Structural Organization and Dynamics of Homodimeric Cytohesin Family Arf GTPase Exchange Factors in Solution and on Membranes

Sanchaita Das,Andrew W Malaby,Agata Nawrotek,Wenhua Zhang,Mahel Zeghouf,Sarah Maslen,Mark Skehel,Srinivas Chakravarthy,Thomas C Irving,Osman Bilsel,Jacqueline Cherfils,David G Lambright

doi:10.1016/j.str.2019.09.007

Abstract

SummaryMembrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through Arf GTPase activation on the plasma membrane and endosomes. In this study, the structural organization of two cytohesins (Grp1 and ARNO) was investigated in solution by size exclusion-small angle X-ray scattering and negative stain-electron microscopy and on membranes by dynamic light scattering, hydrogen-deuterium exchange-mass spectrometry and guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange assays. The results suggest that cytohesins form elongated dimers with a central coiled coil and membrane-binding pleckstrin-homology (PH) domains at opposite ends. The dimers display significant conformational heterogeneity, with a preference for compact to intermediate conformations. Phosphoinositide-dependent membrane recruitment is mediated by one PH domain at a time and alters the conformational dynamics to prime allosteric activation by Arf-GTP. A structural model for membrane targeting and allosteric activation of full-length cytohesin dimers is discussed.

Highlights

Arf GTPases play fundamental roles in vesicle biogenesis and membrane dynamics (Donaldson and Honda, 2005; Donaldson and Jackson, 2011; Hashimoto et al, 2004; Muralidharan-Chari et al, 2009; Nie et al, 2003; Sztul et al, 2019)
Activation is controlled by guanine nucleotide exchange factors (GEFs) containing a Sec7 domain, which catalyzes conversion from the inactive guanosine diphosphate (GDP)-bound state to the active guanosine triphosphate (GTP)-bound conformation (Casanova, 2007; Chardin et al, 1996; Cherfils et al, 1998)
Ab initio envelopes for this monomeric construct give a good fit with the crystal structure of autoinhibited Grp1 (DiNitto et al, 2007) (Figures S1E and S1F), indicating that the Sec7 and PH domains of ARNO likely adopt an autoinhibited conformation in solution similar to Grp1

Summary

Introduction

Arf GTPases play fundamental roles in vesicle biogenesis and membrane dynamics (Donaldson and Honda, 2005; Donaldson and Jackson, 2011; Hashimoto et al, 2004; Muralidharan-Chari et al, 2009; Nie et al, 2003; Sztul et al, 2019). Additional domains mediate membrane recruitment through interactions with phosphoinositides, anionic phospholipids, and proteins including active Arf or Arl GTPases (Cherfils and Zeghouf, 2013; DiNitto and Lambright, 2006; Lemmon, 2004; Nawrotek et al, 2016). The Sec7-PH linker and C-terminal helix/polybasic region (CtH/PBR), strongly suppress GEF activity by occluding the active site in the Sec domain (DiNitto et al, 2007). Mutations in either autoinhibitory element increase GEF activity and truncation of the PBR suffices to render cytohesins constitutively active in vitro (DiNitto et al, 2007), albeit with reduced membrane targeting capacity (Nagel et al, 1998). Binding of membrane-associated Arf6-GTP to an allosteric site centered on the PH domain enhances membrane recruitment and relieves autoinhibition by

Results

Discussion

Conclusion