Structural and Functional Analysis of the Actin Binding Domain of Plectin Suggests Alternative Mechanisms for Binding to F-Actin and Integrin ␤4

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Structure, Vol. 11, 615–625, June, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0969-2126(03)00090-X Structural and Functional Analysis of the Actin Binding Domain of Plectin Suggests Alternative Mechanisms for Binding to F-Actin and Integrin ␤4

Begon˜ a Garcı´a-Alvarez,1,3 Andrey Bobkov,1,3 hemidesmosomes, plectin binds directly to the cyto- Arnoud Sonnenberg,2 and Jose´ M. de Pereda*,1 plasmic moiety of integrin ␣6␤4 (Geerts et al., 1999; 1Program on Cell Adhesion Niessen et al., 1997b; Rezniczek et al., 1998). Thus, plec- The Burnham Institute tin provides a direct link between the integrin and the 10901 North Torrey Pines Road intermediate filament cytoskeleton. Mutations in the La Jolla, California 92037 plectin gene are present in patients suffering from a 2 The Netherlands Cancer Institute skin-blistering disorder termed epidermolysis bullosa Division of Cell Biology simplex, which is frequently associated with a late-onset Plesmanlaan 121 muscular dystrophy (Uitto et al., 1996). A similar pheno- 1066 CX Amsterdam type is observed in mice upon targeted inactivation of The Netherlands the plectin gene (Andra et al., 1997). Hence, plectin plays an essential role to maintain mechanical strength of both skin and muscle. Summary Purified plectin forms dimers and exhibits a dumbbell nm in length, as observed by electron 200ف structure Plectin is a widely expressed cytoskeletal linker. Here microscopy (Foisner and Wiche, 1987). Analysis of the we report the crystal structure of the actin binding plectin sequence predicts a multidomain structure with domain of plectin and show that this region is sufficient three main sections: a central rod domain, flanked by for interaction with F-actin or the cytoplasmic region N- and C-terminal globular regions (Figure 1A). The rod of integrin ␣6␤4. The structure is formed by two cal- domain is predicted to be mainly ␣-helical and to drive ponin homology domains arranged in a closed confor- dimerization by coiled-coil interactions. The C-terminal mation. We show that binding to F-actin induces a globular region contains six sequence repeats. Repeats conformational change in plectin that is inhibited by one to five show sequence similarity with plakin repeat an engineered interdomain disulfide bridge. A two- domain B, while the C-terminal repeat corresponds to step induced fit mechanism involving binding and sub- a plakin repeat domain C. The crystal structures of plakin sequent domain rearrangement is proposed. In con- repeat domains B and C of desmoplakin reveal a com- trast, interaction with integrin ␣6␤4 occurs in a closed mon and novel fold comprising four and a half copies of conformation. Competitive binding of plectin to a 38-residue minimal structural scaffold termed “plakin F-actin and integrin ␣6␤4 may rely on the observed repeat” (Choi et al., 2002). The C-terminal region of plec- alternative binding mechanisms and involve both allo- tin harbors a specific binding site for vimentin and cyto- steric and steric factors. keratins (Nikolic et al., 1996; Steinbock et al., 2000), located next to the fifth plakin repeat domain B. Plectin Introduction association with intermediate filaments is regulated by a mitosis-specific phosphorylation of Thr4542 (Foisner Microtubules, actin microfilaments, and intermediate fil- et al., 1996; Malecz et al., 1996), located in the ␤-hairpin aments constitute the cytoskeleton of eukaryotic cells. of the fourth plakin repeat of the C-terminal plakin repeat The integrated organization of the three systems is es- domain C. sential for cellular functions, including mechanical The N-terminal globular moiety contains a 220-residue strength, adhesion, motility, intracellular trafficking, and region with sequence similarity to the F-actin binding cell division. Network interconnection relies on linker domain (ABD) found in dystrophin, ␣-actinin, spectrins, proteins that interact with multiple filament systems fimbrin, and other plakins (de Arruda et al., 1990; Hem- (Fuchs and Yang, 1999). mings et al., 1992). N-terminal fragments of plectin con- ;kDa) and versatile cytoskeletal taining the ABD interact with actin (Andra et al., 1998 500ف) Plectin is a large linker (Steinbock and Wiche, 1999) and is a member of Fontao et al., 2001) and regulate actin dynamics in vivo the plakin protein family (Leung et al., 2002; Ruhrberg (Andra et al., 1998). Thus, plectin ABD is functional. In and Watt, 1997). Plectin has been shown to interact with addition, fragments carrying the ABD bind to the cyto- several types of intermediate filament proteins (Foisner plasmic moiety of the ␤4 subunit of integrin ␣6␤4 (Geerts et al., 1991, 1988; Pytela and Wiche, 1980), actin fila- et al., 1999; Rezniczek et al., 1998), and binding to integ- ments (Seifert et al., 1992), and microtubules (Herrmann rin ␤4 prevents association with actin filaments (Geerts and Wiche, 1987). In addition to binding individual cy- et al., 1999). toskeletal components, plectin crosslinks intermediate This type of ABD is formed by a tandem pair of cal- filaments with microtubules and actin microfilaments ponin homology (CH) domains (Carugo et al., 1997; Stra- (Svitkina et al., 1996). In epithelial cells, plectin is local- dal et al., 1998) separated by a linker of variable length. ized in desmosomes (Eger et al., 1997) and hemidesmo- The high-resolution structures of single (Banuelos et al., somes (Hieda et al., 1992), two structures that anchor 1998; Bramham et al., 2002; Carugo et al., 1997; Keep intermediate filaments to the plasma membrane. In et al., 1999a) and tandem pairs (Goldsmith et al., 1997; Keep et al., 1999b; Norwood et al., 2000) of CH domains *Correspondence: [email protected] reveal a conserved fold built up of four ␣ helices. The 3These authors contributed equally to the work. two CH domains of fimbrin’s N-terminal ABD make an Structure 616

