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

The mDia_N binds DAD with submicromolar affinity

The N-terminal regulatory domain of mDia1, mDia_N, binds Rho•GTP and the C-terminal DAD in a mutually exclusive manner, where Rho•GTP binds with a low nanomolar affinity to mDia_N (Rose et al, 2005b). In order to compare Rho with DAD binding towards mDia_N, 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 (DAD_1145−1200) (Figure 1A). Binding of the fluorescent F-DAD to mDia_N 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 mDia_N to obtain a K_D of 235 nM (Fig 1B). Furthermore, active site titration using concentrations of reactants above the K_D (3 μM; roughly 13-fold above the K_D) 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 k_on and k_off by stopped-flow. For association, F-DAD was mixed with increasing concentrations of mDia_N, and the observed rate constants k_obs were plotted against the mDia_N concentration. This plot was linear over the concentration range and gives, from the slope, an association rate constant k_on 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•mDia_N complex with an excess of unlabelled DAD_1145−1200 to obtain a k_off of 0.84 s⁻¹ (Figure 1D). The resulting dissociation equilibrium constant K_D (k_off/k_on) 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 mDia_N via ITC, which gives an equilibrium dissociation constant (K_D) 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 mDia_N and F-DAD resulted in an affinity of 249 nM (data not shown). Quite unusually for a protein–protein interaction, the mDia_N–DAD_1145−1200 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.

The core DAD binding site on mDia_N

The N-terminal part of the Rho binding site (GBD_N) is detached from the rest of the ARR of mDia_N (see Figure 2 in Rose et al, 2005b). We thus wondered about the contribution of this fragment to DAD binding, expressed mDia_NΔG, and measured its interaction with DAD_1145−1200. It binds with an affinity of 110 nM (stoichiometry 1:1), very similar to mDia_N, 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 GBD_N makes no apparent contribution to the binding of DAD and mDia_NΔG is thus well suited for structural studies on the DAD–mDia_N interaction (see below).

In contrast to mDia_N, DAD binding to mDia_NΔG 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 mDia_N or mDia_NΔG 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–mDia_N 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•mDia_NΔG 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 mDia_N•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.

Requirements for the DAD–mDia_N 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 mDia_N. Whereas the 17mer DAD_1175−1191 was synthesised with and the 22mer DAD_1175−1196 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 mDia_N. 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 DAD_1151−1200 (K_D=209 nM), DAD_1165−1200 (K_D=194 nM) and DAD_1145–1196 (K_D=1.4 μM) with the fragments DAD_1151−1196 (K_D=1.6 μM) and DAD_1145−1200 (K_D=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 DAD_1145−1191 47mer fragment do not show measurable binding affinity in ITC. Even in a qualitative polarisation assay using an AMCA-labelled DAD_1175−1191 peptide and F-DAD_1145−1191, no increase of polarisation could be detected upon addition of mDia_N, even in a high excess, showing that some of the residues between 1192 and 1200 are essential for binding to mDia_N (data not shown).

Structure of the mDia_NΔG–DAD_1145−1200 complex

The mDia_NΔG•DAD_1145−1200 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 mDia_N 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 (mDia_NΔG) and chain D (DAD) will be used for further discussions (Table III).

The overall structure of mDia_NΔG shows no significant conformational changes for the ARR in comparison to both the structure of mDia_N 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.

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 mDia_N (Table I).

The topology of the complex presented here is in analogy to the interactions of catenin with the competing ligands Tcf, cadherin and APC (Graham et al, 2000; Eklof Spink et al, 2001), and to the binding of Ran to the concave side of the helical HEAT repeat motif of its effector β-importin (Chook and Blobel, 1999; Vetter et al 1999), suggesting that the concave side of armadillo or HEAT repeats is a favoured protein–protein interaction surface.

Mutational analysis of the interface

To analyse the interface in more detail, we introduced point mutations into either DAD or mDia_N and measured the affinities using ITC. The hydrophobic residues Phe1195 and Met1182 make major contributions to affinity as their substitution by Ala leads to roughly 800- and 1400-fold reductions in affinity, respectively (Table I). As a control, the mutants T1179A, S1184A and Q1190A with substitutions of residues on the hydrophilic side of the helix have only a minor effect on affinity. The disruption of the salt bridge between Asp1183 and Lys213 led to a 37-fold reduction in affinity, further supporting the notion that hydrophobic interactions provide the major driving force of the interaction between mDia_N and DAD (Table I).

