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The regulation of mDia1 by autoinhibition and its release by Rho•GTP

2005; Springer Nature; Volume: 24; Issue: 23 Linguagem: Inglês

10.1038/sj.emboj.7600879

1460-2075

Michael Lammers, Rolf Rose, Andrea Scrima, Alfred Wittinghofer,

Protein Kinase Regulation and GTPase Signaling

Article17 November 2005free access The regulation of mDia1 by autoinhibition and its release by Rho•GTP Michael Lammers Michael Lammers Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Rolf Rose Rolf Rose Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Andrea Scrima Andrea Scrima Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Alfred Wittinghofer Corresponding Author Alfred Wittinghofer Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Michael Lammers Michael Lammers Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Rolf Rose Rolf Rose Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Andrea Scrima Andrea Scrima Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Alfred Wittinghofer Corresponding Author Alfred Wittinghofer Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany Search for more papers by this author Author Information Michael Lammers1, Rolf Rose1, Andrea Scrima1 and Alfred Wittinghofer 1 1Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany *Corresponding author. Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany. Tel.: +49 231 133 2100; Fax: +49 231 133 2199; E-mail: [email protected] The EMBO Journal (2005)24:4176-4187https://doi.org/10.1038/sj.emboj.7600879 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Formins induce the nucleation and polymerisation of unbranched actin filaments via the formin-homology domains 1 and 2. Diaphanous-related formins (Drfs) are regulated by a RhoGTPase-binding domain situated in the amino-terminal (N-terminal) region and a carboxy-terminal Diaphanous-autoregulatory domain (DAD), whose interaction stabilises an autoinhibited inactive conformation. Binding of active Rho releases DAD and activates the catalytic activity of mDia. Here, we report on the interaction of DAD with the regulatory N-terminus of mDia1 (mDiaN) and its release by Rho•GTP. We have defined the elements required for tight binding and solved the three-dimensional structure of a complex between an mDiaN construct and DAD by X-ray crystallography. The core DAD region is an α-helical peptide, which binds in the most highly conserved region of mDiaN using mainly hydrophobic interactions. The structure suggests a two-step mechanism for release of autoinhibition whereby Rho•GTP, although having a partially nonoverlapping binding site, displaces DAD by ionic repulsion and steric clashes. We show that Rho•GTP accelerates the dissociation of DAD from the mDiaN•DAD complex. Introduction Cellular processes like cell migration, cytokinesis, maintenance of cell polarity, vesicular trafficking, morphology and phagocytosis are driven by the dynamics of the actin cytoskeleton (Pantaloni et al, 2001; Pollard and Borisy, 2003). Actin filaments are polar structures characterised by a fast-growing barbed and a slow-growing pointed end. They are regulated by extracellular stimuli that activate signalling pathways, including Rho GTP-binding proteins, which in turn, upon activation, can stimulate the actin nucleation and polymerisation machinery (Bishop and Hall, 2000; Pollard and Borisy, 2003; Higgs and Peterson, 2005). So far, three conserved mechanisms of actin nucleation–elongation involving WASP/WAVE-Arp2/3, formins and the recently described Drosophila protein Spire have been identified (Pollard and Borisy, 2003; Quinlan et al, 2005). Formins generate linear, unbranched actin cables and stress fibres (Pruyne et al, 2002; Zigmond, 2004; Higgs, 2005). They are characterised by formin homology (FH) domains, where the FH1 and FH2 domains are responsible for the actin nucleation and polymerisation activity (Evangelista et al, 2003; Zigmond et al, 2003). Most formins have additional domains mediating the regulation of the polymerisation activity and are grouped into seven subfamilies: Diaphanous (Dia), dishevelled-associated activator of morphogenesis (DAAM), formin-related gene in leukocytes (FRL), formin-homology domain-containing protein (FHOD), inverted formin (INF), formin (FMN) and delphilin (Higgs, 2005). Some of these contain an FH3 domain, which is the structurally and functionally least conserved FH domain (Wallar and Alberts, 2003; Zigmond, 2004) and is believed to mediate the subcellular localisation of mDia proteins (Kato et al, 2001; Ozaki-Kuroda et al, 2001). The central catalytic element of formins is the FH2 domain, which nucleates actin filament formation and regulates filament elongation (Higgs and Peterson, 2005). The FH2 domain is the catalytically active element of formins and is sufficient in vitro for actin nucleation (Pruyne et al, 2002; Li and Higgs, 2003; Shimada et al, 2004; Xu et al, 2004; Higgs and Peterson, 2005). In contrast to tight capping proteins, which do not allow any actin dynamics to occur at their binding site, the FH2 domain forms a leaky cap at the barbed end and allows processive elongation of actin filaments (Wear et al, 2003; Zigmond et al, 2003; Harris et al, 2004; Kovar and Pollard, 2004; Kovar et al, 2005). The structures of a dimeric FH2 domain construct of the yeast formin Bni1p and a shorter construct of mDia1 (p140mDia) revealed that the core FH2 domain is all helical (Shimada et al, 2004; Xu et al, 2004). The difference between these structures is a short, unstructured region at the N-terminus, which is of high importance for dimerisation and converts the FH2 domain from a micromolar inhibitor to a nanomolar inducer of actin polymerisation (Shimada et al, 2004), in line with previous findings on the role of FH2 domains (Pring et al, 2003; Zigmond et al, 2003; Harris et al, 2004; Xu et al, 2004). The structure of the dimeric FH2 domain with TMR-actin shows that each dimer of FH2 contacts three actin molecules and suggests a mechanism of processive barbed-end elongation in the presence of FH2 (Otomo et al, 2005a). The proline-rich stretches of the FH1 domain are potential targets for SH3- and WW-domain-containing proteins, but also for profilin (Bedford et al, 1997; Macias et al, 2002; Pring et al, 2003). The actin–profilin complex constitutes the major G-actin pool in the cell and in most cases requires the FH1 domain for efficient polymerisation via the FH2 domain (Otomo et al, 2005a). Binding of the profilin–actin complex to the FH1 domain mediates fast barbed-end elongation by an yet unexplained mechanism that may include high local concentration near the polymerisation site (Zigmond et al, 2003; Romero et al, 2004). It has also been suggested that the increased ATP hydrolysis rate of actin induced by the FH1–FH2 unit decreases the affinity of profilin for actin (Higashida et al, 2004; Romero et al, 2004). In addition to the FH1 and FH2 domains, Diaphanous-related formins (Drfs) contain an N-terminally located GTPase-binding domain (mDiaN) and a C-terminally located Diaphanous-autoregulatory domain (DAD) (Figure 1A) (Watanabe et al, 1997; Alberts, 2001; Otomo et al, 2005b; Rose et al, 2005b). The Drosophila protein Diaphanous, which plays an important role during cytokinesis (Castrillon and Wasserman, 1994), the Saccharomyces cerevisiae proteins Bni1p and Bnr1p, and the mammalian homologues of Diaphanous, mDia1–3, belong to this subgroup (Wallar and Alberts, 2003). In the absence of an activating signal, Drfs are in an inactive autoinhibited conformation due to the interaction between the N-terminal regulatory region and DAD (Alberts, 2001; Li and Higgs, 2005; Otomo et al, 2005b; Rose et al, 2005b). Lacking the N-terminal RhoGTPase-binding domain (GBD) and/or the C-terminal DAD mDia1 is constitutively active and induces formation of stress fibres and SRF-dependent nuclear transcription (Watanabe et al, 1999; Ishizaki et al, 2001; Copeland and Treisman, 2002; Li and Higgs, 2003). The intrasteric inhibition of Drfs is relieved by binding of active Rho proteins to the regulatory region. Rho proteins are Ras-related GTP-binding proteins, which act as molecular switches that cycle between an inactive