSARLFERSRI KALADEREVVQKKTF TKW V N SHLA N-Fimbrin ------SEGTQHSYSSYSEEEKYAF VNW I N KALE (N-terminal and C-terminal ABDs). The exact C-Fimbrin ------PENQDIDWTLLEGETREERTF RNW M N SLGV A secondary structure of the dystrophin structure
40 50 60 70 80 reported here is represented and the ABSs Dystrophin KFGKQH------IENL FSDLQD G RRL LDL LEGLT----GQKL PK----EK GSTRVHALNN VNKAL RV Utrophin KSGKPP------INDM FTDLKD G RKL LDL LEGLT----GTSL PK----ER GSTRVHALNN VNRVL QV are indicated by boxed regions. The positions A-Actinin KAGTQ------IENI EEDFRD G LKL ML L LEVIS----GERL AKP---ER GKMRVHKI SN VNKAL DF B-Spectrin RVSCR------ITDL YKDLRD G RML IKL LEVLS----GEML PKP---TK GKMRI HCL EN VDKAL QF N-Fimbrin NDPDCRHVI PMNPNTDDL FKAVGD G IVL CKM INLSV----PDTI DERAI NKK KLTPFI I QEN LNLAL NS (residues 54, 168, 171 and 231) where C-Fimbrin N----PH------VNHL YADLQD A LVI LQL YERI KVPVDWSKV NKPP- YPK LGANMKKLEN CNYAV EL C E pathogenic dystrophin mutations occur are
90 100 ABS2 110 120 130 shaded yellow. Dystrophin L QN- NNVDL VNI G STDI VDG NHKL T L GL I W N IILHWQVKNV MKNIMAG------LQQTNSEK Utrophin L HQ- NNVEL VNI G GT DI VDG NHKL T L GL L W S IILHWQVKDV MKDVMSD------LQQTNSEK A-Actinin I AS- KGVKL VSI G AEEI VDG NVKM T L GMI W T IILRFAI QDI SVEET------SAKE B-Spectrin LKE- QRVHL ENM G SHDI VDG NHRL V L GL I W T IILRFQIQDI VVQTQEGR------ETRSAKD N-Fimbrin ASA- I GCHV VNI G AEDL RAG KPHL V L GL L W Q IIKI GLFADI ELSRNEALAALLRDGETLEELMKLSPEE C-Fimbrin GKHPAKFSL VGI G GQDL NDG NQTL T L ALVW Q LMRRYTL NVL EDLGDG------QKANDD FG I
140 ABS3 150 160 170 180 190 Dystrophin I LLS W VRQST RNY- PQVNV I N F T- TSWSDGLA LNALI HSHRP ------DLFDWNSVVC----QQSATQ Utrophin I LLS W VRQTTRPY- SQVNV L N F T- TSWTDGLA FNAVLHRHKP ------DLFSWDKVV-----KMSPIE A-Actinin G LLL W CQRKTAPY- KNVNI Q N F H- I SWKDGL G FCALI HRHRP ------ELIDYGKLR-----KDDPLT B-Spectrin A LLL W CQMKTAGY- PHVNV T N F T- SSWKDGLA FNALI HKHRP ------DLIDFDKLK-----DSNARH N-Fimbrin L LLR W ANFHLENS- GWQKI N N F S- ADI KDSKA YFHLLNQI AP KGQKEGEPRIDINMSGFN---ETDDLK C-Fimbrin I IVN W VNRTLSEAGKSTSI Q S F KDKTI SSSLA VVDLI DAI QP ------GCINYDLVKSGNLTEDDKHN ABCD
200 210 220 230 240 Dystrophin RLEHAFNI A RYQL GI EKLLDPED - V DTTYP DKK SI L MYI TSLFQVLPQQVSI E Utrophin RLEHAFSKA QT YL GI EKLLDPED - V AVRLP DKK SI I MYLTSLFEVLPQQVTI D A-Actinin NL NTAFDVA EKYL DI PKMLDAED I V GT ARP DEK AI M TYVSSFYHAFSGAQKAE B-Spectrin NLEHAFNVA ERQL GIIPLLDPED - V FTENP DEK SI I TYVVAFYHYFSKMKVLA N-Fimbrin RAESMLQQA DK- L GCRQFVTPAD - V VSGNP --K LNL AFVANLFNKYPALTKPE C-Fimbrin NAKYAVSMA RR- I GARVYALPED - L VEVKP --K MV M TVFACL MGRGMKRV- - - EFG
Structure
(from different monomers) relative to the other pair. The C-terminal residues of each domain can interact with rotation occurs approximately around helix I, although the each other and the central I helices. The corresponding relative orientation of the helix is altered. This shift interactions cannot occur in utrophin because the CH2 results in the four CH domains in the dystrophin dimer domains are much further apart than they are in dys- adopting a more planar conformation, relative to the trophin. The total area buried at the dystrophin dimer twisted structure of the utrophin dimer. The perturbations interface is 2447 Å2, however, which is a similar value to that occur in the rigid-body shift are localised at the end of that found for utrophin, 2413 Å2. The gap index (as the interconnecting helix I (residues 120–124 at the C ter- briefly described above) is smaller for the dystrophin minus of CH1 and 133–137 at the N terminus of CH2). dimer interface (1.5 Å) than for that of utrophin (1.8 Å). A greater number of potential hydrogen bonds exist In the crystal structure of dystrophin the formation of across the dystrophin dimer interface, with 30 potential the dimer positions the two CH2 domains so that the interactions formed, in contrast to the 18 in utrophin.