Recently, it was shown structurally and in mutational studies that although Rho•GTP and DAD binding to mDia_N are mutually exclusive, their binding sites do not seem to overlap (Otomo et al, 2005b; Rose et al, 2005b). It was shown that the mutations A256D and I259D in mDia_N affected DAD_1145−1200 binding but not RhoA binding, and the mutation N165D affected RhoA binding but not DAD_1145−1200 binding (Figure 4D) (Otomo et al, 2005b; Rose et al, 2005b). The affinities of these and additional mDia_N mutants towards DAD_1145−1200 were further characterised by ITC measurements. A256D and I259D were drastically reduced indeed to approximately 1.4 mM and 232 μM, respectively, as compared to 109 nM for wild type (Table I). In contrast, mutant N165D, which does affect Rho binding (Rose et al, 2005b), showed wild-type affinity towards DAD_1145−1200, with a value 130 nM. While the mutant N217A (K_D=8.4 μM) does influence DAD binding, mutations of residues far away from the Rho binding site, such as R269E, E264K and D366A, do not (K_D=91, 90 and 153 nM) (Table I).

Rho actively displaces DAD from mDia_N

After localising the DAD binding site by both biochemical and structural analysis, we wondered how Rho•GTP would dissociate the mDia_N•DAD_1145−1200 complex, which is a mimic of the autoinhibited full-length mDia1. We and others have shown that Rho•GTP can displace DAD from binding (Figure 3) (Li and Higgs, 2003; Rose et al, 2005b), which is supported by our affinity measurements, that show a 18-fold higher affinity of mDia_N for Rho (K_D=6 nM) (Rose et al, 2005b) compared to DAD_1145−1200–mDia_N (K_D=109 nM). Considering the only slightly overlapping binding sites (see below), the question arises as to why and how Rho•GTP would displace DAD. Does Rho passively compete with DAD for a marginal overlapping binding site on mDia_N and only binds after DAD has dissociated, or does it actively induce DAD dissociation? If the former holds, Rho•GTP binding would be regulated by the kinetics of mDia_N–DAD dissociation, which is 0.84 s⁻¹ for the intermolecular interaction investigated here (Figure 1D), but would be expected to be much slower for the intramolecular interaction of full-length mDia.

Since the structure of the ARR is very similar between the mDia_NΔG•DAD_1145−1200 and the mDia_N•RhoC•GppNHp structure (Figure 4B), we can use the superimpositon of the two structures to show that the Rho and the core DAD binding sites are very close to each other, but overlap only slightly (Figure 5A and B). The N-terminal Gly1180 in DAD, the first residue visible in the structure, would probably clash with GBD_N upon Rho binding. Thr1179, which is not visible in the structure, would definitely clash with RhoC in any reasonable orientation that we can model into the structure (Figure 5B). In addition, the negative charges of Asp1183 and Glu1187 on DAD_1145−1200 would experience electrostatic repulsion from the negative charges of Glu64, Asp65 and Asp67 from Rho. Although this might be sufficient to mediate release of DAD, it might well be that additional residues on the N- or C-terminal extension of the DAD peptide, which we cannot identify in the current structure, would also interfere with Rho. The most likely residues are those on the N-terminal end of DAD, which, although not making any contribution to mDia_N binding (Figure 2), might nevertheless interfere with Rho binding.

For a quantitative analysis of competitive binding by ITC, the mDia_N•DAD_1145−1200 complex (30 μM mDia_N; 60 μM DAD_1145−1200) was titrated with RhoA•GppNHp (Figure 6A). The primary heat changes were fitted using a simple one-site binding model. DAD is released with an apparent affinity IC₅₀ of 29 μM, with a molar ratio of 1:1 for the RhoA•GppNHp mDia_N binding (Figure 6A). The enthalpy of the reaction was –9.8 kcal mol⁻¹ (Figure 6A), which is close to (but with opposite sign) the highly endothermic ΔH (+10.6 kcal mol⁻¹) obtained for the binding of DAD_1145−1200 to mDia_N (Figure 2A). Direct ITC measurements of RhoA•GppNHp binding to mDia_N at different concentrations and temperatures did not lead to a measurable signal. This could be due to the binding of RhoA•GppNHp to mDia_N having an endothermic and an exothermic part, which neutralise each other (e.g. due to conformational change), or that the high-affinity binding reaction is indeed solely entropy driven.