Figure 1. Structure of the ABD of Plectin (A) Domain organization of full-length plectin. (B) Ribbon representation of the ABD structure composed by CH1 (blue) and CH2 (red). (C) Stereo C␣ trace of the ABD in the same orientation as in (B). The sequence is numbered every 10 residues. The exon 2␣ insertion is colored in green. (D) Structure-based sequence alignment of human ABDs from plectin (Plec), utrophin (Utro), dystrophin (Dyst), the N-terminal ABD of fimbrin (Fimb), and the second CH domain of ␤-spectrin (Spec). Secondary structure elements are indicated by boxes and are labeled as in (B); the position of every tenth residue is indicated by dots. The positions of ABS1-3 and the exon 2␣ insertion are indicated by boxes. Utrophin and fimbrin residues previously implicated in direct interaction with F-actin are indicated by green boxes. Molecular figures were generated using the programs MOLSCRIPT (Kraulis, 1991), RASTER3D (Merrit and Bacon, 1997), or SPOCK (Christopher, 1998). extensive interdomain contact; hence, the ABD adopts mains show very weak or negligible affinity for actin a closed or compact conformation. The crystal struc- (Fontao et al., 2001; Way et al., 1992; Winder et al., 1995). tures of the ABD of utrophin (Keep et al., 1999b) and its However, full actin binding capacity is only observed in close homolog dystrophin (Norwood et al., 2000) reveal the presence of both CH domains, supporting the role an antiparallel dimeric arrangement. In these dimers the of tandem CH domains as a functional unit. Within ABDs, N-terminal CH domains (CH1) and the C-terminal CH three conserved actin binding sites (ABS1-3) (Figure 1D) domains (CH2) from different molecules make contacts have been implicated in direct interaction with actin similar to those observed in fimbrin ABD. The crystallo- (Bresnick et al., 1990; Levine et al., 1992). ABS1 and graphic dimer formation may be due to domain swap- ABS2 are in CH1 while ABS3 extends over the N-terminal ping (Liu and Eisenberg, 2002) and suggests that ABDs ␣ helix of CH2. can undergo a conformational switch between open and In order to understand the mechanisms of plectin in- closed states. teraction with F-actin and with the cytoplasmic tail of The two CH domains of plectin are functionally non- integrin ␣6␤4, we have determined the crystal structure equivalent, as observed in other ABDs. Isolated CH1 of the plectin ABD. Using biochemical and biophysical domains support binding to actin; in contrast, CH2 do- methods in combination with structure based mutagen- Plectin Actin Binding Domain Structure 617

Table 1. Summary of Crystallographic Analysis In contrast, the rmsd for the same set of atoms in the CH1 domain of fimbrin is 1.10 A˚ . The nearest structural Data Collection homolog to the CH2 domain of plectin is the CH2 domain

Space group P212121 a ϭ 33.6 A˚ b ϭ 79.3 A˚ c ϭ 82.4 A˚ of ␤-spectrin (rmsd 0.46 A˚ ), and it is closely related Wavelength (A˚ ) 1.5418 to the structure of the CH2 domains of utrophin and Resolution (A˚ ) 32.0–2.15 (2.23–2.15)a dystrophin (rmsd 0.61 A˚ ), while structurally more distant Measured reflections 42883 from the fimbrin CH2 domain (rmsd 1.06 A˚ ). Both CH Unique reflections 11486 Completeness (%) 91.2 (61.4)a domains of plectin show the lowest structural similarity a Rsym (%) 8.2 (31.9) with the CH domain of calponin as indicated by rmsd ϽI/␴IϾ 16.1 (3.9)a of 1.22 A˚ and 1.19 A˚ for the first and second CH domains, Refinement Statistics respectively. Thus, plectin CH domains belong to the spectrin branch of the CH superfamily (Bramham et al., ˚ Resolution range (A) 32.0–2.15 2002; Stradal et al., 1998). Unique reflections work/free 10591/862 Despite the high degree of structural conservation of R (%) 21.2 work the CH fold, the plectin CH1 domain shows some unique Rfree (%) 26.1 Number of residues 243 features. The N-terminal helix A is longer than in other Number of solvent molecules 153 CH structures (38 A˚ in length), with four extra turns at Average B value (A˚ 2) its N terminus, and projects out of the core of the do- Protein 24.2 main. Our construct includes a five-residue insertion, Solvent 31.4 HWRAE, contiguous to helix A, which is coded by exon Rmsd bond lengths (A˚ ) 0.007 ␣ Rmsd angles (Њ) 1.30 2 and subject to alternative splicing (Fuchs et al., 1999). The side chain of the His makes a H-bond with Glu108 a Numbers in parenthesis correspond to the outer shell. in helix C and is buried by the aromatic ring of the Trp. Rsym ϭ⌺|Ii ϪϽIϾ|⌺Ii, where I is the observed intensity and ϽIϾ is the average intensity of multiple observations of symmetry-related The last two residues of the insertion and Ala86 form a reflections. 310 helix, not present in other CH1 domains. The back-

