The structure of the Vps27/Hse1 core fills the last major gap in our understanding of the organization of this complex, and led us to several unexpected observations. First, the region involved in forming the core is more extensive than anticipated and extends beyond the predicted coiled-coil regions in both the N- and C-terminal directions. Second, the core comprises two GAT domains that had not previously been identified in these proteins. Third, the core assembles by the interchange of the homologous C-terminal halves of the α3 helices from each GAT domain. In addition, the resolution of this structure has allowed us to model its function as a scaffold for ubiquitin-binding and ubiquitination-deubiquitination reactions at the endosomal membrane.
The interchange of homologous α3 C-terminal halves from Vps27 and Hse1 is reminiscent of the mechanism of “domain swapping”. As originally defined, domain swapping refers to the oligomerization of identical protomers by interchange of identical regions of subunits (Liu and Eisenberg, 2002
). The assembly of the Vps27/Hse1 core seems to us structurally and functionally equivalent to domain swapping in every respect other than the sequence identity of the exchanged regions.
In order to judge whether the domain-swapped complex represents the bona fide
assembly mechanism for Vps27 and Hse1 in vivo
, we analyzed the structure in the light of mutational analysis in the literature, and carried out additional mutational studies of complex formation. Residues 416-418 of Vps27 (sequence KIS) were critical for complex formation with Hse1 (Bilodeau et al., 2003
). Vps27 Ile-417 within this sequence is deeply buried and almost completely surrounded by hydrophobic residues from Hse1 (), consistent with a critical role in function. We mutated Vps27 residues Leu-410, Ile-417, and Ile-420 individually to Asp, and found that these mutations prevented formation of the complex with Hse1. For each cargo tested, Cps1, Ste3 and CPY, these mutations resulted in a loss of function mirroring that seen in the deletion of Hse1. This establishes that the protein:protein interface observed in the structure is responsible for the assembly of the Vps27/Hse1 complex in yeast.
Despite the fact that Vps27 and Hse1 are subunits of a tightly assembled heterodimer, deletion of the gene encoding each subunit results in a quantitatively different defect in cargo sorting. Deletion of VPS27
causes a much stronger CPY missorting phenotype than deletion of HSE1
(Bilodeau et al., 2002
) (see also ). The missorting of Cps1 and Ste3 is also more severe in VPS27
- than HSE1
-deletion mutants (Bilodeau et al., 2002
disruption phenotypes are more manifest in certain genetic backgrounds, like that of the SF8389D yeast strain used in these studies (Bilodeau et al., 2002
). Our observations shed light on the probable cause for these phenotypic differences. Although the Vps27 core domain prefers to assemble as a heterodimer with the Hse1 core domain, it is nonetheless stable as a monomer when expressed in the absence of the Hse1 core domain. This is likely due to its ability to form an intramolecular GAT fold. In contrast, the Hse1 core domain expressed in isolation tends to aggregate and be degraded. Deletion of the VPS27
gene may thus lead to loss of both the Vps27 and Hse1 proteins, whereas deletion of the HSE1
gene would still leave enough Vps27 protein to sustain a modicum of function. In addition, Vps27 contains the main determinant of attachment of the complex to membranes, the FYVE domain, such that monomeric Hse1 is likely incapable of efficient recruitment to endosomes. Conversely, monomeric Vps27 could bind to membranes independently of Hse1, thus bringing its ubiquitin-, ESCRT-I- and clathrin-binding activities to bear on MVB sorting. Finally, the MC simulations show that the two UIM domains of Vps27 are closer to the membrane and exhibit more cooperativity than the single UIM domain of Hse1.
The human Hrs/STAM complex has been intensively studied, but the structural basis for its assembly remains unknown. The significant sequence homology between STAM, Hse1, and Vps27 allows us to predict that the core region of STAM will adopt the same structural fold as Vps27 and Hse1. The so-called SSM, which is needed for Hrs/STAM complex formation (Mizuno et al., 2004
), corresponds to the C-terminal half of helix α1 and a few residues immediately following α1. Several of the conserved residues in the SSM correspond to key hydrophobic anchor residues in the subunit interface. The sequence of the core region of Hrs diverges from those of Vps27, Hse1, and STAM. However, the region of Hrs corresponding to the Vps27 GAT domain is predicted to be α-helical. Further, the example of Vps27 and Hse1 suggests that the STAM GAT domain requires a complementary GAT domain in Hrs with which to associate.
