Structural and Biochemical Basis for the Inhibitory Effect of Liprin-α3 on Mouse Diaphanous 1 (mDia1) Function

Abstract

Diaphanous-related formins are eukaryotic actin nucleation factors regulated by an autoinhibitory interaction between the N-terminal RhoGTPase-binding domain (mDiaN) and the C-terminal Diaphanous-autoregulatory domain (DAD). Although the activation of formins by Rho proteins is well characterized, its inactivation is only marginally understood. Recently, liprin-α3 was shown to interact with mDia1. Overexpression of liprin-α3 resulted in a reduction of the cellular actin filament content. The molecular mechanisms of how liprin-α3 exerts this effect and counteracts mDia1 activation by RhoA are unknown. Here, we functionally and structurally define a minimal liprin-α3 core region, sufficient to recapitulate the liprin-α3 determined mDia1-respective cellular functions. We show that liprin-α3 alters the interaction kinetics and thermodynamics of mDiaN with RhoA·GTP and DAD. RhoA displaces liprin-α3 allosterically, whereas DAD competes with liprin-α3 for a highly overlapping binding site on mDiaN. Liprin-α3 regulates actin polymerization by lowering the regulatory potency of RhoA and DAD on mDiaN. We present a model of a mechanistically unexplored and new aspect of mDiaN regulation by liprin-α3.

Keywords: Ras homolog gene family, member A (RhoA); actin; cytoskeleton; formin; liprin; mDia1.

FIGURE 1.

Definition of the binding sites in liprin-α3 and mDia1. A, domain organization of liprin-α3 and mDia1 and the constructs used in this study. +, interactions of liprin-α3 fragments with mDia1; −, fragments not interacting. B, thermodynamic characterization of the shortest liprin-α3 and mDia1 fragments needed for full interaction using ITC. Lip(567–587) and DID bind with 6.9 μm, showing that those fragments contain all residues essential for the interaction. C, coiled-coil prediction of mDia1 and mouse liprin-α3 using COILS version 2.1. For mDia1, the prediction shows a high score for aa 460–562 forming a coiled-coil. For liprin-α3, extended coiled-coil regions were predicted in the N terminus covering residues aa 26–493 and a second patch in the C terminus from aa 863 to 889. D, analytical size exclusion chromatography of mDia1 and liprin-α3 fragments as indicated. Shown is the molecular size calculated by calibration (MW calc.) and the molecular weights for the respective monomers (MW mon.). For all proteins, the absorption at 280 nm is shown except for Lip(567–587) (for which A_{220 nm} is shown). Aprotinin (6.5 kDa), ribonuclease A (13.7 kDa), carbonic anhydrase (29.0 kDa), ovalbumin (43.0 kDa), and conalbumin (75.0 kDa) were used for calibration of the S75 10/300, and for S200 10/300, additionally aldolase (158 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa) were used.

FIGURE 2.

Structure of the Lip(567–587)·DID complex. A, ribbon representation of the Lip(567–587)·DID (aa 135–369) complex (left) and a 90° clockwise rotated view (right). Liprin-α3 (green) binds to the ARM3 to -5 region of DID (gray) in an orientation antiparallel to ID α17. B, close-up of the Lip(567–587)·DID structure. Liprin-α3 makes electrostatic interactions with the ID (Arg-575^L–Glu-358^D and Arg-572^L–Glu-362^D) as well as hydrophobic interactions using Leu-573^L, Met-576^L, and other residues. C, schematic presentation of the interactions in the Lip(567–587)·DID structure generated with LigPlot⁺ version 1.4.5. Liprin-α3 makes hydrophobic as well as specificity-creating salt bridges with the mDia1 DID. D, superposition of the Lip(567–587)·DID (gray/green) structure with the mDia_NΔG·DAD (light orange/dark orange) structure (PDB code 2BAP). The liprin-α3 and DAD-binding sites overlap significantly. E, detailed view of the superposition of Lip(567–587)·DID and mDia_NΔG·DAD. The C-terminal half of liprin-α3 overlaps with the C-terminal half of the DCR. Liprin-α3 follows the path of the DBR along the mDia_N ID. F, composition of the putative ternary complexes of liprin-α3·RhoC·mDia_N (green/blue/gray; PDB code 2BAP) and liprin-α3·DAD·DID (green/orange/gray; PDB code 1Z2C). The subdomain GBD_N from the RhoC·mDia_N (PDB code 1Z2C) structure is shown in dark gray. RhoC does not make any obvious steric or electrostatic clashes with liprin-α3. G, structural similarity of DID from the complexes of RhoC·mDia_N (PDB code 1Z2C), DAD·mDia_NΔG (PDB code 2BAP), mDia_N (PDB code 2BNX), and Lip(567–587)·DID. The mDia_N fragments shown were superposed on the DID. The structures are highly similar in the DID with root mean square deviation values ranging from 0.401 to 0.583 Å for the peptide backbone and from 0.367 to 0.541 Å for the C_α atoms. Structural differences are only visible in the ID and the following DD (aa 369–451) not present in the structure presented here. Lip(567–587)·DID resembles most the uncomplexed mDia_N structure (PDB code 2BNX). H, electrostatic surface potential of DID as generated by APBS. RhoC, DAD, and liprin-α3 are shown as they are located on the mDia_N surface. RhoC uses switch I and II to interact with a positively charged groove on the DID surface. As a second binding site, the Rho insert helix is within interaction distance to the ARM4/5 of the DID. Lip(567–587) is located along ID α17 connecting the DID and the DD of mDia_N. Arg-572^L and Arg-575^L make salt bridges toward negatively charged residues Glu-362^D and Glu-358^D of the mDia_N ID. The C-terminal part of the ID is highly negatively charged. Blue, positive charge; red, negative charge. The figure is scaled from −10 to +10 k_bTe_C⁻¹ (k_b, Boltzmann's constant; T, temperature in Kelvin; e_C⁻¹, elementary charge). I, F_O − F_C omit difference map of the N-terminal part of the liprin-α3 peptide covering residues Thr-567 to Arg-572 (chain D) countered at 3σ.