GDP- and an active GTP-bound conformation. In the latter state, they interact with downstream effectors defined as proteins, which bind tightly to the GTP-bound conformation only (Vetter and Wittinghofer, 2001; Etienne-Manneville and Hall, 2002). A common mechanism by which Rho proteins activate effectors is the release of an intramolecular inhibitory interaction, which can be found in many effector proteins such as WASP and the Ser/Thr kinases PAK, ROK and PKN (Maesaki et al, 1999; Millard et al, 2004). In the case of PAK, upon binding of active Rac or Cdc42, an intramolecular regulatory domain is displaced from the active site and thus allows substrate binding (Lei et al, 2000, 2005). For WASP, it is the interaction of the N-terminal CRIB domain with parts of the C-terminal VCA motif that is relieved by Cdc42 (Buck et al, 2004; Leung and Rosen, 2005). In vitro, the interaction of the regulatory N-terminus (mDiaN) with DAD of mDia1 is released by active Rho, thus mimicking the formin activation reaction (Li and Higgs, 2005; Rose et al, 2005b), although some studies suggest that Rho•GTP may not be quite sufficient for full relief of autoinhibition (Li and Higgs, 2003, 2005). Figure 1.N- and C-terminal constructs of mDia1 and their interaction. (A) Schematic representation of the domain structure of mDia1 and the constructs used. mDiaN, the regulatory region; GBDN, GTPase-binding domain; mDiaNΔG, the regulatory region minus GBDN; ARR, armadillo-repeat region; IH, interdomain helix; Dim, dimerisation domain; FH1-2, formin-homology domains 1, 2; DAD, Diaphanous-autoregulatory domain. Shown below is an alignment of the DAD1145−1200 fragment from different genes and organisms (accession numbers in brackets): mDia1–3: mouse (O08808; Q9Z207; O70566); mDam1–2: mouse (Q8BPM0; Q80U19); Dia: Drosophila (P48608); hDia1–3, human (O60610; O60879; Q9NSV4); yBNI1, Saccharomyces cerevisiae (P41832). Black shaded residues are conserved in 100%, dark grey shaded in 80% and bright grey shaded in 60% of the depicted organisms at the specific position. Furthermore, residues with similar physiochemical properties are combined into groups. Residues 1145–1160 might belong to the FH2 domain and have a helical structure as shown previously (Shimada et al, 2004); the structure of the DCR (residues 1180–1195) was determined here. The red and green circles below the alignment represent residues whose mutations do or do not influence affinity towards mDiaN, respectively. (B) Determination of affinity between F-DAD1145−1200 and mDiaN using fluorescence. F-DAD1145−1200 (100 nM) was titrated with increasing concentration of mDiaN and the change in fluorescence obtained is plotted against the concentration of mDiaN. The data were fitted to a quadratic binding equation. (C) Association kinetics of F-DAD1145−1200 and mDiaN as determined by stopped flow. F-DAD is reacted with increasing concentrations of mDiaN and the observed first-order rate constants (kobs) are plotted against the mDiaN concentration. The slope of the linear fit represents kon. (D) The dissociation rate constant koff is determined by incubating 100 nM of a preformed F-DAD•mDiaN complex in the presence of a large excess of unlabelled DAD peptide. The fluorescence transient is fitted to a single-exponential decay. Download figure Download PowerPoint Recently, the structures of mDiaN (residues 69–451) alone and in complex with RhoC•GppNHp were solved by X-ray crystallography (Otomo et al 2005b; Rose et al, 2005b), revealing that the all-helical mDiaN consists of the three subdomains: GBDN (the N-terminal part of the Rho-binding domain), the armadillo-repeat region (ARR) and a C-terminal dimerisation domain (Dim). Surprisingly, mutational and NMR studies showed that the DAD and Rho•GTP binding sites on the mDiaN surface were apparently not overlapping, suggesting that the mutual exclusive binding of Rho and DAD is not simply due to steric exclusion (Otomo et al, 2005b; Rose et al, 2005b). In order to more clearly identify the DAD binding site and to clarify the structural basis for regulation of the Drf subfamily of proteins, we