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Mechanism of IRSp53 inhibition and combinatorial activation by Cdc42 and downstream effectors

Nature Structural & Molecular Biology volume 21, pages 413–422 (2014)Cite this article

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Abstract

The Rho family GTPase effector IRSp53 has essential roles in filopodia formation and neuronal development, but its regulatory mechanism is poorly understood. IRSp53 contains a membrane-binding BAR domain followed by an unconventional CRIB motif that overlaps with a proline-rich region (CRIB–PR) and an SH3 domain that recruits actin cytoskeleton effectors. Using a fluorescence reporter assay, we show that human IRSp53 adopts a closed inactive conformation that opens synergistically with the binding of human Cdc42 to the CRIB–PR and effector proteins, such as the tumor-promoting factor Eps8, to the SH3 domain. The crystal structure of Cdc42 bound to the CRIB–PR reveals a new mode of effector binding to Rho family GTPases. Structure-inspired mutations disrupt autoinhibition and Cdc42 binding in vitro and decouple Cdc42- and IRSp53-dependent filopodia formation in cells. The data support a combinatorial mechanism of IRSp53 activation.

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Figure 1: Autoinhibition and activation of IRSp53 by Cdc42.
Figure 2: Formation of a heterohexameric complex between Cdc42, IRSp53 and Eps8.
Figure 3: Structure of the CRIB–PR bound to Cdc42.
Figure 4: Effect of IRSp53 mutations on filopodia formation in B16F1 melanoma cells as a function of Cdc42 coexpression.
Figure 5: Autoinhibitory interactions involve the CRIB–PR domain.
Figure 6: Autoinhibitory interactions involve the SH3 domain.
Figure 7

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Acknowledgements

This work was supported by the US National Institutes of Health (NIH) grant R01 MH087950 to R.D. D.J.K. was supported by NIH grant T32 AR053461 and American Cancer Society grant PF-13-033-01-DMC. T.S. and C.Y. were supported by NIH grant GM095977. G.S. and A.D. were supported by the Associazione Italiana per la Ricerca sul Cancro grant IG-2013-14104. Use of IMCA-CAT beamline 17-ID was supported by the Industrial Macromolecular Crystallography Association through a contract with the Hauptman-Woodward Medical Research Institute. The Advanced Photon Source was supported by the US Department of Energy Contract DE-AC02-06CH11357. We thank P. Leavis (Tufts University) for the synthesis of the CRIB–PR peptide.

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Authors and Affiliations

  1. Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    David J Kast, Malgorzata Boczkowska, Yadaiah Madasu & Roberto Dominguez

  2. Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Changsong Yang & Tatyana Svitkina

  3. FIRC Institute of Molecular Oncology, Milan, Italy

    Andrea Disanza & Giorgio Scita

  4. Department of Health Sciences, University of Milan School of Medicine, Milan, Italy

    Giorgio Scita

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  1. David J Kast
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Contributions

D.J.K. designed and purified proteins, determined crystal structure, designed and performed FRET and ITC experiments, analyzed filopodia from cell images and participated in the writing of the manuscript. C.Y. transfected and imaged cells. A.D. prepared Eps8 and performed cosedimentation assays. M.B. and Y.M. participated in construct design and protein preparation. G.S. and T.S. participated in project design and data analysis. R.D. was responsible for the overall design of the project and participated in structure determination, data analysis and preparation of the manuscript.

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Correspondence to Roberto Dominguez.

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Integrated supplementary information

Supplementary Figure 1 Purification of IRSp53 constructs and design of FRET reporters.

(a) SDS–PAGE showing purified IRSp53 fragments and GTPases (see also Supplementary Fig. 2 for gels of IRSp53 FL and Eps8). (b) Design of the FL and BAR–SH3 FRET reporters. Endogenous Cys230 and a C-terminal cysteine added by mutagenesis (Cys519 or Cys452) were first under-labeled with the donor probe fluorescein maleimide and then labeled to saturation with the acceptor probe tetramethylrhodamine maleimide (Methods). In the structure of the BAR domain (PDB ID: 1WDZ) Cys230 falls 53 Å apart from its counterpart in the second molecule of the dimer. The BAR domain has a rigid structure that is not expected to change much upon binding to targets or the membrane. Only reporters containing a donor-acceptor pair will show a change in energy transfer. Molecules that compete with autoinhibitory interactions (GTPases, effectors and the SH3 domain of IRSp53 itself) would be expected to induce a conformational change in the reporter, resulting in a decrease in energy transfer. (c) Peptide mass fingerprinting analysis of tryptic peptides derived from wild type IRSp53 after labeling with fluorescein maleimide. Based on a fluorescein-to-protein ratio of 1:1, IRSp53 was found to be labeled at a single cysteine residue. MS/MS fragmentation of a tryptic peptide with m/z 635.74 containing Cys230 (229QCAVAKNSAAYHSK242) produces a series of b-ions. The masses of all the b-ions are 427.36 Da higher than their theoretical masses, corresponding to the mass of the fluorescein maleimide probe.

