Structural characterization of the ATPase reaction cycle of endosomal AAA protein Vps4

The multivesicular body (MVB) pathway functions in multiple cellular processes including cell surface receptor down-regulation and viral budding from host cells. An important step in the MVB pathway is the correct sorting of cargo molecules, which requires the assembly and disassembly of endosomal sorting complexes required for transport (ESCRTs) on the endosomal membrane. Disassembly of the ESCRTs is catalyzed by ATPase associated with various cellular activities (AAA) protein Vps4. Vps4 contains a single AAA domain and undergoes ATP-dependent quaternary structural change to disassemble the ESCRTs. Structural and biochemical analyses of the Vps4 ATPase reaction cycle are reported here. Crystal structures of Saccharomyces cerevisiae Vps4 in both the nucleotide-free form and the ADP-bound form provide the first structural view illustrating how nucleotide binding might induce conformational changes within Vps4 that lead to oligomerization and binding to its substrate ESCRT-III subunits. In contrast to previous models, characterization of the Vps4 structure now supports a model where the ground state of Vps4 in the ATPase reaction cycle is predominantly a monomer and the activated state is a dodecamer. Comparison with a previously reported human VPS4B structure suggests that Vps4 functions in the MVB pathway via a highly conserved mechanism supported by similar protein-protein interactions during its ATPase reaction cycle.     

Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs)

The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly.     

Biochemical and structural studies of yeast Vps4 oligomerization

The ESCRT (endosomal sorting complexes required for transport) pathway functions in vesicle formation at the multivesicular body, the budding of enveloped RNA viruses such as HIV-1, and the final abscission stage of cytokinesis. As the only known enzyme in the ESCRT pathway, the AAA ATPase (ATPase associated with diverse cellular activities) Vps4 provides the energy required for multiple rounds of vesicle formation. Like other Vps4 proteins, yeast Vps4 cycles through two states: a catalytically inactive disassembled state that we show here is a dimer and a catalytically active higher-order assembly that we have modeled as a dodecamer composed of two stacked hexameric rings. We also report crystal structures of yeast Vps4 proteins in the apo- and ATPγS [adenosine 5′-O-(3-thiotriphosphate)]-bound states. In both cases, Vps4 subunits assembled into continuous helices with 6-fold screw axes that are analogous to helices seen previously in other Vps4 crystal forms. The helices are stabilized by extensive interactions between the large and small AAA ATPase domains of adjacent Vps4 subunits, suggesting that these contact surfaces may be used to build both the catalytically active dodecamer and catalytically inactive dimer. Consistent with this model, we have identified interface mutants that specifically inhibit Vps4 dimerization, dodecamerization, or both. Thus, the Vps4 dimer and dodecamer likely form distinct but overlapping interfaces. Finally, our structural studies have allowed us to model the conformation of a conserved loop (pore loop 2) that is predicted to form an arginine-rich pore at the center of one of the Vps4 hexameric rings. Our mutational analyses demonstrate that pore loop 2 residues Arg241 and Arg251 are required for efficient HIV-1 budding, thereby supporting a role for this “arginine collar” in Vps4 function.     

Structural basis for autoinhibition of ESCRT-III CHMP3

Endosomal sorting complexes required for transport (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III) are selectively recruited to cellular membranes to exert their function in diverse processes, such as multivesicular body biogenesis, enveloped virus budding, and cytokinesis. ESCRT-III is composed of members of the charged multivesicular body protein (CHMP) family—cytosolic proteins that are targeted to membranes via yet unknown signals. Membrane targeting is thought to result in a membrane-associated protein network that presumably acts at a late budding step. Here we provide structural evidence based on small-angle X-ray scattering data that ESCRT-III CHMP3 can adopt two conformations in solution: a closed globular form that most likely represents the cytosolic conformation and an open extended conformation that might represent the activated form of CHMP3. Both the closed and open conformations of CHMP3 interact with AMSH with high affinity. Although the C-terminal region of CHMP3 is required for AMSH interaction, a peptide thereof reveals only weak binding to AMSH, suggesting that other regions of CHMP3 contribute to the high-affinity interaction. Thus, AMSH, including its MIT (microtubule interacting and transport) domain, interacts with ESCRT-III CHMP3 differently from reported Vps4 MIT domain-CHMP protein interactions.     

Solution structure of UIM and interaction of tandem ubiquitin binding domains in STAM1 with ubiquitin

STAM1 and Hrs are the components of ESCRT-0 complex for lysosomal degradation of membrane proteins is composed of STAM1 Hrs and has multiple ubiquitin binding domains. Here, the solution structure of STAM1 UIM, one of the ubiquitin binding motif, was determined by NMR spectroscopy. The structure of UIM adopts an α-helix with amphipathic nature. The central hydrophobic residues in UIM provides the binding surface for ubiquitin binding and are flanked with positively and negatively charged residues on both sides. The docking model of STAM1 UIM-ubiquitin complex is suggested. In NMR and ITC experiments with the specifically designed mutant proteins, we investigated the ubiquitin interaction of tandem ubiquitin binding domains from STAM1. The ubiquitin binding affinity of the VHS domain and UIM in STAM1 was 52.4 and 94.9 μM, and 1.5 and 2.2 fold increased, respectively, than the value obtained from the isolated domain or peptide. The binding affinities here would be more physiologically relevant and provide more precise understanding in ESCRT pathway of lysosomal degradation.     