To see if Rho has an active dissociating effect on the dissociation of the mDia_N•DAD complex, a preformed complex between mDia_N and F-DAD was mixed with increasing concentrations of either RhoA•GppNHp or RhoA•GDP, in the presence of a 100-fold excess nonlabelled DAD_1145−1200 to silence the back reaction. Dissociation was followed by stopped-flow. RhoA•GppNHp does indeed accelerate the dissociation of the mDia_N•F-DAD complex, with a threefold increase over the concentration range used, while RhoA•GDP does not (Figure 6B). This argues for an active displacement mechanism by the binding of RhoA•GTP in the release of autoinhibition.

Recombinant proteins

Proteins were expressed as GST fusions using the vector pGEX-4T1 (Amersham Biociences). The bicistronic mDia_NΔG–DAD_1145−1200 construct was generated using an overlapping PCR. For expression, E. coli BL21DE3 cells were grown to an OD₆₀₀ of 0.7 (37°C, 160 r.p.m.), induced with 100 μM of isopropyl-β-D-thiogalactopyranoside (IPTG) overnight at 20°C. The cells were harvested, suspended in buffer A (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM β-mercaptoethanol) containing 0.1 mM phenylmethylsulphonyl fluoride (PMSF) and 2 mM EDTA. Cells were lysed by sonication or microfluidizing, the extract applied to a GSH-Sepharose column (Amersham Biosciences) and the column washed with buffer B (50 mM Tris–HCl, pH 7.5, 300 mM NaCl, 2 mM β-mercaptoethanol). The fusion protein was digested with 5 U thrombin (serva) per mg of GST-fusion protein on the column overnight at 4°C. The final purification was carried out with size exclusion chromatography in buffer B using a Superdex S75 for mDia_N or S200 for the DAD fragments. For the mDia_NΔG–DAD_1145−1200 complex used for crystallisation, the buffer used for gel filtration contained 20 mM HEPES, pH 7.1, 300 mM NaCl and 2 mM β-mercaptoethanol. For selenomethionine labelling, mDia_NΔG–DAD_1145−1200 was expressed in LeMaster medium containing 0.1 g/l Se–Met. The gel filtration buffer contained 20 mM HEPES, pH 7.1, 300 mM NaCl and 10 mM β-mercaptoethanol.

Point mutations were introduced into DAD by the QuickChange protocol (Stratagene) or the PCR mutagenesis protocol of Picard and Bock (1997). Mutant proteins were purified like the wild-type proteins. Concentrations of mDia_N and mDia_NΔG were determined by absorption at 280 nm using the extinction coefficient deduced from the sequence. Rho was purified and loaded with GppNHp as described elsewhere (Rose et al, 2005b).

Synthesised DAD peptides and constructs

DAD peptides comprising residues 1175–1196 and the N-terminally AMCA-labelled (7-amino-4-methylcoumarin-3-acetic acid (AMCA)) peptide were synthesised by Biosynthan, Berlin. The Iaedans labelling of 100 μM of DAD_1145−1200 carrying a C-terminal Cys in buffer C (50 mM Tris–HCl, pH 7.5, 300 mM NaCl, 10 mM ascorbic acid) was carried out by incubating it overnight (4°C) with a 3–5-fold excess of the fluorophore (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1.5-Iaedans)) and exchanging the buffer to buffer B. Concentrations of the DAD fragments were determined by the methods of Ehresmann et al (1973) and Ellman (1958).

