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Double Inhibition and Activation Mechanisms of Ephexin Family Rhogefs Double inhibition and activation mechanisms of Ephexin family RhoGEFs Meng Zhanga,1, Lin Linb,1, Chao Wanga,2, and Jinwei Zhub,2b,,2 aMinistry of Education Key Laboratory for Membraneless Organelles and Cellular Dynamics, Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, 230027 Hefei, China; and bBio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai 200240, China Edited by Alfred Wittinghofer, Max Planck Institute of Molecular Physiology-Dortmund, Dortmund, Germany, and accepted by Editorial Board Member Brenda A. Schulman January 20, 2021 (received for review December 2, 2020) Ephexin family guanine nucleotide exchange factors (GEFs) trans- functions of Ephexin2 and Ephexin3 remain elusive although fer signals from Eph tyrosine kinase receptors to Rho GTPases, they are known to activate RhoA (8). Therefore, the Ephexin which play critical roles in diverse cellular processes, as well as family RhoGEFs serve as the regulatory hubs that link Ephrin- cancers and brain disorders. Here, we elucidate the molecular basis Eph signaling with cytoskeletal dynamics through spatiotemporal underlying inhibition and activation of Ephexin family RhoGEFs. regulation of Rho GTPases. Dysfunctions of Eph-Ephexin–mediated The crystal structures of partially and fully autoinhibited Ephexin4 Rho signaling have been associated with a variety of diseases, ranging reveal that the complete autoinhibition requires both N- and from cancers to brain disorders (18–22). C-terminal inhibitory modes, which can operate independently to Each member of the Ephexin family proteins contains a Dbl impede Ras homolog family member G (RhoG) access. This double homology (DH) domain, responsible for catalyzing guanine nu- inhibition mechanism is commonly employed by other Ephexins cleotide exchange, and an adjacent regulatory pleckstrin ho- and SGEF, another RhoGEF for RhoG. Structural, enzymatic, and mology (PH) domain. In addition, all members possess an Src cell biological analyses show that phosphorylation of a conserved homology 3 (SH3) domain C-terminal to the DH–PH domain tyrosine residue in its N-terminal inhibitory domain and associa- tandem, except Ephexin5 (Fig. 1A). Ephexin4 contains an ad- tion of PDZ proteins with its C-terminal PDZ-binding motif may ditional type-I PDZ-binding motif (PBM) at its very C terminus respectively relieve the two autoinhibitory modes in Ephexin4. (Fig. 1A). Previous work has demonstrated that elimination of BIOCHEMISTRY Our study provides a mechanistic framework for understanding the C-terminal SH3 domain in Ephexin4 can significantly in- the fine-tuning regulation of Ephexin4 GEF activity and offers pos- crease its GEF activity toward RhoG, suggesting that Ephexin4 sible clues for its pathological dysfunction. may adopt an autoinhibited conformation via SH3-mediated intra- or intermolecular interactions (23, 24). Intriguingly, such Ephexin | RhoGEF | autoinhibition | crystal structure an SH3-mediated autoinhibition mechanism may be also appli- cable to Ephexin1 and Ephexin3 (25). In addition to the ho GTPases are master regulators of cytoskeletal dynamics C-terminal inhibition, several lines of evidence have suggested Rand play pivotal roles in diverse cellular processes, including that an evolutionarily conserved helix preceding the catalytic DH cell polarity, cell motility, cell division, and synaptic signaling – (1 3). Typically, Rho GTPases function as molecular switches Significance that cycle between an active guanosine triphosphate (GTP)- bound form and an inactive guanosine diphosphate (GDP)- Ephexin family guanine nucleotide exchange factors (GEFs) act bound form. Upon activation, they interact with a wide range of downstream of Eph signaling to regulate cytoskeletal dynam- downstream effectors, such as actin cytoskeletal regulators, ki- ics, playing critical roles in diverse cellular processes. To achieve nases, and scaffold proteins, to drive essential changes in cyto- precise regulation of Eph signaling, the GEF activities of skeletal architecture necessary for corresponding physiological Ephexins need to be tightly controlled. We here present the functions (4, 5). Rho GTPases are activated by guanine nucle- crystal structures of autoinhibited Ephexin4, which reveal that otide exchange factors (GEFs) and inactivated by GTPase- both N- and C-terminal fragments directly contact with and activating proteins (GAPs) (6, 7). These regulatory proteins inhibit the DH catalytic domain of Ephexin4. This double inhi- are precisely controlled so that Rho GTPase activities are spa- bition mechanism may be commonly utilized by other Ephexins tiotemporally