solved the three-dimensional structure of the complex between mDiaNΔG (residues 136–451) and DAD encompassing residues 1145–1200. Using a number of biochemical and biophysical measurements, we can propose a two-step binding mechanism for the release of autoinhibition by Rho•GTP. Results The mDiaN binds DAD with submicromolar affinity The N-terminal regulatory domain of mDia1, mDiaN, binds Rho•GTP and the C-terminal DAD in a mutually exclusive manner, where Rho•GTP binds with a low nanomolar affinity to mDiaN (Rose et al, 2005b). In order to compare Rho with DAD binding towards mDiaN, we determined the kinetic and equilibrium constants of DAD binding using isothermal titration calorimetry (ITC) and fluorescence-based equilibrium and kinetic measurements. For these, a fluorescently labelled DAD fragment (F-DAD) was made by coupling 1,5-Iaedans to an extra cysteine on the C-terminus of the DAD fragment encompassing residues 1145–1200 (DAD1145−1200) (Figure 1A). Binding of the fluorescent F-DAD to mDiaN leads to an increase in fluorescence, presumably due to transfer of the fluorescence label to a more hydrophobic environment. This fluorescence change was used for equilibrium binding measurements, by titrating F-DAD with increasing concentrations of mDiaN to obtain a KD of 235 nM (Fig 1B). Furthermore, active site titration using concentrations of reactants above the KD (3 μM; roughly 13-fold above the KD) shows that DAD binds with a clear 1:1 stoichiometry (data not shown). To analyse the dynamics of the reaction, we measured the association and dissociation rate constants kon and koff by stopped-flow. For association, F-DAD was mixed with increasing concentrations of mDiaN, and the observed rate constants kobs were plotted against the mDiaN concentration. This plot was linear over the concentration range and gives, from the slope, an association rate constant kon of 3.5 μM−1 s−1 (Figure 1C). The dissociation rate constant was obtained by following the fluorescence decrease on incubating the preformed F-DAD•mDiaN complex with an excess of unlabelled DAD1145−1200 to obtain a koff of 0.84 s−1 (Figure 1D). The resulting dissociation equilibrium constant KD (koff/kon) is 238 nM, in good agreement with the equilibrium titration experiment (Figure 1C). For an independent measurement of affinity and to get more information on the thermodynamics of binding, we measured the interaction of DAD with mDiaN via ITC, which gives an equilibrium dissociation constant (KD) of 109 nM and a stoichiometry of 1:1 (Figure 2A and Table I). The affinity is twofold higher than determined by fluorescence, which is due to the fluorescence label at the C-terminus of DAD, since an ITC experiment using mDiaN and F-DAD resulted in an affinity of 249 nM (data not shown). Quite unusually for a protein–protein interaction, the mDiaN–DAD1145−1200 interaction is highly endothermic, with an enthalpy change ΔH of 10.2 kcal mol−1. The thermodynamically unfavourable enthalpy of the reaction is compensated by the highly favourable (positive) entropic contribution (T ΔS=19.5 kcal mol−1), which is the driving force of the reaction. Figure 2.ITC analysis of the DAD–mDiaN interaction. ITC of the interaction between DAD1145−1200 and either mDiaN (A) or mDiaNΔG (B), measured by titrating 40 μM/30 μM mDiaN/mDiaNΔG in the chamber with 940 μM/400 μM DAD1145−1200 in the syringe. Top panels, raw heating power over time; bottom panels, fit of the integrated energy values normalised for injected protein. (C) Result of affinity analysis of different DAD fragments for mDiaN, measured by ITC as in (A, B) and indicated as affinity reduction relative to the 'full-length' DAD1145−1200 fragment. Download figure Download PowerPoint Table 1. Thermodynamic properties of binding of mDiaN to DAD1145–1200, as determined by ITC Mutation KD (nM) ΔH (kcal mol−1) T ΔS (kcal mol−1) N WT 109±10 10.16±0.05 19.5 0.9 N165D 130±6 10.17±0.03 19.4 1.0 N217A 8470±697 8.01±0.28 14.8 1.0 A256D ⩾1 440 000a I259D ⩾232 000a E264K 91±9 10.16±0.05 17.9 1.1 D366A 153±6 7.81±0.02 16.9 1.0 R269E 90±5 9.5±0.03 18.9 1.1 K1152A 117±7 8.69±0.03 