Stopped-flow kinetics

The experiments were performed at 20°C using a SX18 MW Applied Photophysics apparatus (Leatherhead, UK). The Aedans fluorophore (Molecular Probes) was excited at 336 nm with a bandpass of 6.4 nm. Emission (at 490 nm) was recorded using a 408 nm cut-off filter. All measurements were carried out with a final concentration of 100 nM of F-DAD and mDia_N–F-DAD. Increasing concentrations of mDia_N (1–15 μM) and RhoA•GppNHp/RhoA•GDP (1–10 μM) were used in the experiments using pseudo-first-order conditions. The observed fluorescence transients were fitted to a single-exponential function to obtain k_obs. The association rate constant k_on was derived from the slope of k_obs versus protein concentration. The dissociation rate constants were obtained by mixing a preformed 100 nM mDia_N–F-DAD complex with a 100-fold excess of unlabelled DAD_1145−1200. When investigating the dissociation acceleration by RhoA, RhoA•GppNHp or RhoA•GDP was used together with a 100-fold excess of unlabelled DAD_1145−1200. All measurements were carried out in buffer B, additionally containing 5 mM MgCl₂ in the RhoA experiments. GraFit 3.0–GraFit 5.0 was used for data analysis.

ITC measurements

The interaction of mDia_N and DAD, characterisation of DAD and mDia_N mutants, and analysis of different DAD constructs was performed by ITC based on Wiseman et al (1989) (MicroCal™; VP-ITC microcalorimeter). All measurements were carried out in buffer B. Concentrations of 0.02–3.7 mM DAD or 400 μM RhoA•GppNHp (syringe) were stepwise injected to the 20–280 μM mDia_N or to the 30 μM mDia_N–60 μM DAD_1145−1200 complex solution (cell), respectively. The heating power per injection was observed over the reaction time until equilibrium was reached. The data were analysed using the software provided by the manufacturer.

Fluorescence equilibrium titration and polarisation measurements

All experiments were performed by using a FluoroMax II spectrofluorimeter at 20°C. Polarisation experiments were carried out using a polarisation filter (SPEX Instruments, NJ), buffer B plus 5% DMSO plus 5 mM MgCl₂ if RhoA was used. The AMCA fluorophore was excited at 353 nm, the Aedans fluorophore at 336 nm. The emission was detected at 442 nm for the AMCA and at 490 nm for the Aedans fluorophore. Fluorescence titration experiments were performed in buffer B plus 0.001% Tween20. The AMCA- or Aedans-labelled DAD fragments (100 nM) were preincubated until the baseline was stable. In addition, mDia_N was added stepwise (0.017–67 μM), mixed rapidly and the fluorescence was followed until the signal did not change anymore. The relative fluorescence yield or polarisation was plotted against the protein concentration and the obtained curve was analysed by fitting a quadratic function to the data as described by Herrmann and Nassar (1996). For data analysis, Grafit 3.0–Grafit 5.0 was used.

Crystallisation and structure determination

Selenomethionine crystals of the GBD_136−451–DAD_1145−1200 complex were grown in buffer D containing 3.7 M Na formate, pH 7.1, 100 mM HEPES, pH 7.1 and 4.0% PEG5000MME, using the hanging drop/vapour diffusion method. The protein was solved in buffer containing 20 mM HEPES, pH 7.1, 300 mM NaCl and 10 mM β-mercaptoethanol. In all, 1 μl of protein solution of various concentrations (2.5 μg/μl, 5 μg/μl, 10 μg/μl) was mixed with the reservoir solution (buffer D). After 1 day, crystals that had a size of 200 × 100 × 100 μM grew at 20°C. These crystals were flash frozen in liquid nitrogen using buffer D as cryoprotectant.

Data-set collection has been performed at the Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland (Table II). A Se–Met data set was collected at 100 K on beam line PXII at a wavelength of 0.95 Å, using a Mar225 CCD detector. The detector distance was 350 mm, the oscillation range was 0.5° and 404 frames were collected. Data were indexed, integrated and scaled with the XDS package (Kabsch, 1993).

Crystals belonged to the hexagonal space group P6₁22 and contained two mDia_NΔG–DAD_1145−1200 heterodimers in the asymmetric unit. Initial phases were determined with the program Phaser (McCoy et al, 2005) using residues 132–369 of chain B of the mDia_N•RhoC structure (PDB: 1Z1C) as a template and searching for two molecules. The program Coot (Emsley and Cowtan, 2004) was used to build the model into the 2F_o–F_c and F_o–F_c maps in iterative rounds of NCS refinement with REFMAC (Table III) (Murshudov et al, 1999). The final model has a good geometry with 98% of all residues in the allowed regions of the Ramachandran plot, as judged by the program Procheck (Laskowski et al, 1993). All structure figures were prepared with PyMOL (DeLano, 2002).