initiated or suppressed in response to various up- and SGEF. Phosphorylation of a conserved tyrosine at its N stream signals from cell-surface receptors, such as integrins, terminus and association of PDZ protein(s) to its C-terminal growth factors, and tyrosine kinase receptors, among others (4). PDZ-binding motif may relieve the autoinhibited Ephexin4. Eph-interacting exchange protein (Ephexin) family RhoGEFs These results reveal versatile autoinhibitory mechanisms that activate Rho GTPases, including RhoA, Rac, Cdc42, and RhoG fine-tune the GEF activities of Ephexin family RhoGEFs. (8). This family consists of five known members in most verte- brate species (Ephexin1 to -5). A common feature of the Author contributions: C.W. and J.Z. designed research; M.Z. and L.L. performed research; Ephexin family proteins is that they all associate with and act C.W. contributed new reagents/analytic tools; M.Z., L.L., C.W., and J.Z. analyzed data; and downstream of Eph receptors, the largest subfamily of tyrosine J.Z. wrote the paper. kinase receptors that are activated by Ephrins and participate in The authors declare no competing interest. various cellular processes (8–11). Specifically, Ephexin1 (also This article is a PNAS Direct Submission. A.W. is a guest editor invited by the known as NGEF or ARHGEF27) regulates axon growth cone Editorial Board. dynamics and spine morphogenesis via binding to EphA4 and Published under the PNAS license. activation of RhoA (9, 12–14). Ephexin4 (also named as ARH- 1M.Z. and L.L. contributed equally to this work. GEF16) activates RhoG by interacting with EphA2, which pro- 2To whom correspondence may be addressed. Email: [email protected] or jinwei. motes RhoG/ELMO/DOCK/Rac signaling and regulates cell [email protected]. migration (15, 16). Ephexin5 (also known as Vsm-RhoGEF or This article contains supporting information online at https://www.pnas.org/lookup/suppl/ ARHGEF15) functions together with EphB2 to regulate excit- doi:10.1073/pnas.2024465118/-/DCSupplemental. atory synapse development (17). Notably, the biological Published February 17, 2021. PNAS 2021 Vol. 118 No. 8 e2024465118 https://doi.org/10.1073/pnas.2024465118 | 1of10 Downloaded at Diane Sullenberger on February 18, 2021 A autoinhibited DPSH 1 261 713 Ephexin4 IH DH PH SH3 HC ETDV IH: Inhibitory helix (ARHGEF16) Type-I PBM DH: Dbl homology SGEF IH DH PH SH3 HC ETNV PH: Pleckstrin homology (ARHGEF26) SH3: Src homology 3 HC: Helix at C-terminus Ephexin1/2/3 IH DH PH SH3 HC PBM: PDZ-binding motif Ephexin5 IH DH PH BCHC SH3 HC SH3 PH PH q DH 180 DH D RhoA E RhoG binding site PH C SH3 H DH PBM PH DH Ephexin4 DH-PH -SH3 -HC autoinhibited Ephexin4 DPSH ARHGEF11 DH-PH/RhoA (PDB code: 1XCG) Fig. 1. Crystal structure of Ephexin4DPSH.(A) Schematic diagrams showing the conserved domain organizations of Ephexins and SGEF. The PBM sequences of Ephexin4 and SGEF are shown. The domain color coding is consistent throughout this paper. The domain keys are also shown here. (B) Ribbon diagram representation of the Ephexin4DPSH structure. (C) Surface representation showing the overall architecture of the Ephexin4DPSH.(D) Superposition of ARHGEF11 DH-PH/RhoA (PDB ID code: 1XCG) and the Ephexin4DPSH (this study) structures. (E) Schematic representation of autoinhibited Ephexin4DPSH. The circle indicates the DH active site. domain (referred to as inhibitory helix [IH]) (Fig. 1A) also Ephexin4DH-end fragment (amino acids 261 to 713) (Fig. 1A). DH-end contributes to the inhibitory functions in Ephexin1 to -3, by Ephexin4 was crystallized in the P3112 space group with binding directly to DH (9, 25, 26). Phosphorylation of a con- four molecules in the asymmetric unit. Single-wavelength anomalous served tyrosine residue within the IH could potentially relieve dispersion (SAD) data were collected using selenomethionine- the autoinhibition and activate Ephexin GEF activity (9, 25). substituted protein crystals (Table 1). The structure was determined However, the molecular basis underlying these autoinhibition and refined at 2.39 Å resolution (Table 1 and Fig. 1 B and C). and activation events is not well understood. The topic of In the structure, Ephexin4DH-end consists of four major struc- whether a common regulatory mechanism is shared by all tural elements: DH, PH, SH3, and a previously undefined α-helix Ephexins remains an intriguing and potentially informative as- C-terminal to the SH3 domain (amino acids 693 to 707; referred pect of Rho GTPase biology. to as HC; this four-element fragment is hereafter abbreviated as In the present study, we report crystal structures of Ephexin4 Ephexin4DPSH) (Fig. 1B). HC binds tightly to SH3, forming the in its partially and fully
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