18.0 1.1 R1166A 288±13 7.92±0.03 16.7 0.9 T1179A 180±8 9.51±0.04 18.6 0.9 T1179D 500±19 9.85±0.05 18.3 1.1 M1182A 88 500±3370 17.92±1.22 23.3 1.0 M1182D ⩾200 000a D1183R 4100±81 9.03±0.04 16.3 1.5 D1183N 934±24 12.09±0.49 20.2 1.0 S1184D 216±17 6.77±0.06 15.7 0.9 L1186A 1830±91 8.10±0.08 15.8 1.0 L1189A 9804±865 9.06±0.52 15.8 1.1 Q1190A 114±7 8.19±0.04 17.5 1.2 F1195A ⩾150 000a mDiaNΔG DADWT 110±7 7.43±0.03 16.8 1.0 N is the stoichiometry of binding. The last row shows the characteristics concerning the mDiaNΔG–DAD1145–1200WT interaction. a Affinity of A256D, I259D, F1195A and M1182D is too low for accurate measurements of enthalpy, entropy and stoichiometry. The core DAD binding site on mDiaN The N-terminal part of the Rho binding site (GBDN) is detached from the rest of the ARR of mDiaN (see Figure 2 in Rose et al, 2005b). We thus wondered about the contribution of this fragment to DAD binding, expressed mDiaNΔG, and measured its interaction with DAD1145−1200. It binds with an affinity of 110 nM (stoichiometry 1:1), very similar to mDiaN, and the enthalpy is likewise unfavourable, with ΔH 7.4 kcal mol−1 and a compensating entropy term of 16.8 kcal mol−1 (Figure 2B). Thus, the three-helix motif GBDN makes no apparent contribution to the binding of DAD and mDiaNΔG is thus well suited for structural studies on the DAD–mDiaN interaction (see below). In contrast to mDiaN, DAD binding to mDiaNΔG is not released by RhoA, as shown by a fluorescence polarisation assay. Here, 0.5 μM of a DAD peptide encompassing residues 1175–1196 labelled at the N-terminus with a fluorescent AMCA label (from now: A-DAD) was treated with 2 μM of either mDiaN or mDiaNΔG and the development of polarisation was followed. Due to complex formation and the corresponding increase of mass, the mobility of the fluorophore decreases, which leads to an increase in the polarisation signal (Figure 3). When adding 4 μM of active RhoA•GppNHp to the fluorescent A-DAD–mDiaN complex, polarisation returns to the starting level, whereas addition of RhoA•GDP does not lead to dissociation of the complex, as shown previously under slightly different conditions (Rose et al, 2005b). However, with the A-DAD•mDiaNΔG complex, neither the active nor the inactive Rho can release DAD from the complex, even after addition of a 100-fold excess (data not shown). Using this polarisation assay, the same results could be obtained using the longer F-DAD fragment, which includes the basic residues in the patch 1197–RKRG–1200, which also demonstrates that the nature of the fluorescence label does not influence the results (Supplementary Figure 1). Thus, although part of the Rho binding site is on the ARR region, as shown by the structure of the mDiaN•RhoC complex, the affinity of Rho•GTP for this region is too weak to effectively release DAD, or the mode of binding is inappropriate for such a release. Figure 3.Fluorescence polarisation assay to analyse competitive binding of RhoA or DAD towards mDiaN and mDiaNΔG, respectively. Fluorescently (AMCA)-labelled A-DAD peptide (residues 1175–1196) is incubated with either mDiaN (A, B) or mDiaNΔG (C, D) and leads to increase of the polarisation signal due to the decreased mobility of A-DAD upon complex formation. RhoA•GppNHp (B, D) and RhoA•GDP (A, C) are added as indicated. Download figure Download PowerPoint Requirements for the DAD–mDiaN interaction The DAD regions of Drfs can be aligned and show different levels of conservation (Figure 1A), with a central portion that is conserved between Bni1p and mammalian Dia. In UniProt (www.uniprot.org), it is defined as a stretch of 15 amino acids (mDia1: aa 1180–1194) with a large number of identical and conserved residues. In order to sort out the requirements for efficient intramolecular interaction, we screened various DAD constructs and mutants for interaction with mDiaN. Whereas the 17mer DAD1175−1191 was synthesised with and the 22mer DAD1175−1196 peptide with and without a fluorescent label, all the other DAD fragments were recombinantly expressed as GST fusion proteins and cleaved. Using ITC, values for affinity, stoichiometry, enthalpy