R ϭ⌺|Fobs Ϫ Fcalc|/⌺Fobs. bone of helix A and the AC loop downstream of the Rfree is calculated using 7.5% of reflections that were not included insertion adopt a similar conformation to the equivalent in the refinement. region of dystrophin and utrophin. Thus, the 2␣ insertion has a limited local effect and does not propagate structural changes to other regions of the ABD. esis, we demonstrate that binding to actin induces a The approximate overall dimensions of the ABD are conformational change in plectin ABD. We show evi- 67 ϫ 40 ϫ 35 A˚ , not including the N terminus of helix A dence for alternative mechanisms of interaction with from CH1. The CH domains are arranged in a closed actin and ␤4. These observations are discussed in the conformation forming extensive intramolecular contacts context of a possible allosteric competition. that bury 1550 A˚ 2 of solvent accessible surface. Helix G of CH2 packs against the cleft formed between helices Results and Discussion A and G from CH1. Additional contacts in CH2 involve residues from the EF loop, the short helix F, and the N Overall Structure terminus of helix A. The nature of the interface is mixed We have crystallized a fragment of the N-terminal region with both hydrophobic and polar interactions. Polar con- of human plectin, residues 59–293, corresponding to the tacts include six H-bonds and two salt bridges formed ABD. The structure was solved by molecular replace- by Lys73 with Asp264 and Asp267. The side chain of ment and refined to 2.15 A˚ resolution (Table 1). The Lys276 in helix G of CH2 projects toward the CH1 do- crystal contains a single molecule in the asymmetric main and makes a cation-␲ interaction with the aromatic unit, and interpretable electron density was observed ring of Trp165. Comparison with other tandem pairs of for all residues. CH domains reveals an intramolecular domain organiza- The ABD is formed by two CH domains (Figures 1B– tion very similar to the first ABD of fimbrin (Goldsmith 1D). Each domain adopts the common fold observed in et al., 1997) and to the intermolecular CH1-CH2 arrange- other members of the CH domain superfamily; they are ment in the crystallographic head-tail dimers of utrophin built around a conserved nucleus formed by four ␣ heli- (Keep et al., 1999b) and dystrophin ABDs (Norwood et ces A, C, E, and G that enclose a hydrophobic core. The al., 2000). The differences are limited to a small change 310 helix B and short ␣ helix F cap the N-terminal end in the angle between CH1 and CH2. The linker region of the parallel helical bundle formed by helices C, E, between CH domains in plectin ABD does not adopt a and G. The CH2 has an additional short ␣ helix D in the defined secondary structure. Clear but weak electron CE loop not present in CH1. density is observed for this linker region, and its B fac- As observed in other tandem pairs of CH domains, tors refine to higher values than those of the CH do- each CH domain of plectin is structurally more similar mains, indicating a significant degree of conformational to the equivalent CH domains from other proteins than variability. This is reminiscent of the CH1-CH2 linker of to the companion CH domain. Superposition of other fimbrin, which is 16 residues longer than in plectin and CH domain structures reveal that plectin’s CH1 is more is partially disordered in the crystal structure (Goldsmith similar to the CH1 domain of utrophin and dystrophin, et al., 1997). In contrast, the linker regions of utrophin with a root mean square deviation (rmsd) of 0.43–0.50 A˚ and dystrophin adopt an ␣-helical conformation and are for 47 equivalent C␣ atoms used in the superpositions. two residues longer than in plectin (Keep et al., 1999b; Structure 618

Norwood et al., 2000). Thus, in plectin the interdomain arrangement is likely to be governed by the large interaction surface, not by specific orientations dictated by the very flexible interdomain linker. The plectin ABD is a monomer in solution as determined by size exclusion chromatography and sedimen- tation equilibrium (data not shown), and residues in the interdomain interface are not accessible under native conditions (see below). Thus, the crystal structure most likely corresponds to the conformation in solution.

Actin Binding Induces a Conformational Change in Plectin ABD N-terminal fragments of plectin containing the ABD have been shown to colocalize with actin filaments in vivo and to bind directly to actin in vitro (Andra et al., 1998; Fontao et al., 2001; Geerts et al., 1999). We confirmed that the ABD supports binding to F-actin (data not

shown). The observed dissociation constant (KD)ofthe interaction is 22.3 Ϯ 2 ␮M, similar to that of the isolated ABD of utrophin and dystrophin (Moores and Kendrick- Jones, 2000; Winder et al., 1995). The plectin N-terminal fragment 1–339, fused to maltose binding protein, has

