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Updated: May 27, 2026

Comparing the Affinity of GTPase-binding Proteins using Competition Assays
Published on: October 8, 2015
Insights regarding guanine nucleotide exchange from the structure of a DENN-domain protein complexed with its Rab
Xudong Wu1, Michael J Bradley, Yiying Cai
1Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
Abstract:
Rab GTPases are key regulators of membrane traffic pathways within eukaryotic cells. They are specifically activated by guanine nucleotide exchange factors (GEFs), which convert them from their "inactive" GDP-bound form to the "active" GTP-bound form. In higher eukaryotes, proteins containing DENN-domains comprise a major GEF family. Here we describe at 2.1-Å resolution the first structure of a DENN-domain protein, DENND1B-S, complexed with its substrate Rab35, providing novel insights as to how DENN-domain GEFs interact with and activate Rabs. DENND1B-S is bi-lobed, and interactions with Rab35 are through conserved surfaces in both lobes. Rab35 binds via switch regions I and II, around the nucleotide-binding pocket. Positional shifts in Rab residues required for nucleotide binding may lower its affinity for bound GDP, and a conformational change in switch I, which makes the nucleotide-binding pocket more solvent accessible, likely also facilitates exchange.
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