Figure 3
Stereoview of the dystrophin I helix and 225 225 C-terminal dimer interface.
114 114
V123 V123 M124 M124
L131 L131 I127 148 I127 148 246 246 A129 L243 A129 L243 I245 M128 I245 M128 M128 I245 M128 I245 A129 A129
I127 L243 246 I127 L243 246 L131 L131
M124 M124 V123 V123 148 148
114 114
225 225 Structure Research Article Crystal structure of the dystrophin actin-binding domain Norwood et al. 485
Figure 4
(a) (b) (c) BCH2 ACH1 BCH2 ACH1 CH2 CH1
ACH2
BCH1 ACH2
BCH1 Structure
Ribbon representation of (a) dystrophin, (b) utrophin and (c) fimbrin. The molecules were oriented by superposition on all conserved Cα atoms of the upper CH1–CH2 pair.
These two factors would suggest that the dystrophin during equilibrium centrifugation a monomer–dimer equi- dimer is more stable than utrophin. The majority of librium exists (with a relatively weak Kd of approximately residues in the region of helix I are conserved between 4 µM; FLMN and JK-J, unpublished observations). The dystrophin and utrophin (Figure 2) and there does not conservation of the CH1–CH2 interface suggests that the appear to be an obvious single mechanism for the confor- dystrophin ABD might adopt a compact fimbrin-like mational rearrangement. It is not possible to determine structure in its monomeric form whereas the dimeric state whether or not the C-terminal interactions present in is similar to that found in the crystal structure. This idea dystrophin, but absent in utrophin, are a result of the highlights the importance of the linker region between dimer structure or contribute to its formation. The sub- the CH domains, which was previously revealed by stitution of sulphur with selenium in the methionines of genetic analysis of mutants of the yeast fimbrin, Sac6p utrophin, which is necessary for its structure determina- [32]. The longer linker polypeptide (an additional 13 tion [25], might have contributed to the dimer conforma- residues relative to dystrophin; Figure 2) between the CH tion observed in that structure. Two of these domains in the fimbrin N-terminal ABD might favour the methionines in the equivalent positions in helix I of dys- monomeric structural conformation, whereas the shorter trophin (residues 124 and 128) are buried within the dystrophin/utrophin linker might inhibit the monomer structure; between the two I helices of the dimer and the folding back on itself in the crystal structure. Apart from C-terminal residues. The additional bulk of the sele- dystrophin and utrophin there is no detectable sequence nium atoms might have contributed to structural similarity in this region in the ABDs of related proteins. rearrangements in this region in utrophin. The fimbrin ABD crystal structure [24] indicates that con- formational flexibility can exist in the CH1–CH2 linker A striking feature of the crystal structure of dimeric dys- region. Eight residues cannot be located in the electron- trophin compared with the monomeric fimbrin structure is density map as a result of being disordered and a further the conservation of the interface between the CH1 and seven residues on either side of this region have no CH2 domains. The dystrophin interface is formed sidechains included in the model. In order for dystrophin between CH domains from different monomers, whereas to adopt a similar monomeric structure the linker region in fimbrin the structurally homologous interface is formed would have to adopt a quite different conformation to that with CH1 and CH2 from the same monomer (Figure 4). observed in the crystal structure. In the dystrophin ABD Analytical ultracentrifugation and gel filtration experi- crystal structure the C terminus of the CH1 domain from ments have revealed that the dystrophin ABD can exist in monomer A and the N terminus of the CH2 domain from solution as relatively stable monomers or dimers and monomer B are located in close proximity to one another 486 Structure 2000, Vol 8 No 5
