PDBsum entry 3eie

Protein transport

PDB id: 3eie

Contents

Protein chain

303 a.a. *

Ligands

SO4 ×2

Waters ×35

* Residue conservation analysis

PDB id:

3eie

Name:

Protein transport

Title:

Crystal structure of s.Cerevisiae vps4 in the so4-bound state

Structure:

Vacuolar protein sorting-associated protein 4. Chain: a. Fragment: unp residues 122-437. Synonym: protein end13, doa4-independent degradation protein 6, vacuolar protein-targeting protein 10. Engineered: yes. Mutation: yes

Source:

Saccharomyces cerevisiae. Baker's yeast, yeast. Organism_taxid: 4932. Gene: vps4, csc1, did6, end13, grd13, vpl4, vpt10, ypr173c, p9705.10. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.70Å R-factor: 0.244 R-free: 0.287

Authors:

M.D.Gonciarz,F.G.Whitby,D.M.Eckert,C.Kieffer,A.Heroux,W.I.Sundquist, C.P.Hill

Key ref:

M.D.Gonciarz et al. (2008). Biochemical and structural studies of yeast Vps4 oligomerization. J Mol Biol, 384, 878-895. PubMed id: 18929572 DOI: 10.1016/j.jmb.2008.09.066

Date:

15-Sep-08 Release date: 30-Sep-08

Protein chain

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

Seq: 437 a.a.

Struc: 303 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

DOI no: 10.1016/j.jmb.2008.09.066

J Mol Biol 384:878-895 (2008)

PubMed id: 18929572

Biochemical and structural studies of yeast Vps4 oligomerization.

M.D.Gonciarz, F.G.Whitby, D.M.Eckert, C.Kieffer, A.Heroux, W.I.Sundquist, C.P.Hill.

ABSTRACT

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 ATPgammaS [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.

Selected figure(s)

Figure 2.
Fig. 2. Vps4 nucleotide binding pockets. (a) Stereo view of the Vps4–ATPγS nucleotide binding site of molecule A in crystal form 2. Active-site Vps4 residues are color coded according to their functional roles in Mg^2+coordination/ATP hydrolysis (cyan, S180, D232, and E/Q233), adenine ring stacking (magenta, Y181 and M307), and phosphate sensing (yellow, K179 and N277) with the “arginine finger” residue R288 from an adjacent molecule in the modeled hexamer shown in green. Note that the E233Q mutant was used here and throughout to allow ATP/ATPγS binding while inhibiting hydrolysis. (b) Electron density for the ATPγS nucleotides in molecule A of Vps4[ΔMIT] crystal form 2. The densities show (F[o] – F[c]) omit maps contoured at 2.5σ.

Figure 5.
Fig. 5. Mutational analyses of crystallographic Vps4 dimer interfaces. (a) Crystallographic Vps4 dimer interfaces. Interface 1 is a symmetric interface between two large ATPase domains (residue Q216 is shown in cyan), interface 2 is a symmetric interface between two small ATPase domains (residue L407 is shown in blue), and interface 6 is an asymmetric interface between the large and small ATPase domains (residues L151 and W388 are shown in orange and green, respectively). (b) Gel-filtration chromatograms of Vps4[ΔMIT] proteins with the following mutations: Q216A (interface 1), L407D (interface 2), L151D (interface 6), and W388A (interface 6). Vps4[ΔMIT] proteins used here and elsewhere contained the E233Q mutation, which allowed ATP binding but inhibited hydrolysis. For reference, the elution profile of the “wild-type” Vps4[ΔMIT],[E233Q] protein is shown in red in each panel, elution positions for monomeric (1) and dimeric (2) proteins are shown as dotted vertical lines, and the elution positions of molecular weight standards are shown below the chromatograms. Vps4 protein concentrations were 150 μM in all cases. Note that at low micromolar concentrations, the dimeric proteins exhibited concentration-dependent mobilities (not shown), indicating that appreciable concentrations of monomers could accumulate under these low-protein and nonequilibrium conditions.

The above figures are reprinted from an Open Access publication published by Elsevier: J Mol Biol (2008, 384, 878-895) copyright 2008.

Figures were selected by an automated process.