Saturday, 16 June 2012

Conformational changes in the AAA ATPase p97–p47 adaptor complex by Beuron


The AAA+ATPase p97/VCP, helped by adaptor proteins, exerts its essential role in cellular events such as endoplasmic reticulum-associated protein degradation or the reassembly of Golgi, ER and the nuclear envelope after mitosis. Here, we report the three-dimensional cryo-electron microscopy structures at approx20 Å resolution in two nucleotide states of the endogenous hexameric p97 in complex with a recombinant p47 trimer, one of the major p97 adaptor proteins involved in membrane fusion. Depending on the nucleotide state, we observe the p47 trimer to be in two distinct arrangements on top of the p97 hexamer. By combining the EM data with NMR and other biophysical measurements, we propose a model of ATP-dependent p97(N) domain motions that lead to a rearrangement of p47 domains, which could result in the disassembly of target protein complexes.
  • Keywords:

    • AAA ATPase, 
    • conformational change, 
    • Cryo-EM, 
    • p47, 
    • p97

INTRODUCTION

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  • p 97 belongs to AAA+ family of proteins
  • AAA+ proteins have 1 or 2 conserved AAA domains (D1 and D2 domains)
  • contain Walker A and B motifs
  • AAA+ proteins are oligomeric
  • p97 binds Ufd-Np41 adaptor complex
  • mediate events during ER-associated degradation(ERAD), nuclear envelope formation and spindle disassembly after mitosis
  • p07 functions in membrane fusion
         --- use p47 adaptor protein to mediate reformation of Golgi membranes
         --- and reassembly of nuclear envelope at end of mitosis
  • p97=p47 complex is proposed to disassemble t-t SNARE
  • during golgi regrowth, it is proposed p47 mediates binding of p97 to t-SNARE syntaxin 5 post fusion
  • Add protein, VCIP 135 binds p97-p47 syntaxin 5 complex
  • dissoc it using ATP hydrolysis events in p97
  • prepare syntaxin 5 for further rounds of membrane fusion
  • NSF–alpha-SNAP complexes and the p97–p47–VCIP135 complexes were shown to act sequentially in cell cycle-dependent reformation of the ER network 
  • involve t-SNARE syntaxin 18
  • p47 binds ubiquitin via its N terminal UBA domain
  • this binding essential for efficient p97-p47 mediated reassembly of Golgi cisternae
  • p97 comprises 3 domains
          --- N terminal cofactor binding domain,
          ---  2 AAA ATPase domains (D1 and D2)
  • 3D reconstructions of p97 in diff nt state obtained by cryo-EM shows N domains are flesible
  • together with D1 and D2 domains can undergo significant conform change during ATP binding and hydrolysis
  • N domains interact with D1 in D1 hexamer plane
  • adaptor protein p47 has 3 domains connected by long flexible regions
  • p47 interact with p97 N subdomains via a conserved loop in UBX domain (S3/S4(
          --- inserts into a hydrophobic pocket between 2 p97 N subdomains
  • p97 provides mechanisl force cia ATPase activity
          --- disassemble or unfold target proteins or protein complexes
          --- force though to be mediated thru adaptor proteins 
  • p97-p47 complex resembles NSF 20S complex structurally

RESULTS AND DISCUSSION

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p47 trimerisation and stoichiometry of the p97–p47 complex


Taken together our data firmly establish that three p47 molecules bind to a single p97 hexamer and indicate that p47 trimerisation mediated by the SEP domain and parts of the N-terminal linker are important determinants for this stoichiometry.

Cryo-EM reconstructions of the p97–p47 complex

  • complex adopts barrel-shaped arrangement
  • formed by stacking of D1 and D2 hexameric rings
  • top and bottom rings have diamter of 110 A
  • comdined height of 95A
  • corresponds to diameter of p97 and height of stacked D1 and D1 rings
  • In presence of AMPPNP, D2 ring has no central pore
  • small densities portrude outwards from outer tip of D1 ring
  • Connected antennae densities emerge from 3 points on central plug
  • contiguous ---> give rise to ringlike appearance
  • In presence of ADP, D2 ring has pore of 20 A diameter
  • at end of a funnel-shaped conduit
  • particle is wider at D1-D2 interface
  • 3 arms emerge from central plug
  • form a 3 fold symmetric propeller-like str that links onto top of 3 D1 domains

Similarities between the cryo-EM reconstructions of the p97–p47 and NSF–-SNAP–SNARE complexes