,.␮M) (Fontao et al 0.3ف a higher affinity for F-actin (KD 2001). The observed variability may be due to different experimental conditions, including pH and ionic strength. Alternatively, the presence of plectin regions additional to the ABD may increase the affinity, as observed in utrophin (Moores and Kendrick-Jones, 2000). The low-resolution structures of several CH domains bound to F-actin have been obtained by electron microscopy and image reconstruction of F-actin decorated filaments (Galkin et al., 2002; Hanein et al., 1997; Hodg- kinson et al., 1997; Moores et al., 2000; Volkmann et al., 2001). Docking of high-resolution structures into the electron microscopy maps yielded detailed pseu- doatomic models of the CH domains bound to F-actin; these include the N-terminal ABD of fimbrin (Hanein et al., 1998), the ABD domain of utrophin (Galkin et al., Figure 2. Conformational Change of Plectin ABD upon Binding to 2002; Moores et al., 2000), and the CH domain of cal- F-Actin ponin (Bramham et al., 2002). The ABD of fimbrin bound DSC scans obtained for F-actin complexes with wild-type plectin to actin has been docked using the closed conformation ABD (A), plectin T74C/S277C reduced (B) and oxidized (C). For each observed in the crystal structure; it binds to F-actin in panel curves 1 and 2 represent melting profiles of 18.0 ␮M plectin ␮ the cleft between two adjacent actin monomers (Hanein ABD in the absence and presence of 18.0 M F-actin, respectively. Curve 3 is for 18.0 ␮M F-actin alone. The peak corresponding to et al., 1998). In contrast, utrophin ABD has been pro- plectin ABD or the reduced T74C/S277C mutant shifts to lower posed to bind F-actin in extended conformations (Galkin temperature in the presence of actin. This effect is inhibited in the et al., 2002; Moores et al., 2000) similar to the one ob- disulfide crosslinked T74C/S277C mutant. served in the crystal structure (Keep et al., 1999b). Opti- mal docking of utrophin ABD requires interdomain reori- 2A; Table 2). The ABD shows a denaturation profile with entation, suggesting an induced fit mechanism. The CH -64ЊC. In the presفa thermal transition maximum (Tm)of domain of calponin bound to F-actin has been docked in ence of F-actin, the ABD is destabilized and its Tm is a utrophin-like orientation in order to satisfy biochemical reduced to 59ЊC, whereas the Tm of F-actin is increased constraints (Bramham et al., 2002). by 1ЊC in the presence of plectin. Thus, plectin ABD The conformation displayed by the plectin ABD when undergoes a conformational change upon interaction. bound to F-actin is unknown. Our crystal structure corre- The strong effect of F-actin on plectin ABD thermal sta-

5ЊC decrease in Tm value) indicates substantialف) sponds to the closed conformation of the plectin ABD bility that is compatible with a fimbrin-like interaction with conformational change in ABD upon F-actin binding, actin. On the other hand, a conformational change would which is consistent with an induced fit mechanism. Ther- be required if plectin ABD binds to F-actin in an ex- mal destabilization is not a typical behavior for actin tended conformation. We used differential scanning cal- binding proteins. For example, there is an increase in orimetry (DSC) to assess the effect of interaction with the thermal stability of myosin and tropomyosin upon actin on the thermal stability of both proteins (Figure binding to F-actin (Levitsky et al., 1998, 2000). Plectin Actin Binding Domain Structure 619

Table 2. Thermodynamic Parameters Obtained from DSC Scans for Plectin-Actin, Plectin-␤4 Interactions

Tm1 (ЊC) Tm2 (ЊC) ⌬Hcal (kcal/mol) Plectin ABD 63.9 — 115.0 Plectin ABD T74C/S277C reduced 65.7 — 111.2 Plectin ABD T74C/S277C oxidized 68.0 — 110.9 F-actin 69.4 — 125.0 Plectin ABD ϩ F-actin 59.1 70.3 — Plectin ABD T74C/S277C reduced ϩ F-actin 61.8 69.7 — Plectin ABD T74C/S277C oxidized ϩ F-actin — 69.2 — ␤4 59.0 — 116.8 Plectin ABD ϩ␤4 59.6 63.9 — Plectin ABD T74C/S277C reduced ϩ␤4 59.7 65.7 — Plectin ABD T74C/S277C oxidized ϩ␤4 59.9 68.0 —

The absolute error in Tm values did not exceed Ϯ0.2ЊC; the relative error in ⌬Hcal values did not exceed Ϯ10%.

To analyze the nature of the conformational change interdomain disulfide increases the electrophoretic mobil- linked to actin binding, we have created the double- ity of the ABD and covalently links the N- and C-terminal mutant T74C/S277C (Figure 3A). Thr74 is located in helix fragments produced by trypsin digestion (Figure 3B). A of CH1 and S277 in helix G of CH2; the distance The T74C/S277C mutant behaves as a monomer in solu- between their C␤ atoms is 4.1 A˚ . The introduced sulfhy- tion in both the oxidized and the reduced form, as judged dryl groups of the mutant are at optimal distance and by size exclusion chromatography. Wild-type and mu- orientation to form a disulfide bond without disrupting tant proteins show the same secondary structure con- the interdomain orientation observed in the crystal tent as estimated by the far-uv circular dichroism spec- structure. Both Thr74 and Ser277 are buried in the inter- tra (data not shown). domain interface; therefore, these mutations do not alter Thiol group titration of the reduced mutant yielded the accessible surface of the protein. The formation of the 0.3 Ϯ 0.3 free accessible sulfhydryl groups per molecule

Figure 3. The Double Mutant T74C/S277C Locks ABD in the Closed Conformation (A) Ribbon representation of the ABD structure; the view corresponds to a 180Њ rotation around a vertical axis with respect to Figure 1B. The side chains of C74 and C277 were modeled forming the interdomain disulfide bridge and are shown in detail. Position of the trypsin digestion point between R121–M122 (Garcı´a-Alvarez and de Pereda, unpublished data) in the CE loop of CH1 is indicated by an arrow. (B) Analysis by SDS-PAGE under nonreducing (left lanes) or reducing conditions (right lanes) of the T74C/S277C mutant in the two oxidation states: disulfide bond opened and formed. Tryptic fragments ABD-Tryp1 and ABD-Tryp2 remain covalently linked under non-reducing SDS-PAGE when the C72– C277 disulfide bridge is formed. Position of molecular weight markers is indicated on the left side. Structure 620