Competitive binding of UBPY and ubiquitin to the STAM2 SH3 domain revealed by NMR

To date, the signal transducing adaptor molecule 2 (STAM2) was shown to harbour two ubiquitin binding domains (UBDs) known as the VHS and UIM domains, while the SH3 domain of STAM2 was reported to interact with deubiquitinating enzymes (DUBs) like UBPY and AMSH. In the present study, NMR evidences the interaction of the STAM2 SH3 domain with ubiquitin, demonstrating that SH3 constitutes the third UBD of STAM2. Furthermore, we show that a UBPY-derived peptide can outcompete ubiquitin for SH3 binding and vice versa. These results suggest that the SH3 domain of STAM2 plays versatile roles in the context of ubiquitin mediated receptor sorting.     

Identification of a novel ubiquitin binding site of STAM1 VHS domain by NMR spectroscopy

Interaction between the signal-transducing adapter molecule 1 (STAM1) Vps27/Hrs/Stam (VHS) domain and ubiquitin was investigated by nuclear magnetic resonance (NMR) spectroscopy. NMR evidence showed that the structure of STAM1 VHS domain resembles that of other VHS domains, especially the homologous domain of STAM2. We found that the VHS domain binds to ubiquitin via its hydrophobic patch consisting of N-terminus of helix 2 and C-terminus of helix 4 in which Trp26 on helix 2 plays a pivotal role in the binding. The binding between VHS and ubiquitin seems to be very similar to that between ubiquitin associated domain (UBA) and ubiquitin, however, the direction of α-helices involved in the ubiquitin binding is opposite. Here, we propose a novel ubiquitin binding site and the manner of ubiquitin recognition of the STAM1 VHS domain.

Structured summary

MINT-6804185:STAM1 (uniprotkb:Q92783) binds (MI:0407) to ubiquitin (uniprotkb:P62988) by nuclear magnetic resonance (MI:0077)     

Validity and applicability of membrane model systems for studying interactions of peripheral membrane proteins with lipids

The cell membrane serves, at the same time, both as a barrier that segregates as well as a functional layer that facilitates selective communication. It is characterized as much by the complexity of its components as by the myriad of signaling process that it supports. And, herein lays the problems in its study and understanding of its behavior — it has a complex and dynamic nature that is further entangled by the fact that many events are both temporal and transient in their nature. Model membrane systems that bypass cellular complexity and compositional diversity have tremendously accelerated our understanding of the mechanisms and biological consequences of lipid–lipid and protein–lipid interactions. Concurrently, in some cases, the validity and applicability of model membrane systems are tarnished by inherent methodical limitations as well as undefined quality criteria. In this review we introduce membrane model systems widely used to study protein–lipid interactions in the context of key parameters of the membrane that govern lipid availability for peripheral membrane proteins. This article is part of a Special Issue entitled Tools to study lipid functions.     

The PI3 Kinase Complex II–PI3P–Vps27 Axis on Vacuolar Membranes is Critical for Microautophagy Induction and Nutrient Stress Adaptation

Phosphatidylinositol 3-phosphate (PI3P), a scaffold of membrane-associated proteins required for diverse cellular events, is produced by Vps34-containing phosphatidylinositol 3-kinase (PI3K). PI3K complex I (PI3KCI)-generated PI3P is required for macroautophagy, whereas PI3K complex II (PI3KCII)-generated PI3P is required for endosomal sorting complex required for transport (ESCRT)-mediated multi-vesicular body (MVB) formation in late endosomes. ESCRT also promotes vacuolar membrane remodeling in microautophagy after nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase in budding yeast. Whereas PI3KCI and macroautophagy are critical for the nutrient starvation response, the physiological roles of PI3KCII and microautophagy during starvation are largely unknown. Here, we showed that PI3KCII-produced PI3P on vacuolar membranes is required for microautophagy induction and survival in nutrient-stressed conditions. PI3KCII is required for Vps27 (an ESCRT-0 component) recruitment and ESCRT-0 complex formation on vacuolar surfaces after TORC1 inactivation. Forced recruitment of Vps27 onto vacuolar membranes rescued the defect in microautophagy induction in PI3KCII-deficient cells, indicating that a critical role of PI3P on microautophagy induction is Vps27 recruitment onto vacuolar surfaces. Finally, vacuolar membrane-associated Vps27 was able to recover survival during nutrient starvation in cells lacking PI3KCII or Vps27. This study revealed that the PI3KCII–PI3P–Vps27 axis on vacuolar membranes is critical for ESCRT-mediated microautophagy induction and nutrient stress adaptation.