  • NSF is close homologue of p97
  • dissoc v-t SNARE complexes
  • reactivates them
  • mediate further rounds of membrane fusion
  • 3D reconstruction of NSF 20S complex is similar to p97-p47 in size and overall shape
  • residues implicated in a-SNAP binding in NSF(N) are on opposite side of p47 binding site on p97 (N)
  • diff in ATPase domain arrangements between NSF and p97
  • in complex with adaptor protein, overall similarity

Distinct nucleotide-dependent conformations of the p97–p47 complex

  • p47 is on top of D1 ring (plug and antennae_
  • Dinstinct diff
  • in AMPPMP conforms, D1 and D2 rings have similar diameters
  • with D1 stacking on top of D2
  • in p97-p47 the D2 ring is closed
  • it is open in p97 alone
  • in ADP conforms, D1 and D2 rings show good agreement
  • D2 ring is open in p07-47
  • but closed in p97 alone
  • in ADP state, p97 adp[ts a barrel shape with widest point between D1 and D2 domains
  • D1 ring in ADP state has slightly wider diameter: 120 A
  • D1 and D2 domains in AMPPMP state have similar diameters
  • D1 domain stacks directly on top of D2
  • cause CCW rotation in D2 or 20 degrees in ADP map compared to AMPPMP map

Proposed p47 domain arrangements in the p97–p47 complex

  • syntaxin 5 and ubiquitin (p97 target proteins) bind top of p97-p47 complex
  • p47 has a propensity to form trimers in solution
  • mediated by p47 (SEP) domain and flanking regions

A speculative model for ATP-dependent p97 driven domain rearrangements in p47

  • after incubation with ADP, onform changes of p97-p47 omplex
  • conform change in D2 domain in ADP state
  • reorganisation of densities attributed to p47 on top of D1 ring
  • it is possible during ATP hydrolysis, p97 (N) domains form contats with p47 (UBX) domains at periphery of hexamer
  • ATP hydrolysis in D2 causes conform changes
  • induce rearrangement of p97 (N)-p47(UBX) domains and p47 (SEP-IBX) linker
  • form part of propellet str
  • SEP-UBX linker relocation could affect position and properties of SEP trimer
  • could reorder UBA-SEP linker
  • promote conform changes in target proteins (fig 7)
linker to promote conformational changes in target proteins (Figure 7).

fig 7. conformational changes in AAA ATPase p97-p47 adaptor  complex. Schematic representation of the proposed p47 domain rearrangements within the p97–p47 complex. p97(D1–D2) protomers are represented in light blue, with N-domains in dark blue. The p47 domains are shown as ellipses with N-terminal UBA domain coloured red, SEP orange and UBX yellow. Left shows the domain arrangements as interpreted in the AMPPNP p97–p47 EM reconstruction. The p97(N) domains sit above the D1 hexamer with three out of the six in direct contact with p47(UBX). The p47 SEP–UBX linker (grey) makes contact with the adjacent p97(N) before connecting to p47(SEP). A p47 SEP trimer sits centrally on top of the p97 hexamer with UBA domains shown vertically above connected by the UBA–SEP linker (grey). Right shows the domain arrangements as interpreted in the ADP p97–p47 EM reconstruction. The p97(N)–p47(UBX) domain complex has rotated approx100° in the plane to form the extremities of the propeller structure (yellow arrow, left) breaking contact between the SEP–UBX linker and the adjacent p97(N) domain. p47(SEP) domain and p47 UBA–SEP linker rearrangements (grey arrow, left) result in the p47(UBA) domain experiencing a lateral displacement (red arrow, left). This nucleotide-dependent conformational change would result in mechanical force being applied to target proteins bound to p47 via UBA domains and may represent part of the p97–p47 disassembly activity.
  • p97 (N) domain motion is flexibitlity it important for p97 function
  • Conserved glycines in p97 (N-D1) linker could form pivoting points
  • allow N-D1 linker to rotate and relocate
  • N-D1 linker residues are involved in D1 nt binding
  • connects nt binding and hydrolysis to potential N-D1 linker and N domain relocations
  • D1-D2 linker contains conserved glycines
  • may play a simialr role in transmitting nt binding/hydrolysis events in D2 to conform changes in D1 and N domains
  • Changes during nt binding in hydrolysis in D2 domain of p97 transmitted and applied to p97(N) and p47 adaptor domains

 We suggest a possible interplay between the two p47 linkers mediated by an SEP trimer rearrangement and p97(N) domain relocation, which would exert conformational changes on proposed p97 target proteins such as SNARE complexes or ubiquitylated substrates bound to the p47(UBA) domains. To address how these conformational changes disassemble protein complexes or act on substrates, further structures of p97 adaptor complexes bound to defined target proteins will be required. We also cannot exclude that p47 or its orthologues could have additional adaptor functions where nucleotide hydrolysis in p97 is not required.






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