PDBsum entry 2qp9

Protein transport

PDB id: 2qp9

Contents

Protein chain

288 a.a. *

Ligands

SO4

Metals

_CD ×2

* Residue conservation analysis

PDB id:

2qp9

Name:

Protein transport

Title:

Crystal structure of s.Cerevisiae vps4

Structure:

Vacuolar protein sorting-associated protein 4. Chain: x. Fragment: residues: 83-437. Synonym: protein end13, doa4-independent degradation protein 6, vacuolar protein-targeting protein 10. Engineered: yes. Mutation: yes

Source:

Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Gene: vps4, csc1, did6, end13, grd13, vpl4, vpt10. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.90Å R-factor: 0.260 R-free: 0.288

Authors:

J.Xiao,Z.Xu

Key ref:

J.Xiao et al. (2007). Structural characterization of the ATPase reaction cycle of endosomal AAA protein Vps4. J Mol Biol, 374, 655-670. PubMed id: 17949747 DOI: 10.1016/j.jmb.2007.09.067

Date:

23-Jul-07 Release date: 09-Oct-07

Protein chain

P52917  (VPS4_YEAST) -  Vacuolar protein sorting-associated protein 4 from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 437 a.a.

Struc: 288 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

DOI no: 10.1016/j.jmb.2007.09.067

J Mol Biol 374:655-670 (2007)

PubMed id: 17949747

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

J.Xiao, H.Xia, K.Yoshino-Koh, J.Zhou, Z.Xu.

ABSTRACT

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.

Selected figure(s)

Figure 5.
Fig. 5. Structural flexibility within Vps4. (a) Superimposition of the four independently determined Vps4 structures in the current study. The three molecules (ADP-bound) in the asymmetric unit of the P2[1]2[1]2[1] space group are colored white, cyan, and red, respectively. The one molecule (nucleotide-free) in the asymmetric unit of the P6[5]22 space group is colored yellow. The magnitude of the en bloc motions between the large AAA subdomain and small AAA subdomain and between the small AAA subdomain and the β domain is illustrated schematically. (b) Two potential hinge regions within Vps4 structure. The conserved proline residues in the hinge regions are shown as spheres and highlighted in brown.

Figure 6.
Fig. 6. Nucleotide binding induces conformational change within the N-terminal region of Vps4. (a) The ESCRT-III subunits interact with Vps4 in the presence of ATP. ESCRT-III subunits Vps2, Vps20, Vps24 and Snaf7 were expressed as GST-tagged fusion proteins and bound to glutathione–agarose beads (left panel). Purified Vps4^E233Q was loaded onto the ESCRT-III subunit-bound matrix either in the absence (−) or presence (+) of ATP. Proteins retained on the matrix after extensive washes were separated on 12% SDS-PAGE gel and stained with Coomassie blue (right panel). (b) The N-terminal domain of Vps4 interacts with the ESCRT-III subunits. Vps2 and Vps20 were expressed as GST-tagged fusion proteins and bound to glutathione–agarose beads. Cell lysate containing His[8]-Vps4^1–82 or His[8]-Vps4^1–120 was loaded onto GST-Vps2-or GST-Vps20-bound matrix. Proteins retained on the matrix after extensive washes were separated on the SDS-PAGE gel and detected by either Ponceau S staining (top panel) or anti-His antibody (bottom panel). (c) Vps4^1–120 competes with full-length Vps4 for binding to the ESCRT-III subunit Vps2. GST-Vps2 was bound to glutathione–agarose beads. Purified Vps4^E233Q was loaded onto Vps2-bound matrix in the presence of ATP and increasing amounts of bovine serum albumin or Vps4^1–120. Proteins retained on the matrix were separated on SDS-PAGE gel and detected by Western blotting with anti-Vps4 antibody. The amount of Vps4^E233Q was quantified by program ImageJ and shown in a bar diagram (the amount in the first lane was set as 100%). (d) Vps4 undergoes conformational change at the linker region upon ATP binding. Vps4^E233Q was incubated with increasing amounts of subtilisin at 4 °C for 30 min with different nucleotides. Digestion products were separated on 15% SDS-PAGE, followed by Coomassie staining.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 374, 655-670) copyright 2007.

Figures were selected by an automated process.