(the distance between A123 Cα and B136 Cα is 5.6 Å). G (Figure 5a,b). The substitution Arg54→Leu would be The dystrophin linker region, although shorter than the expected to destabilise the domain fold by the introduc- fimbrin linker, can span this distance but would require tion of a large charged sidechain into this hydrophobic some unwinding of the interconnecting helix to adopt the environment. This residue is conserved as leucine or compact fimbrin-like conformation. Current evidence sug- isoleucine in CH1 domains (Figure 2), with the exception gests that full-length dystrophin is unlikely to dimerise in of the CH1 domains from human T fimbrin and yeast a similar manner to that of the dystrophin ABDs in the Sac6p in which this residue is tyrosine. Both of these pro- crystal structure. Gel filtration experiments with dys- teins contain double ABDs and appear to be more evolu- trophin-bound to DAG [33], and blot overlay assays using tionarily divergent than dystrophin [17]. dystrophin fragments [34] suggest that full-length dys- trophin is monomeric, although oligomeric as well as The other three missense mutations occur in BMD monomeric dystrophin was observed in rotary-shadowed patients. Ala168→Asp is located on the internal face of micrographs [35]. helix C in a hydrophobic pocket formed by Val154 and Val156, and Trp164 (Figure 5c). An aspartate sidechain Structural basis of DMD/BMD phenotypes could be accommodated in its most favoured rotamer but Solving the structure of the dystrophin ABD makes it pos- the introduction of a charged residue into an internal sible to examine the structural basis of some of the muta- hydrophobic pocket would be expected to destabilise the tions detected in Duchenne and Becker muscular protein fold. Ala171 also lies in helix C (Figure 5c); a dystrophy patients. Gene rearrangements, comprising large mutation to a proline residue would be expected to intro- deletions and duplications, account for two-thirds of the duce a kink into the helix [41,42] possibly disrupting the mutations found in these patients (e.g., see [36]). In addi- tertiary fold of the CH2 domain. Tyr231→Asn is located tion, a number of small deletions and missense mutations on the internal surface of helix G (Figure 5c), with the aro- have been characterised (reviewed in [14]), some of which matic ring packing against the sidechains of Phe200, occur in the ABD. These include four missense mutations Leu213 and Leu234. This mutation results in a sidechain and two single-exon deletions in which the reading frame with decreased steric bulk and hydrophobic nature. A is maintained. However, the background information sup- mutation responsible for suppressing actin-binding at an plied on these patients is limited. BMD patients with the equivalent position in yeast fimbrin (Phe381→Cys) has mutations Ala168→Asp and Tyr231→Asn are reported also been characterised [32]. The location of the mutation only as components of a tabulated summary [14]. Most sites within the core of CH domains suggests that these details are available for a DMD patient with a Leu54→Arg mutations affect protein folding, and do not replace amino mutation [37]. This patient is unusual in that he appears to acid sidechains that are crucial for actin binding. produce 20% of the normal level of dystrophin; most DMD patients produce little or none. In addition, the protein is Pathogenic in-frame deletions of exons 3 (residues 32–62) reported as being of normal size and correctly localised at and 5 (residues 89–119; Figure 5d) of the dystrophin gene the sarcolemma. Despite these findings, the phenotype is have also been characterised [40]. Removal of these large severe. Recently, a new mutation has been reported in a regions of the ABD would be expected to have a cata- point mutation database [38]; which is Ala171→Pro in a strophic effect on protein folding and function. BMD patient, and lies only three residues distal to Ala168→Asp. Several studies have reported deletions of Mechanism of actin binding one or more exons in this region (e.g., see [39]); these are Attempts have been made to define the residues in the summarised in a review [40]. In-frame deletions of each of dystrophin ABD involved in binding to F-actin, and three the single exons 3 (residues 32–62) and 5 (residues 89–119) areas have been proposed. Nuclear magnetic resonance are rare. The phenotype resulting from the deletion of (NMR) studies of the interaction of synthetic peptides exon 3 is variable (DMD to BMD) and that for exon 5 corresponding to short stretches of dystrophin sequence intermediate to severe. Exon 3 deletion patients have little suggested two regions (ABS1 residues 17–26 and ABS3 detectable protein whereas exon 5 deleted patients have residues 131–148), which might be involved in binding to reduced levels of dystrophin. actin residues 83–117 and 350–375, respectively, on differ- ent sides of the outer surface of the actin filament [27,28]. Examination of the position of these mutations in the ter- Fusion proteins with combinations of deletions of each of tiary structure is of interest. The mutation