The mechanism of activation of the actin binding protein EHBP1 by Rab8 family members

Amrita Rai,Ingrid R. Vetter,Roger S. Goody,Nathalie Bleimling

doi:10.1038/s41467-020-17792-3

Amrita Rai, Ingrid R. Vetter + Show 2 more

Open Access

https://doi.org/10.1038/s41467-020-17792-3

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Abstract

EHBP1 is an adaptor protein that regulates vesicular trafficking by recruiting Rab8 family members and Eps15-homology domain-containing proteins 1/2 (EHD1/2). It also links endosomes to the actin cytoskeleton. However, the underlying molecular mechanism of activation of EHBP1 actin-binding activity is unclear. Here, we show that both termini of EHBP1 have membrane targeting potential. EHBP1 associates with PI(3)P, PI(5)P, and phosphatidylserine via its N-terminal C2 domain. We show that in the absence of Rab8 family members, the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domain forms an intramolecular complex with its central calponin homology (CH) domain and auto-inhibits actin binding. Rab8 binding to the bMERB domain relieves this inhibition. We have analyzed the CH:bMERB auto-inhibited complex and the active bMERB:Rab8 complex biochemically and structurally. Together with structure-based mutational studies, this explains how binding of Rab8 frees the CH domain and allows it to interact with the actin cytoskeleton, leading to membrane tubulation.

Highlights

EHBP1 is an adaptor protein that regulates vesicular trafficking by recruiting Rab[8] family members and Eps15-homology domain-containing proteins 1/2 (EHD1/2)
Our biochemical and structural data suggest that the interaction between the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domain and the central calponin homology (CH) domain has a regulatory role in the function of EHBP1 and binding of Rab[8] family members to the bMERB domain releases the CH domain, which can interact with the actin cytoskeleton
EHBP1 consists of an N-terminal C2 domain, a central CH, a C-terminal bMERB domain, and a CaaX box at the C-terminus that is a substrate for FTase[4]

Summary

Introduction

EHBP1 is an adaptor protein that regulates vesicular trafficking by recruiting Rab[8] family members and Eps15-homology domain-containing proteins 1/2 (EHD1/2). We show that in the absence of Rab[8] family members, the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domain forms an intramolecular complex with its central calponin homology (CH) domain and auto-inhibits actin binding. 1234567890():,; EHBP1 was originally identified as an Eps15-homology domain-containing protein 1/2 (EHD1/2) interacting partner that plays a central role in GLUT4 transport and couples endocytic vesicles to the actin cytoskeleton[1,2]. Our biochemical and structural data suggest that the interaction between the C-terminal bMERB domain and the central CH domain has a regulatory role in the function of EHBP1 and binding of Rab[8] family members to the bMERB domain releases the CH domain, which can interact with the actin cytoskeleton

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