under native conditions and 3.1 Ϯ 0.1 groups per molecule under denaturing conditions. The third sulfhydryl group corresponds to C207; it is located in the AB loop of CH2 and is not exposed to the solvent. The inaccessi- bility of the cysteine residues engineered in the interdomain interface is consistent with a closed conformation for the ABD in solution, as the one observed in the crystal structure. The thermal stability of the T74C/S277C mutant in the reduced form is slightly higher than for the wild-type (Figure 2B; Table 2), possibly due to new hydrophobic interactions made by the introduced sulfhydryl groups. Formation of the disulfide bond results in an additional 2.3ЊCف stabilization of the ABD that is reflected in a increase of the Tm. This effect can be rationalized in terms of additional stabilization of plectin ABD by the covalent crosslinking of the two CH domains. We pro- pose that the engineered disulfide bond introduces a constraint between the two CH domains and prevents opening of the ABD without altering the structure of the individual domains or their arrangement. Compared to the wild-type protein, the reduced T74C/

S277C mutant has equivalent affinity for F-actin (KD ϭ 23.1 Ϯ 0.8 ␮M) and displays a similar thermal destabilization upon binding to F-actin (Figure 2B). The oxidized T74C/S277C mutant, with the disulfide bond formed, has lower affinity for F-actin (KD ϭ 59.3 Ϯ 10.7 ␮M), but its Tm does not change in the presence of F-actin (Figure 2C; Table 2). That is, locking of the closed conformation prevents the thermal destabilization of the ABD linked to the interaction with actin. This suggests that reduction in the Tm of wild-type ABD upon binding to actin reflects a conformational change that involves rearrangement of the CH domains. We termed the final conformation “open” to illustrate the disruption of the CH1-CH2 interface. The locked closed T74C/S277C mutant retains the Figure 4. Model of ABD Opening Linked to F-Actin Binding ability to bind to F-actin, indicating that opening of the Surface representation of plectin ABD in the closed conformation ABD is not essential for interaction with actin filaments. observed in the crystal structure (upper panel), in the same orienta- This suggests that the conformational change observed tion as in Figure 1B. Residues in equivalent positions as the ones upon interaction with actin may occur subsequent to implicated in fimbrin binding to F-actin are colored in green. The the binding. The oxidized T74C/S277C mutant prevents ABS2, ABS3, and the linker are in the same face. The observed an interdomain rearrangement and permits a distinction conformational change is illustrated in a hypothetical extended conformation based on the structure of utrophin ABD bound to actin between the two steps. (lower panel). CH1 is rotated 90Њ around a vertical axis to orient The two-step mechanism of plectin ABD binding to the ABS1 toward F-actin (viewer point of view). Disruption of the actin can be interpreted on the basis of the previously interdomain interface would allow the orientation of ABS3 in contact described models of ABDs bound to F-actin (Figure 4). with F-actin. Residues corresponding to the regions of utrophin In a first step, plectin ABD binds to F-actin in a closed suggested to interact with F-actin are colored in green, and those conformation compatible with a fimbrin-like model in coded by exon 2␣ are in red. The positions of T74 and S277 are which ABS-2, ABS-3, and the interdomain linker region indicated. form a continuous surface interface with F-actin. In this conformation the ABS-1 is hidden by the interdomain interface and does not contribute to the binding. In a Interaction with Integrin ␣6␤4 second step, actin may act as a wedge triggering the Plectin N-terminal fragments containing the ABD inter- ABD opening by alteration of the hinge angle between act with the cytoplasmic region of the integrin ␤4 subunit the CH domains. Disruption of the interdomain interface (Geerts et al., 1999; Rezniczek et al., 1998). The minimal may lead to dissociation of the two plectin CH domains ␤4 binding region in plectin has been mapped, using a and exposure of the masked ABS-1. Direct contribution yeast two-hybrid assay, to residues 36–302 (Geerts et 90Њ al., 1999), which roughly corresponds to the crystallizedف of ABS-1 to the interaction interface requires a rotation of CH1, while the conformational flexibility of ABD. The integrin ␤4 cytoplasmic region 1115–1355 is the linker may allow the correct repositioning of ABS-3 the minimal fragment needed to interact with N-terminal toward the interface with actin. This final stage may be fragments of plectin containing the ABD (Geerts et al., described by a model similar to the proposed structures 1999; Koster et al., 2001). This region includes the first of utrophin ABD bound to F-actin (Galkin et al., 2002; tandem pair of FnIII domains and part of the connecting Moores et al., 2000). segment that links the second and third FnIII domains. Plectin Actin Binding Domain Structure 621

Table 3. Affinity of Plectin ABD to Integrin ␤4 (1126–1355)

Plectin KD (␮M) ABD 24.8 Ϯ 4.9 ABD/2␣ 37.9 Ϯ 18.4 ABD T94C/S277C reduced 57.5 Ϯ 1.7 ABD T94C/S277C oxidized 42.9 Ϯ 2.6