Leu54→Arg in these sites and of a highly conserved site (ABS2 residues a DMD patient, is located on a hydrophobic face of the 88–116) involved in actin binding in α-actinin, showed, CH1 domain C helix where its leucine sidechain, in con- however, that actin-binding could take place with a con- junction with leucines 50, 51, 53 and 57, contributes to the struct containing only the most N-terminal 90 residues core of the domain. This region of the C helix packs and that, within this sequence, the highly conserved against Ala80, Val83 and Leu84 on the hydrophobic amino acid sequence KTFT (in single-letter amino acid surface of helix E, as well as Ile114 and Trp118 from helix code) was not essential for binding [29]. Research Article Crystal structure of the dystrophin actin-binding domain Norwood et al. 487
Figure 5
Pathogenic missense mutations in the (a) dystrophin ABD. (a) Stereoview of the environment of Leu54. (b) Stereoview of the 2Fo–Fc electron-density map in the region of 14 14 Leu54. The electron-density map is contoured at 1.0σ. (c) Stereoview of the environment of Ala168, Ala171 and Tyr231. The mutated residues are coloured red, with neighbouring L53 L53 hydrophobic sidechains coloured green. I114L50 I114 L50 (d) Schematic representation of the dystrophin ABD sequence. The upper representation 122 L57 122 L57 W118 W118 shows the actin-binding and mutation sites L54 L51 L54 L51 and the lower shows the exon boundaries. L84 A80 L84 A80 V83 V83
(b)
Leu 57 Leu 57
Leu 53 Leu 53
Leu 54 Leu 54
Leu 50 Leu 50
Leu 51 Leu 51
(c)
240 240 A171 A171 T148 T148 L172 L172 V154 I209 V154 I209 V156A168 V156A168 L234 F200 L234 F200 Y231 Y231 I232 I232 W163 W163 L212 L212 L213 L213 136 136
(d) ABS1 ABS2 ABS3 118 27 88 116 131 147 246
L54RA168D A171P Y231N
12 3 4 5 6 7 8 32 62 89 119 Structure
From the crystal structure, the positions of these proposed and ABS3 are located on helix A of the CH1 and CH2 actin-binding sites on dystrophin can be assessed. ABS1 domains, respectively, whereas ABS2 is located on the 488 Structure 2000, Vol 8 No 5
E and F helices of CH1. These three ABSs do not form a density peak at the same site as fimbrin on the actin fila- surface together that could dock dystrophin onto an actin ment [44]. The structure of the utrophin ABD–F-actin filament (Figure 6a), suggesting that a conformational complex has also been recently determined by helical change brings the ABS regions into closer proximity to form reconstruction of electron micrographs [45]. A model of the the binding surface. Within the ABD, CH1 appears to form complex was constructed by docking the crystal structure of the major contacts with actin because a cleaved CH1 a utrophin ABD into the difference density peaks with the domain is able to bind actin, whereas an isolated CH2 most satisfactory fit obtained with a model in which the rel- domain cannot [12]. The CH2 domain does contribute to ative positions of the CH1 and CH2 domains were altered actin binding, however, because the binding affinity of the within the ABD. This rearrangement occurs at the C-termi- complete ABD is greater than that of the isolated CH1 nal end of the interconnecting helix, helix I. The different domain [12]. It has been proposed, therefore, that the actin- conformations of the dystrophin and utrophin ABD crystal binding mechanism involves the CH1 domain forming the structures result from a rotation centered about the same principal binding surface, complemented by additional region of the protein. This model of the complex places interactions from the CH2 domain. In the crystal structure ABS1, ABS2 and the hydrophobic face of helix I of the the major region of ABS1 is buried at the dystrophin dimer utrophin ABD in contact with the actin filament. The interface, whereas ABS2 and ABS3 are more solvent acces- reconstructions of the ABDs of utrophin, fimbrin and sible. Dimerisation of the dystrophin ABD might provide a α-actinin ABDs reveal differences in their structures when mechanism in which the hydrophobic surface of helix I is bound to actin, although the binding site on the filament kept buried from solvent until actin binding occurs. (between subdomains 1 and 2 of actin monomer n and sub- domain 1 of monomer n + 2) is the same. Differences in A model for fimbrin binding to F-actin has been proposed modes of actin-binding are also revealed by examination of on the basis of fitting the fimbrin ABD crystal structure into the actin filament structure in these reconstructions, with an electron-difference map