Supplementary Figure 2 IRSp53 and Eps8 form a 2:2 complex.

(a) Size exclusion chromatography elution profiles of IRSp53, Eps8, IRSp53–Eps8 complex compared to protein standards with known Stokes radii (1, thyroglobulin; 2, aldolase; 3, albumin; 4, ribonuclease-A). IRSp53 and Eps8 are both elongated in solution, and migrate with apparent MW higher than expected from their theoretical masses. SDS–PAGE analysis identifies the fraction volume corresponding to the peak for each protein. The gel filtration fraction volumes of IRSp53, Eps8, and IRSp53–Eps8 are plotted against those of the protein standards to determine their Stokes radii. (b) Glycerol gradient (10–40%) sedimentation of IRSp53, Eps8, and IRSp53–Eps8 compared to protein standards (1', chymotrypsinogen; 2', albumin; 3', aldolase; 4', catalase) with known Svedberg coefficients. The sedimentation fraction volumes of IRSp53, Eps8, and IRSp53–Eps8 are plotted against those of the protein standards to determine their sedimentation velocities. The buffer used in both experiments was 100 mM Tris-HCl pH 8, 500 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA. The table compares the theoretical masses of the samples to the experimentally determined values. The Stokes radii and the sedimentation velocities were used to calculate the MW of each sample according to the equation1: MW = α × SR × Sv.

Supplementary Figure 3 Localization of IRSp53 and Cdc42G12V in B16F1 mouse melanoma cells.

(a–d) Cells coexpressing GFP and RFP, GFP and mCherry–Cdc42G12V, FL–GFP and RFP, FL–GFP and mCherry–Cdc42G12V. The intensity profiles of actin, GFP (or FL–GFP) and RFP (or mCherry–Cdc42G12V) along line-scans (indicated by a white lines) are shown on the right. Note that when expressed alone, IRSp53 localizes in patches along the leading edge, whereas its plasma membrane localization becomes more uniform when coexpressed with Cdc42G12V.

Supplementary Figure 4 Mutations of CRIB–PR residues disrupt binding to Cdc42G12V.

ITC titration of 400 μM GMPPNP–Cdc42G12V into 15 μM BAR–SH3 constructs carrying mutations within the CRIB and PR regions that disrupt binding.

Supplementary Figure 5 Mutations that disrupt autoinhibitory interactions.

(a) In the structure of the CRIB–PR bound to Cdc42G12V, the canonical 278PxxP281 site is partially exposed, potentially allowing for competitive binding of the SH3 domain of IRSp53 itself or a binding partner. Such mechanism of competitive binding is illustrated here by superimposing a SH3–peptide complex (PDB ID: 1W702) onto the structure of the CRIB–PR bound to Cdc42G12V. SH3 residue P428 and CRIB–PR residues P278 and P281 were mutated to test this model. (b) Cells coexpressing FL–GFP mutant P428L with RFP or with mCherry–Cdc42G12V, and quantification of the number of filopodia per cell, filopodia length and percentage of filopodia filled with actin (as described in Fig. 4, main text). The data for filopodia per cell are presented as mean ± SEM. The total number of cells quantified is indicated inside each bar. *P < 0.005 by two-tailed unpaired Student's t test. Zoomed-in regions (white box) are shown separately for Cdc42G12V, IRSp53 and actin. Scale bars: 10 μm.

Supplementary Figure 6 Nucleotide and GTPase specificity of the CRIB–PR domain.

(a) The CRIB–PR can sense the nucleotide state of Cdc42. Comparison of GMPPNP–Cdc42G12V (gray) and GDP–Cdc42 (slate blue; PDB ID: 1A4R3) showing that switch I (residues 30–38) is open (green) in the GDP-bound state and closed (yellow) in the GMPPNP- and CRIB–PR-bound state. The conformation of switch II (residues 57–72) remains mostly unchanged in the GDP-bound (magenta) and GMPPNP-bound (purple) states. (b) Specificity of the CRIB–PR for Cdc42. Residue conservation scores were calculated with the programs ConSurf4 based on the sequences of the two human isoforms of Cdc42 (UniProt ID: P60953-1 and -2) and the three human isoforms of Rac (Rac1, P63000; Rac2, P15153; Rac3, P60763). The conservation scores were displayed on the surface of the structure of Cdc42 using the program Pymol (Schrödinger). The close-up on the bottom (boxed region) highlights sequence variation between Cdc42 and Rac3 (PDB ID: 2QME) at the CRIB–PR interface. Side chains that differ between the two GTPases are highlighted in the structure and in a sequence alignment (red/salmon).

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Kast, D., Yang, C., Disanza, A. et al. Mechanism of IRSp53 inhibition and combinatorial activation by Cdc42 and downstream effectors. Nat Struct Mol Biol 21, 413–422 (2014). https://doi.org/10.1038/nsmb.2781

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