The unexpected observation of GAT domains in Vps27 and Hse1 highlights the parallel roles of these proteins with other GAT-domain-containing trafficking adaptors, the GGAs and Tom1 and Tom1-like proteins. The GGAs are modular proteins that contain a receptor-binding VHS domain, an Arf-binding helical hairpin domain, a ubiquitin-, Rabex-5, and ESCRT-I-binding GAT domain, an unstructured region containing autoinhibitory and clathrin-binding domains, and a GAE domain that binds to various accessory proteins (Bonifacino, 2004
). Tom1 and its relatives Tom1L1 and Tom1L2 have a similar modular structure to the GGAs (), and bind to ubiquitin via their GAT domains (Katoh et al., 2004
; Yamakami et al., 2003
) and to ESCRT-I (Puertollano, 2005
). Collectively, the GGAs, Tom1 and the Tom1-like proteins, and the Vps27/Hse1 and Hrs/STAM complexes are a class of endosomal clathrin-binding proteins that sort ubiquitinated cargo proteins into the ESCRT pathway (Raiborg et al., 2006
). These similarities highlight the GAT domain proteins collectively () as a family of proteins that sort ubiquitinated cargo into the ESCRT system.
The GAT domains of GGA1, GGA3, and Tom1 bind ubiquitin (Katoh et al., 2004
; Puertollano and Bonifacino, 2004
; Scott et al., 2004
; Shiba et al., 2004
) with affinities ranging from 180-410 μM (Akutsu et al., 2005
; Kawasaki et al., 2005
; Prag et al., 2005
). However, no ubiquitin binding to the Vps27/Hse1 core was detected by isothermal titration calorimetry or surface plasmon resonance at concentrations of up to 8.0 mM and 2.0 mM, respectively (data not shown). Known ubiquitin-binding GAT domains contain two ubiquitin binding sites. Ubiquitin binds to the GGA and Tom1 GAT domains at site 1 on helices α1 and α2, and site 2 on helices α2 and α3. These sites are incompletely conserved in Vps27 and Hse1 (). Unlike Vps27 and Hse1, the GGAs and Tom1 do not contain ubiquitin-binding UIM motifs. If the family of GAT-domain containing adaptors evolved from a common ancestor, the GAT domains have served multiple purposes in ubiquitin binding, dimerization, and other functions. By the same token, different ubiquitin-binding, GAT domain-containing proteins acquired different mechanisms for binding ubiquitinated cargo, some binding through the GAT domain, and others via their UIMs.
The structures and the ubiquitin and membrane affinities of individual domains from the Vps27/Hse1 and Hrs/STAM complexes are known (Diraviyam et al., 2003
; Fisher et al., 2003
; Hirano et al., 2006
; Kaneko et al., 2003
; Mao et al., 2000
; Misra and Hurley, 1999
; Stahelin et al., 2002
; Swanson et al., 2003
). Despite a wealth of information on individual domains, it has not been possible to integrate this knowledge into a unified model of the Vps27/Hse1 complex. The structure determination of the Vps27/Hse1 core provides the missing link that allows the integration of the domain information for the first time. The simulations show that cooperativity between the Vps27 and FYVE and UIM domains in membrane and ubiquitinated membrane protein binding offsets steric constraints imposed in the complex. The simulations portray the complex as open and dynamic. Vps27/Hse1 traffics a variety of ubiquitinated cargo. The molecular weight, the size of the cytosolic domain, and the location of the ubiquitination sites on these cargoes vary widely. An open, dynamic complex such as Vps27/Hse1 can adapt to these differences in cargo in ways that a rigid complex could not. Finally, the simulations show that the Hse1 SH3 domain, which targets DUBs that potentially deubiquitinate cargo, samples conformational space that frequently approaches within 20 Å of ubiquitinated cargo. This suggests that the action of the Hse1 UIM and SH3 domains coordinates ubiquitinated cargo binding and deubiquitination reactions.
A model has been proposed for Hrs, based on a 16 Å resolution cryo-EM structure of an Hrs hexamer determined in the absence of STAM (Pullan et al., 2006
). In the Hrs model, three sets of membrane and ubiquitin binding domains are located 175 Å apart from each other at two sets of end caps. It is difficult to compare these models given the substantial differences in the Hrs and Vps27 core sequences and the presence of a 1:1 heterodimer in one structure vs. a homohexamer in the other.
We have visualized the core of the Vps27/Hse1 complex, which uses an elegant variation on the GAT domain, not to bind ubiquitin directly, but instead to coordinate ubiquitinated cargo binding, and ubiquitination and deubiquitination reactions. The structure of the Vps27/Hse1 complex shows how the complex can spatially confine ubiquitinated cargo and coordinate the action of DUBs (e.g., Ubp7) against a tightly localized subpopulation of substrate. Such a mechanism could help account for the biological specificity of this deubiquitinating enzyme, given the large number of potential substrates in the cell. Most importantly, the structure of the Vps27/Hse1 core complex has provided us with a unifying framework for understanding the integrated action of the many modular domains of these two proteins.