FIGURE 3.

Stopped-flow analysis of the influence of liprin-α3 on the mDia_N·RhoA interaction dynamics. A, association rate constant. Mant-GppNHp-loaded RhoA was titrated with increasing concentrations of mDia_N together with/without different liprin-α3 fragments. The observed association rate constants (k_obs) were plotted against the mDia_N concentration. The association rate constants (k_a) correspond to the slope of the linear fit. B, dissociation rate constant. A preformed mDia_N·RhoA·mant-GppNHp complex was mixed with a 100-fold excess of active RhoA Q63L, and the increase in fluorescence was followed over time. The data followed a single exponential behavior. Error bars, S.D.

FIGURE 4.

Competition of RhoA and DAD(1145–1200) with liprin-α3 for binding toward mDia_N. A, the competition of RhoA Q63L with Lip(567–587) for mDia_N binding was studied by ITC. RhoA Q63L was titrated on a preformed complex of Lip(567–587) and mDia_N. The interaction showed a negative reaction enthalpy, ΔH, mainly driven by the favorable entropy, TΔS. The presence of liprin-α3 reduced the RhoA affinity toward mDia_N from 4 to 45 nm. B, competition of DAD(1145–1200) and Lip(561–587) for DID binding. The presence of liprin-α3 reduced the binding of DAD(1145–1200) to DID 4-fold (37 versus 153 nm). C, the ITC cell content from B analyzed by analytical gel filtration (S75 10/300). DAD(1145–1200) quantitatively displaces Lip(561–587) from DID. The SDS-PAGE (inset) shows that the high molecular weight peak at 11.06 ml contained only DID·DAD(1145–1200), whereas liprin-α3 eluted in the second peak. D, fluorescence polarization assay. The addition of 10 μm mDia_N to 100 nm F-Lip(567–587) and F-Lip(567–582) led to an increase in the polarization signal, reflecting complex formation. The addition of 15 μm active RhoA Q63L dissociates the Lip·mDia_N complexes shown by the decrease of the polarization signal to the starting level. No ternary Lip·mDia_N·RhoA complexes are formed. Values are shown as mean ± S.D. (error bars) of three replicates.

FIGURE 5.

The minimal liprin-α3 fragment Lip(567–582) reduces the actin filament content in HeLa cells overexpressing constitutively active RhoA Q63L. A, HeLa cells were transiently transfected with mCherry-C1-liprin-α3 and pEGFP-N3-RhoA G14V expression constructs as indicated. Filamentous actin was stained with CF647-phalloidin. The presence of liprin-α3 fragments reduced the cellular actin filament content in a RhoA G14V overexpression background. Images for selected liprin-α3 fragments are shown; all fragments are depicted in the quantification diagram. The images are shown as a single 0.2-μm-thick optical section of the bottom. For full-length liprin-α3, a close-up representing the merged image of DAPI, EGFP-N3-RhoA G14V, and mCherry-liprin-α3 fluorescence is shown. Scale bars, 50 μm. B, quantification of filamentous actin in HeLa cells. The expression of full-length liprin-α3 did not influence the actin filament content. Of the N-terminal fragments compared, Lip_1–817 had the mildest effect on filamentous actin content. C, quantification of F-actin in N2a cells. Full-length liprin-α3 and Lip(561–582) and Lip_{(3GS)567–587} significantly reduced the F-actin content in RhoA G14V-expressing cells. For B and C, the counted cell numbers are shown in parentheses. The experiment was repeated independently at least two times, and one representative experiment is shown. The data are represented as mean ± S.D. (error bars). **, p < 0.01; ***, p < 0.001 for the indicated comparison; ###, p < 0.001 compared with mock (one-way analysis of variance). a.u., arbitrary units. D, full-length liprin-α3 is localized to the cell periphery in N2a cells (inset of merged image as in A). N2a cells were transfected with mCherry-C1-liprin-α3 full-length and EGFP-N3-RhoA G14V. F-actin was stained with CF647-phalloidin. E, working model for the inhibitory effect of liprin-α3 on mDia1 function. The LCR (aa 567–582) binds to the DID only if the formin is in the open conformation. Binding to the DID lowers the DAD affinity moderately, whereas the affinity toward RhoA is more strongly affected. In this way, the DAD is able to compete with RhoA to re-establish the autoinhibited conformation. Moreover, Rho regulators, such as RhoGAPs, could compete with mDia_N for RhoA binding, leading to subsequent RhoA and thereby mDia1 inactivation. The mDia1 membrane translocation could be achieved by liprin-α3 competing for DID/formin interaction partners, such as IQGAP, and by RhoA dissociation and inactivation. The LCR might also interfere with the interplay of the DBR, PIP₂, and ID, resulting in formin inhibition and reduction in cellular filamentous actin. For simplicity, the model does not include the subcellular localization of mDia1/liprin-α3 and the dimeric state of mDia1 and possible oligomeric (homo- and/or heterooligomers) states of liprin-α3. a.u., arbitrary units.