calculated from helical recon- utrophin [45] and fimbrin [46] inducing different conforma- structions of fimbrin ABD decorated and undecorated actin tional changes in the actin filament and α-actinin [44] filaments [43]. The fimbrin ABD is modelled as the having no effect. These electron microscopy reconstruc- compact crystal structure bound with ABS2, ABS3 and the tions, in conjunction with the crystal structures of the ABDs CH1–CH2-connecting region interacting with actin. ABS1 from dystrophin, utrophin and fimbrin, have raised several remains buried at the interface between the CH1 and CH2 intriguing questions about the mechanism of actin binding domains (Figure 6b). Helical reconstructions of α-actinin of dystrophin, and the structure of the dystrophin ABD–F- ABD decorated F-actin reveal that the ABD occupies a actin complex. The dystrophin ABD binds actin in a 1:1 bell-shaped (of dimensions 38 × 42 Å) electron-difference stoichiometry [5], and is expected to bind to the same site
Figure 6
(a) (b)
ABS3
ABS3 ABS3
ABS1
ABS2
ABS1 ABS1 ABS2 ABS2 Structure
Actin-binding sites of the dystrophin and fimbrin ABDs. (a) Ribbon and The right surface view is rotated 180° about the vertical axis. surface representations of the dystrophin ABD. The ABSs are colour- (b) Ribbon representation of the fimbrin ABD. The ABSs are colour- coded: ABS1 (green), ABS2 (cyan) and ABS3 (red). The left surface coded: ABS1 (green), ABS2 (cyan) and ABS3 (red). view is shown in the same orientation as the white ribbon monomer. Research Article Crystal structure of the dystrophin actin-binding domain Norwood et al. 489
on the filament as utrophin, fimbrin, α-actinin and other via a membrane-associated glycoprotein complex. The F-actin-binding proteins [47]. The conservation of the N-terminal actin-binding domain (ABD, residues 1–246) domain-swapped CH1–CH2 interface between the of dystrophin contains two modules (called calponin monomers in the dimeric dystrophin crystal structure sug- homology domains, CH1 and CH2) that are found in gests that monomeric dystrophin ABD in solution might other cytoskeletal proteins such as utrophin, fimbrin and adopt a compact globular conformation similar to that of the α-actinin. Two dystrophin ABDs crystallise as an fimbrin ABD crystal structure. The hydrophobic patch antiparallel dimer similar to utrophin, with each ABD formed by the I helix and C terminus, which is buried upon adopting an extended structure of two CH domains con- dimer formation, would appear to make it unlikely that the nected by an α helix. In dystrophin, compared with elongated monomeric dystrophin species exists in exactly utrophin, a conformational change has occurred within the same conformation in solution as found in the crystal the ABD resulting in different orientations of the two CH dimer. Because of the high level of sequence homology domains relative to one another, although the structures between the dystrophin and utrophin ABD it would be of the individual CH domains remain very similar. The expected that they would bind actin in a similar way, with a different relative conformations of the CH domains binding mechanism involving rearrangement of the CH within the ABDs of dystrophin, utrophin and fimbrin domains to form a binding surface. At present, however, we suggest a mechanism in which the ABDs might undergo cannot completely exclude an alternative model in which structural rearrangements upon actin-binding. Three the dystrophin ABD binds actin in a similar way that regions within the ABD have been proposed as actin- fimbrin binds actin, adopting a compact monomeric struc- binding sites (ABSs). Mapping these regions on the ABD ture with the ABS2 and ABS3 regions forming the majority structure does not appear to present any continuous of the binding surface. surface that could be obviously docked onto an actin fila- ment. Several pathogenic mutations identified in cases of Differences in modes of actin binding between dystrophin, Duchenne and Becker muscular dystrophy have been utrophin, fimbrin and α-actinin might be related to the dif- localised within the dystrophin ABD structure. Missense ferent biological roles the proteins play within the cell, as mutations Leu54→Arg, Ala168→Asp, Ala171→Pro and well as interactions with the other domains of the full- Tyr231→Asn are located within the hydrophobic core of length proteins. Dystrophin appears to be primarily a struc- the protein fold and none of them lies within the proposed tural protein linking the actin cytoskeleton