The binding affinity of the plectin ABD for integrin ␤4 fragments was determined by isothermal titration calorimetry (Table 3). All binding data were best described by a one to one stoichiometry model. The insertion of five residues coded by exon 2␣ does not modify the affinity for ␤4 significantly, suggesting that the AB loop and the short helix B of CH1 do not participate directly in the interaction. The effect of the interaction between plectin ABD and ␤4 (1126–1355) on the conformation of both proteins was analyzed by DSC (Figure 5; Table 2). Binding of plectin ABD to integrin ␤4 does not produce any change in the Tm of plectin. A small and consistent increase of 0.6ЊC in the thermal stability of the ␤4 fragment isف observed upon binding to plectin ABD. Therefore, interaction with ␤4 is not linked to a detectable conformational change in the plectin ABD. To further evaluate the role of the interdomain conformational flexibility in the interaction with ␤4, we have measured the binding of the reduced and oxidized forms of the T74C/S277C ABD mutant. In both oxidation states, the affinity of the T74C/ S277C mutant showed the same order of magnitude as that of the wild-type. In either the presence or absence of the disulfide bridge, the mutants showed a similar behavior in DSC to the wild-type protein. Thus, the plectin ABD interacts with ␤4 in a closed conformation repre- sented by the crystal structure. In contrast to the interaction with actin, individual plectin CH domains do not support binding to ␤4 (Geerts et al., 1999). The requirement of the complete plectin ABD for interaction with ␤4 suggests direct participation Figure 5. Interaction with Plectin ABD Induces Thermal Stabilization of both CH domains in binding. In addition, the domain of ␤4 (1126–1355) organization may be essential for the correct presenta- DSC scans obtained for ␤4 complexes with wild-type ABD (A), ABD tion of the interaction surface. The binding interface may mutant T74C/S277C reduced (B) and oxidized (C). For each panel be formed by discontinuous binding sites in each of the curves 1 and 2 represent melting profiles of 18.0 ␮M plectin ABD in CH domains or by a single site formed in the interdomain the absence and presence of 18.0 ␮M ␤4 (1126–1355), respectively. area. Curve 3 is for 18.0 ␮M ␤4 (1126–1355) alone. No change was observed in the position of the plectin ABD peak, but a moderate The crystal structure of the first pair of tandem FnIII ␤ ␤ temperature increase was observed for the 4 denaturation peak domains of integrin 4 reveals an elongated arrangement in the presence of plectin proteins. of the two domains (de Pereda et al., 1999), also observed in solution (Chacon et al., 2000). A missense mutation in integrin ␤4, R1281W, has been described in a patient that this region may be implicated in the interaction. suffering from a nonlethal form of epidermolysis bullosa Interestingly, plectin ABD has a basic belt around the with pyloric atresia (Pulkkinen et al., 1998). This mutation interdomain seam and the helix G of CH1. This belt is disrupts binding of ␤4 1115–1457 to an N-terminal plec- surrounded by acidic residues (Figure 6) and has good tin fragment containing the ABD (Geerts et al., 1999). shape complementarity with the ␤4 interdomain acidic R1281 is located exposed to the solvent in the CЈE loop groove. We speculate that the plectin-␤4 interaction in- of the second FnIII domain spatially close to E1242. volves interdomain surfaces in both pairs of tandem CH The R1281W mutation is not likely to produce a general and FnIII domains, and, therefore, binding to ␤4 may alteration of the FnIII fold. Lack of plectin binding to induce a conformational lock of plectin ABD. the R1281W mutant may be due to the loss of a polar interaction with a positively charged group from the Biological Implications ABD. R1281 lies at the edge of an acidic V-shaped The ability to interact with multiple components of the groove formed at the interface between the first and cytoskeleton makes plectin a highly versatile cross- second FnIII domain (de Pereda et al., 1999), suggesting linker. The N-terminal moiety of plectin contains an ABD Structure 622

suggests that binding of wild-type plectin to F-actin induces rearrangement of the CH domains. Conforma- tional changes are not observed during binding to a cytoplasmic fragment of integrin ␤4, providing evidence that binding to F-actin or integrin ␣6␤4 occurs through different molecular mechanisms. The alternative modes of interaction of the ABD may explain the cellular localization of plectin. For example, in epithelial cells the cytoplasmic moiety of integrin ␤4 recruits plectin into hemidesmosomes and prevents association with actin filaments (Niessen et al., 1997a). Interaction with ␤4 may induce a conformational lock of the ABD that would prevent proper interaction with F-actin. However, the locked closed ABD mutant retains the ability to bind to F-actin with an affinity in the same order of magnitude as the wild-type. Therefore, the competitive binding of plectin ABD to integrin ␣6␤4 and F-actin may involve both allosteric and steric components.

Experimental Procedures

Protein Expression and Purification The cDNA sequence corresponding to residues 59–293 of human plectin (EMBL accession number U53204) was amplified by polymerase chain reaction (PCR) and cloned into the bacterial expression vector pET15b (Dubendorff and Studier, 1991) using NdeI and BamHI restriction sites. The five residue insertion coded by the exon 2␣ was introduced by PCR. The absence of errors in the sequences was confirmed by DNA sequencing. Proteins were expressed in Escherichia coli strain BL21(DE3). Cell cultures were induced with 0.2 mM isopropylthiogalactoside (IPTG) for 3 hr at 37ЊC and pellets frozen at Ϫ80ЊC. Upon thawing, cells were sonicated on ice and cell debris removed by centrifugation. The proteins were purified using a nickel-chelating affinity column (Amersham Biosciences, Piscataway, NJ). The His-tag was cleaved by thrombin digestion and removed by extensive dialysis against 20 mM TRIS (pH 7.0), 150 mM NaCl. The purified proteins include four residues (Gly-Ser- His-Met) from the His-Tag at the N terminus. Integrin ␤4 fragment 1126–1355 was amplified by PCR, cloned into pET15b, expressed, and purified as for the plectin proteins.