and entropy of the binding were obtained and are summarised in Table I and Figure 2C. The 22mer containing the highly conserved 15 amino acids of DAD (Figure 1A) has micromolar affinity and is essential for binding. N- and C-terminal elongations of this DAD core lead to an increasing affinity towards mDiaN. The 56mer peptide covering the complete DAD region shows a 138-fold increase in affinity, with the C-terminus having a much larger influence. Comparison of DAD1151−1200 (KD=209 nM), DAD1165−1200 (KD=194 nM) and DAD1145–1196 (KD=1.4 μM) with the fragments DAD1151−1196 (KD=1.6 μM) and DAD1145−1200 (KD=109 nM) demonstrates that the C-terminal residues 1197–RKRG–1200 are mostly responsible for the increased affinities, while the N-terminal residues 1145–1165 have nearly no effect. This is consistent with the findings of Li and Higgs (2005), who found that a 1177–1200 DAD peptide shows a 250 nM affinity towards mDia1 1–548 and 129–548. The synthesised DAD 17mer fragment 1175–1191 and the recombinantly expressed DAD1145−1191 47mer fragment do not show measurable binding affinity in ITC. Even in a qualitative polarisation assay using an AMCA-labelled DAD1175−1191 peptide and F-DAD1145−1191, no increase of polarisation could be detected upon addition of mDiaN, even in a high excess, showing that some of the residues between 1192 and 1200 are essential for binding to mDiaN (data not shown). Structure of the mDiaNΔG–DAD1145−1200 complex The mDiaNΔG•DAD1145−1200 complex was purified using a bicistronic expression system in Escherichia coli. The soluble complex could be purified using a GSH-Sepharose column, subsequent cleavage and size exclusion chromatography (S200 16/60). It eluted with an apparent molecular mass of about 80 kDa (data not shown), corresponding to a 2:2 hetero-tetramer, which was crystallised. While no molecular replacement solution could be found for a native data set, the better diffracting Se–Met crystals and the structure of mDiaN in complex with RhoC•GppNHp (Table II; Rose et al, 2005a, 2005b; PDB 1Z2C) were used for phasing by molecular replacement. The anomalous signal of Se–Met1182 was used to identify DAD. A ribbon model of the complete structure shows that the DAD binding site is located on the concave site of the ARR (Figure 4A), as predicted from previous experiments (see Figure 3; Rose et al, 2005b; see also Otomo et al, 2005b). As the site chain density of the DAD peptide corresponding to chain D of the hetero-tetramer was better defined, chain B (mDiaNΔG) and chain D (DAD) will be used for further discussions (Table III). Figure 4.Structure of the mDiaNΔG–DAD complex. (A) Ribbon diagram of the structure, where mDiaNΔG is grey and DAD green. Only residues 1180–1195, the DCR, are visible in the structure. (B) Superimposition of the mDiaNΔG from this structure (grey) and the structures of unbound (yellow) and Rho-bound (blue) mDiaN (Otomo et al, 2005b; Rose et al, 2005b), leaving out RhoC and GBDN. (C) Stereo view of the interface between the ARR and DAD, highlighting residues mentioned in the text. (D) Conservation of residues, where the intensity of red indicates the degree of conservation (accession numbers of compared proteins: mDia1 O08808, mDia2 Q9Z207; mDia3 O70566; hDia1 O60610; hDia2 O60879; DAAM1 human Q9Y4D1; DAAM1 mouse Q8BPM0; DAAM2 human Q86T65; DAAM2 mouse Q80U19; Gallus gallus Dia Q9DEH3; E. histolytica Dia Q514T8; DroMe Dia P48608); dashed lines show the possible paths of the N- and C-terminal extensions of the polypeptide chain; Asp366, Ile259, Ala256, Lys213 and Asn217 are highlighted. (E) Electrostatic potential of the mDiaNΔG surface as calculated with APBS (Baker et al, 2001), position of the DCR and the suspected polypeptide path from N to C as indicated. Download figure Download PowerPoint Table 2. Data collection statistics Wavelength (Å) 0.950 Resolution (Å) 3.4 Space group P6122 Unit-cell parameters a=138.7, b=138.7, c=210.9 VM (Å3 Da−1) 3.6 Total measurements 437 327 Unique reflections 33 924 Average redundancy 11.51 I/σ(I) 21.24 (6.92) Completeness (%) 99.4 (99.4) Wilson B-factor (Å2) 77 Rsym (%) 8.4 Table 3. Refinement statistics Resolution (Å) 20–3.3 Reflections (working/test set) 17 565/925 Number of atoms 4966 Rwork (%)a 28.8 Rfree (%)b 36.4 Mean B-value (Å2) 92.2 R.m.s. deviations from standard geometryc Counts/r.m.s.d./weight Bond length (Å) 5031/0.010/0.022 Bond angle (deg) 6770/1.248/1.990 Torsion angle (deg) 615/5.907/5.0 Isotropic thermal factor restraints Counts/r.m.s.d./weight Main-chain bond refined atoms (Å2) 3192/0.411/1.500 Main-chain angle refined atoms (Å2) 4974/0.718/2.000 Side-chain bond refined atoms (Å2) 2018/0.694/3.000 Side-chain angle refined atoms (Å2) 1796/1.132/4.500 a Rwork=∑∣Fo−Fc∣/∑Fo where Fo and Fc are the observed and calculated structure factor amplitudes. b Rfree is claculated similarly to Rwork using the test set reflections. c For definition, see REFMAC5 (www.ysbl.york.ac.uk/~garib/refmac/index.html). The overall structure of mDiaNΔG shows no significant conformational changes for the ARR in comparison to both the structure of mDiaN in complex with RhoC•GppNHp and one subunit of the unliganded form of the protein (Figure 4B and Table IV) (Otomo et al, 2005b; Rose et al, 2005b). The long interdomain helix and the dimerisation domain appear to be more flexible and show the largest differences. In the structure of the unbound form (Otomo et al, 2005b), the long interdomain helix of one of the monomers is interrupted and causes a large distortion of the dimerisation domain, which cannot be observed in the two other structures and the other monomer of the unbound structure (Figure 4B), suggesting that the deviation is due to the extensive crystal contacts at the N-terminus. Table 4. RMSD scores of compared mDia-structures mDia Molecules compared Compared Number of atoms compared R.m.s.d. (Å) DAD-bound/Rho-bound Cα 246 0.87 DAD-bound/unbound Cα 226 0.7 Rho-bound/unbound Cα 241 0.8 DAD-bound/Rho-bound All atoms 2108 1.514 DAD-bound/unbound All atoms 2015 2.286 Rho-bound/unbound All atoms 2064 2.078 Only 16 (1180–1195) of the 56 residues of the DAD fragment used for crystallisation are visible in the structure (Figure 4C, Supplementary Figure 2). The first 12 of these form an amphipathic helix that makes extensive hydrophobic contacts to the second helices of armadillo-repeats (ARM) two, three and four. At residue 1192, the polypeptide makes a 90° bend and seems to form another helical turn (Figure 4A and C). Apart from the hydrophobic interactions, Asp1183 forms a salt bridge with Lys213 of ARM2 (Figure 4C). The same aspartate is also in contact distance of Asn217 (Table I), which is important for the correct orientation of the 'arginine wedge' (Arg68) of Rho, being crucial for the binding of the latter (Rose et al, 2005b). Although the precise orientation of the motif SGAA directly C-terminal of the core DAD was difficult to trace at this resolution, Phe1195 following the SGAA motif is clearly defined. It is wedged into the space between the interdomain helix and ARM4 (Figure 4C). In the structure, DAD is situated on a patch of surface exposed residues with very high conservation between different Drfs down to Entamoeba histolytica (Figure 4D). It is close to, but only marginally overlapping with the Rho binding site (see below), as had been predicted from NMR and biochemical data (Otomo et al, 2005b; Rose et al, 2005b). We have shown above that the basic residues (1196–RRKRG–1200) following the 1191–SGAAF–1195 motif are essential for high-affinity binding (Figure 2C and Table I), but are not visible in the structure of the complex, most likely due to the high salt conditions used in crystallisation, which tend to mask ionic interactions in the crystal. We wondered where this basic stretch of residues could bind and can identify two negatively charged grooves on the interdomain helix or between ARM3 and ARM4, with conserved residues as the possible binding sites (Figure 4D and E). However, since mutation of Asp366 to Ala did not lead to a significant decrease of affinity towards DAD, we are uncertain about the path of the DAD polypeptide on mDiaN (Table I). The topology of the complex presented here is in analog