with the plasma ABS1–3. The internal location of these residues implies membrane, whereas fimbrin is involved in the bundling of that these mutations alter protein function by destabilisa- actin filaments. The identification of an actin-binding site tion of the domain fold as opposed to the substitution of within the triple helical repeat region of dystrophin has led residues essential for actin binding. to a model of full-length dystrophin binding actin in a side- on fashion [48,49]. This method of binding cannot be Materials and methods shared by fimbrin, which lacks the helical repeat region A construct containing the N-terminal region (residues 1–246) of dys- and contains two ABDs that cross-link actin filaments, and trophin was generated by the polymerase chain reaction (PCR) using utrophin, in which this actin-binding site is missing [50]. Pfu DNA polymerase and a human dystrophin cDNA clone (a gift from Sequence analysis has revealed that the fimbrins are the G Dickson, Royal Holloway College, London) as template. The cysteine residues at positions 10 and 188 in this construct were changed to most divergent members of the ABD-containing protein serine residues using a PCR-based mutagenesis protocol [51]. The family and consequently have been placed into a separate resulting insert was ligated into the EcoR1 restriction sites of the pGEX subgroup [17]. Formation of antiparallel dimers allows 4T2 expression vector (Pharmacia) and overexpressed in Escherichia coli BL21(DE3) cells. When the A from a single colony α-actinin to cross-link actin filaments, illustrating another 600 cultured at 37°C reached 0.8, the cells were induced with 0.5 mM iso- mechanism by which CH domains are positioned and propyl-β-D-thiogalactoside (IPTG) at 25°C. After 3 h, cells were col- utilised for actin-binding. The dystrophin ABD crystal lected and suspended in a buffer containing 140 mM NaCl, 2.7 mM structure has revealed a different conformation to that of KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 (PBS) and the protease utrophin and fimbrin, indicating that rearrangement of the inhibitor cocktail (Complete Boehringer Mannheim) and sonicated at 4°C. The resulting lysed cell suspension was centrifuged at 10,500 g CH domains within the ABD is possibly an important (8,000 rpm in Sorvall GSA rotor) for 20 min and the lysate loaded onto factor for actin binding. a 3 ml glutathione-Sepharose 4B affinity matrix column (Pharmacia). After washing with 3 × 10 bed volumes of PBS buffer, the column Biological implications matrix was sealed and incubated with 500 units of thrombin protease (Pharmacia) dissolved in 5 mls of PBS for 16 h at 4°C to cleave the The X-chromosome-linked Duchenne and Becker mus- dystrophin portion from the N-terminal glutathione-S-transferase cular dystrophies are severe progressive muscle wasting domain. The flow through after thrombin digestion containing the disorders caused by pathogenic mutations that result in expressed dystrophin was immediately loaded onto a Sephacryl HR the absence of functional dystrophin. Dystrophin is a 200 gel-filtration column (80 × 2.6 cm) equilibrated in 20 mM Tris-Cl large multidomain protein localised to the sarcolemma pH 8.0, 1 mM DTT, 50 mM NaCl for further purification and to remove the thrombin. The peak fractions containing the dystrophin ABD were that appears to function primarily in a structural role, pooled, concentrated in a Centricon-10 concentrator (Amicon) and linking the actin cytoskeleton to the extracellular matrix dialysed against a buffer containing 5 mM Tris-Cl pH 8.0 and 1 mM 490 Structure 2000, Vol 8 No 5
Table 1 Table 2
Data statistics. Refinement and model statistics.
All data Outermost shell R factor 40.0–2.60 Å 0.232 (0.265)
Rfree 40.0–2.60 Å 0.268 (0.314) Resolution (Å) 40.0–2.6 2.69–2.60 Bond length rmsd (Å) 0.013 No. unique reflections 36,284 3448 Bond angle rmsd (Å) 0.043 Completeness (%) 95.4 91.6 Temperature factor rmsd (Å2)2.8 Multiplicity 1.7 1.6 NCS rmsd (all atoms) (Å) 0.012
Rmerge* 0.051 0.25 Residues in most favoured region Mean I/σ I 12.7 2.6 of Ramachandran plot*(%) 97 Space group P1 Average mainchain temperature factor (Å2) 37.5 Cell parameters a = 59.69 Å, b = 79.33 Å, c = 81.95 Å Average sidechain temperature factor (Å2) 37.7 α = 61.08°, β = 78.22°, γ = 70.54° Average solvent temperature factor (Å2) 38.4 Wilson B factor (Å2)52 Number of protein atoms in model 7543 Number of solvent atoms in model 74 Σ Σ *Rmerge = |Ii—
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