Insights regarding guanine nucleotide exchange from the structure of a DENN-domain protein complexed with its Rab GTPase substrate

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.I n eukaryotic cells, material is transported between membranebound organelles or the plasma membrane by vesicles. These bud from the membrane of the donor organelle, travel to and are recognized at the target compartment, and finally fuse with it to deliver their cargo. Vesicle traffic is orchestrated by a large number of proteins, including coat proteins that facilitate budding, tethering complexes involved in vesicle recognition, SNARE proteins that drive membrane fusion, and small GTPases in the Rab/Ypt family that have regulatory roles (1). Different subsets of these proteins are involved in different transport pathways.Rab GTPases are key determinants of organelle identity and hence in ensuring that vesicular cargo is delivered to the correct destination (2-4). The Rabs themselves are activated at the appropriate membranes by Rab-specific guanine nucleotide exchange factors (GEFs), which facilitate the conversion of the Rab from its inactive, GDP-bound, to the active, GTP-bound state. The GEF interacts with its Rab substrate to lower its nucleotide-binding affinity, accelerating the departure of bound GDP and allowing GTP, which is 10-fold more abundant in the cell than GDP, to bind (5, 6). In their GTP-bound membrane associated form, Rabs then recruit additional proteins to mediate vesicle recognition, tethering, and fusion events.Mammals have more than 60 different Rabs (7), with GEFs that activate most of these yet to be identified. Recent data suggest that a large subset of mammalian Rabs, or at least 10 different Rab GTPases, are activated by proteins containing a DENN domain (8). In humans, eighteen different DENN-domain proteins have been identified, including members of the DENND1 through DENND5 families, the myotubularin-related proteins MTMR5 and 13, and the MAP-kinase activating death domain MADD (8, 9). Studies of the DENND1 family have shown that the DENN domain itself is sufficient for GEF activity (10, 11), where both DENND1A and 1B activate Rab35 for its role in the endocytic pathway (8, 10, 11). The substrate specificity of DENND1C rema...

Rab Proteins and the Compartmentalization of the Endosomal System

“…Studies in the yeast secretory pathway have shown that Rab GTPases can be sequentially activated via GEF/effector complexes (Ortiz et al 2002). A subset of GEFs could be identified via bioinformatics approaches based on the presence of a conserved DENN domain (Yoshimura et al 2010;Wu et al 2011); however, there are GEF family members that are structurally distinct (Esters et al 2001;Guo et al 2013). Clearly, more factors such as GEFs, GAPs, and effectors coupling the activities of individual and sequentially acting Rab proteins need to be identified and characterized to fully understand how cargo flows between compartments on the endocytic pathway and endocytosis is coordinately up-regulated to address physiological demands.…”

“…First, Rab GTPases undergo cycles of GTP binding and hydrolysis to GDP, which serves to drive a reversible conformational change that is decoded by interacting proteins (Wittinghofer et al 1993). Guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) catalyze the exchange and hydrolysis reactions and, therefore, act as regulators of the GTP-GDP cycle (Yoshimura et al 2010;Wu et al 2011;Barr 2013;Guo et al 2013;Kotting and Gerwert 2013). Second, Rab GTPases behave both as soluble and specifically localized, integral-membrane proteins.…”

Multiple Types of Guanine Nucleotide Exchange Factors (GEFs) for Rab Small GTPases

“…The interaction between a nucleotide-free Rab and a GEF, which is an intermediate step in the activation process, is usually stable, and thus allows their crystal structures to be determined. Thus far, at least five crystal structures of Rab-GEF complexes, i.e., of Rabex5-Rab21, Sec2p-Sec4p, Rabin8-Rab8, TRAPP I-Ypt1p, and DENND1B-Rab35, have been determined, and they provide the structural basis for Rab activation by these GEFs (Delprato and Lambright, 2007;Dong et al, 2007;Sato et al, 2007;Cai et al, 2008b;Wu et al, 2011;Guo et al, 2013). The stable interaction between GEFs and their substrate Rabs can be applied to identifying the substrate specificity of GEFs, e.g., the Rab21-specific GEF Varp specifically interacts with a constitutively negative form of Rab21 and does not interact with constitutively negative forms of 59 other Rabs (Tamura et al, 2009;Mori et al, 2013).…”

“…It is therefore possible that such uncharacterized longin proteins (e.g., DENN-related protein; see next section) function as In general, a GDP-bound form of Rab is present in the cytosol through interaction with GDI (top). A GEF, a Rab activation enzyme, forms a high-affinity complex with a nucleotide-free form of the Rab (bottom right), and such complexes are often used for crystallographic analysis because of their stability (Delprato and Lambright, 2007;Dong et al, 2007;Sato et al, 2007;Cai et al, 2008b;Wu et al, 2011;Guo et al, 2013). When the Rab binds to GTP, the GEF is released from the Rab (bottom left).…”

“…A subsequent bioinformatic analysis has revealed the existence of a number of DENN homologous (referred to as "classical DENN proteins") in mammals, and their homologous regions (i.e., DENN domains) are divided into three subdomains, upstream DENN (u-DENN), core DENN (c-DENN), and downstream DENN (d-DENN) (Levivier et al, 2001). The crystal structure of DENND1B has demonstrated that the u-DENN domain and an N-terminal region of the c-DENN domain constitute a longin fold, which contributes an interaction surface for its substrate Rab35 (Wu et al, 2011). Interestingly, DENN-related domains, whose secondary and 3-dimensional structures are quite similar to classical DENN domains despite the two domains showing little conservation at the primary sequence level, have also been identified in several DENN-related proteins (Table I) (Nookala et al, 2012;Zhang et al, 2012;Levine et al, 2013a).…”