Crystallization and Structure Determination Crystals of the fragment 59–293/2␣ were obtained at room temperature by vapor diffusion using 0.1 M TRIS (pH 7.0), 15% (v/v) Figure 6. Molecular Surface of Plectin ABD 2-propanol as precipitant. Protein at 20 mg/ml in 20 mM TRIS (pH Two views of the solvent-accessible surface colored according to 7.0), 150 mM NaCl, was mixed with an equal volume of precipitant. electrostatic potential: blue for positive (20 kT/e), red for negative Crystals belong to the space group P212121 with cell dimensions a ϭ (Ϫ20 kT/e). In the upper panel the ABD is in the same orientation 33.6 A˚ , b ϭ 79.3 A˚ , c ϭ 82.4 A˚ . Each asymmetric unit contains a as in Figure 1B. A basic cleft extends along the interdomain contact, single molecule corresponding to a solvent content of 33%. Prior and it is flanked by acidic residues. to data collection, crystals were transferred into cryoprotectant solutions containing 0.1 M TRIS (pH 7.0), 17% (v/v) 2-propanol, and composed of a tandem pair of CH domains. Plectin 20% (v/v) MPD and were frozen by direct immersion in liquid nitro- gen. Data from a single crystal were collected at 100 K using a interacts through the ABD with actin or the cytoplasmic Rigaku FR-D X-ray generator and an R-Axis IVϩϩ image plate. moiety of integrin ␣6␤4, and binding to these two part- Data were indexed with DENZO and reduced with SCALEPACK ners is competitive (Geerts et al., 1999). We have solved (Otwinowski and Minor, 1997). the structure of the ABD and designed biophysical and The structure was solved by molecular replacement using CNS mutational approaches to probe the different mecha- (Brunger et al., 1998). The CH1 domain of dystrophin (PDB code ␤ nisms responsible for the ABD dual function. 1DXX) (Norwood et al., 2000) and the CH2 domain of -spectrin (PDB code 1AA2) (Carugo et al., 1997) were used as search models. The crystal structure of plectin ABD reveals a closed The rotation function search only showed a clear peak for the CH2 conformation with an extensive interaction surface be- domain, which was further confirmed in the translation function. The tween the two CH domains. This is likely to be the pre- rotation function search with the CH1 domain of dystrophin did not dominant arrangement in solution. A conformational yield any outstanding solution. Once the correct translation solution change in plectin ABD linked to F-actin binding was for the CH2 domain was fixed, it was possible to find a clear transla- inferred from differential scanning calorimetry analysis. tion function solution for one of the rotation function solutions of the CH1 domain. A plectin ABD mutant containing an engineered interdo- The model was refined with the package CNS version 1.1 (Brunger main disulfide bond still binds to F-actin although it does et al., 1998) against data to 2.15 A˚ resolution. The refinement was not undergo a measurable conformational change. This started using the search models, in which residues not identical to Plectin Actin Binding Domain Structure 623

the plectin sequence were mutated to Ala. After initial rigid body tively. Fluorescence intensities of actin alone and plectin ABD alone refinement of the two independent domains followed by simulated were subtracted from the titration data before fitting. annealing, the R factor (Rwork) was 37.0% and the Rfree (calculated with 7% of reflections omitted from refinement) was 43.8%. Extra Differential Scanning Calorimetry density corresponding to plectin residues not present in the initial DSC experiments were performed on a N-DSC II differential scan- model was unequivocally observed in fobs Ϫ fcalc sigmaA maps. Fur- ning calorimeter (Calorimetry Sciences Corp, Provo, UT) with a cell ml. All experiments were performed at a scanning 0.33ف ther refinement included cycles of conjugate gradient minimization volume of and individual B factor refinement, alternated with manual model rate of 1 K/min under 3.0 atm of pressure. Prior to measurements, building. The model converged to an Rwork of 21.2% and an Rfree plectin and ␤4 protein samples were dialyzed against 20 mM PIPES of 26.1%. The final model includes residues 59–293 of the plectin (pH 7.0) and 150 mM NaCl. Alternatively, experiments with rabbit sequence, the five residues coded by exon 2␣, three additional skeletal muscle F-actin were performed in 20 mM PIPES (pH 7.0), residues at the N terminus coded by the expression vector, and 153 150 mM NaCl, 1.0 mM MgCl2, and 0.2 mM ATP. The dialysis buffer solvent molecules. The main chain torsion angles (91.1%) of the was used as the reference solution. The reversibility of the thermal nonglycine residues lie in the most favored regions and 8.4% in the transitions was checked by a second heating of the sample immedi- additional allowed regions of the Ramachandran plot, as defined ately after cooling, following the first scan. All thermal transitions in PROCHECK (Laskowski et al., 1993). Only Thr211 falls in the were irreversible under the conditions used thereby permitting use generously allowed region of the Ramachandran plot; similar torsion of only simple thermodynamic parameters and terms to interpret angles have been observed for residues occupying the equivalent the results. The thermal stability of the proteins was described by the position in other CH domain structures. temperature of the thermal transition maximum (Tm). The calorimetric

enthalpy (⌬Hcal) was calculated as the area under the excess heat Design and Production of the T74C/S277C ABD Mutant capacity function. Because these parameters can be obtained di- Identification of residues in optimal position to engineer an interdo- rectly from experimental calorimetric traces after subtraction of the main disulfide bridge was done with the program SSBOND (Hazes chemical baseline and concentration normalization, they can be and Dijkstra, 1988). The double-mutant T74C/S277C of the plectin used for the description of the irreversible thermal denaturation. 59–293 fragment was made with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The correct sequence of Isothermal Titration Calorimetry the mutant was confirmed by DNA sequencing. The mutant plectin ITC was performed on a VP-ITC calorimeter from Microcal (North- protein was expressed in Escherichia coli and affinity purified as ampton, MA). Fourteen microliters of plectin solution (1.4–1.7 mM) for the wild-type. Formation of the engineered disulfide bond was were injected into the cell containing 90–180 ␮M ␤4 solution. Re- obtained by incubation of the protein at 1 mg/ml in 20 mM PIPES versed experiments where 150 ␮M plectin solution in the cell was ␤ (pH 7.0), 150 mM NaCl with 0.1 mM CuSO4 and 0.1 mM o-phenan- titrated with 4 (1126–1355) solution (1.1–1.4 mM) were carried out throline (Lee et al., 1994) at room temperature for 4 hr. Oxidant was as well. In each experiment 20 injections were made. The experi- removed by extensive dialysis against 20 mM PIPES (pH 7.0), 150 ments were performed at 23ЊC. Prior to ITC titrations all protein mM NaCl, and the protein was concentrated to the desired concen- samples were dialyzed against 20 mM PIPES (pH 7.0) and 150 mM tration in an Amicon ultrafiltration cell (Millipore, Bedford, MA). Sulf- NaCl. Experimental data were analyzed using Microcal Origin soft- hydryl groups were titrated with 5,5Ј-dithio-bis(2-nitrobenzoic acid) ware provided by the ITC manufacturer (Microcal, Northampton, (DTNB) (Riddles et al., 1983) in 20 mM TRIS (pH 8.0), 150 mM NaCl, in MA). Fitting of ITC data has been previously described (Freire et al., the absence or presence of 6 M guanidinium hydrochloride. Correct 1990). formation of the disulfide bond was checked by SDS-PAGE under reducing and nonreducing conditions. Alternatively, the mutant was Acknowledgments subject to limited proteolysis with 1% (w/w) Trypsin (Sigma, St. Louis, MO) for 1 hr at room temperature, and digested samples We thank Robert C. Liddington for essential support and discussion, were analyzed in parallel with undigested ones. Nuria Assa-Munt, Carolyn Moores, Kenneth A. Taylor, and Clare McCleverty for critical comments on the manuscript, and Heidi Er- F-Actin Binding Assay landsen and Ray Stevens for access to the X-ray facility. This work Binding of plectin ABD to F-actin was characterized using a cosedi- was supported by grants from the National Institutes of Health. mentation assay as described (Moores and Kendrick-Jones, 2000). Briefly, polymerized actin at 8 ␮M (Cytoskeleton, Denver, CO) was Received: December 2, 2002 titrated with a range of plectin ABD concentrations, 2–120 ␮M, in Revised: March 12, 2003 Accepted: March 20, 2003 10 mM PIPES (pH 7.0), 0.2 mM CaCl2, 2 mM MgCl2, 1 mM ATP, 50 mM KCl. Samples were incubated for 1 hr at room temperature Published: June 3, 2003 and centrifuged at 100,000 ϫ g for 30 min at 25ЊC. Pellets were resuspended in twice the initial volume of SDS sample buffer, and References supernatants were diluted with an equal volume of SDS sample buffer. Samples were analyzed by SDS-PAGE, stained with Coomas- Andra, K., Lassmann, H., Bittner, R., Shorny, S., Fassler, R., Propst, sie blue and quantified by gel densitometry. The dissociation con- F., and Wiche, G. (1997). Targeted inactivation of plectin reveals stant and stoichiometry were calculated by fitting a hyperbolic essential function in maintaining the integrity of skin, muscle, and model to the plot of moles of bound ABD per mole of monomeric heart cytoarchitecture. Genes Dev. 11, 3143–3156. actin versus concentration of free ABD. Andra, K., Nikolic, B., Stocher, M., Drenckhahn, D., and Wiche, G. In addition, binding of plectin ABD to F-actin was monitored via (1998). Not just scaffolding: plectin regulates actin dynamics in cul- changes in the intrinsic tryptophan fluorescence. We established tured cells. Genes Dev. 12, 3442–3451. that the tryptophan fluorescence intensity of plectin ABD-F-actin Banuelos, S., Saraste, M., and Carugo, K.D. (1998). Structural com- complex was lower than the arithmetic sum of the fluorescence parisons of calponin homology domains: implications for actin bind- intensities of plectin ABD and F-actin alone. Thus, binding of plectin ing. Structure 6, 1419–1431. ABD to F-actin causes partial quenching of the tryptophan fluores- Bramham, J., Hodgkinson, J.L., Smith, B.O., Uhrin, D., Barlow, P.N., cence of plectin ABD and/or F-actin. We have used this phenome- and Winder, S.J. (2002). Solution structure of the calponin CH do- non to measure the affinity of plectin ABD to F-actin. Six micromolar main and fitting to the 3D-helical reconstruction of F-actin:calponin. of rabbit skeletal muscle F-actin (generous gift from Dr. Reisler, Structure 10, 249–258. UCLA) was titrated with increasing amounts of plectin ABD in 20 Bresnick, A.R., Warren, V., and Condeelis, J. (1990). Identification mM PIPES (pH 7.0), 150 mM NaCl, 1.0 mM MgCl2, and 0.2 mM ATP. 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