Saturday, 16 June 2012

Structures of Asymmetric ClpX Hexamers Reveal Nucleotide-Dependent Motions in a AAA+ Protein-Unfolding Machine Glynn


INTRODUCTION

  • E coli ClpXP is a hexameric AAA+protein unfolding machine
  • can function alone or with ClipP peptidase
  • In ClpXP protease,  a ring hexamer of ClpX mediates ATP-depenednt unfolring of spec proteins
  • translocates denatured pp into lumen of ClpP for degradation
  • each subunit of ClpX hexamer is indentical in seq 
  • axial pore of ClpX hexamer is translocation channel into ClpP
  • 3 pore loops, CYVG. pore 2 and RKH play roles in binding ssrA tag
  • Some loops mediate binding and communication with ClpP
  • needed for protein unfolding and/or translocation
  • models suggest GYVG loops grip pp substrates
  • pull or drag mols into pore due to nt-dependent loop mvements
  • pulling mech could generate force
  • unfold natuve substartes that cannot enter pore
  • translocate pp once unfolding occurs
  • ATP binding and hydrolysis fuel protein unfolding and translocation by ClpX

Asymmetric Ring Hexamers

  • ClpX formed an aymmetric ring hexamer in nt-free and nt-bound str's
  • in 6 fold symmetric ring hexamer, corresponding atoms of all 6 subunits lie in planes perpendicular to symmetry axis
  • axial positions of equivalent aa's in ClpX small AAA+ domains were staggered in bt-bound and nt-free hexamers
  • non-planarilty of equivalent residue positions in diff subunits in large AAA+ domains and linkers that connect 2 domains

Structural Origins of Asymmetry

  • both ClpX hexamers have similar backbone conform
  • folds of smalAAA+ domains were identical
  • str changes in interfaces between diff subunits or domains in same subunit must generate asymmetry in hexamers and conform rearrangements between nt-free and nt-bound hexamers
  • distinct types of subunits present in both ClpX hexamers
  • roation between domains in type 1 subunits creates a confom compativle with nt-binding in interdomain cleft
  • rotations between domains in type 2 subunits results in a conform that destroys nt-binding pocket
  • 2 interfaces contribute to packing between neighbouring subunits in ClpX ring
  • major interface is formed by packing each small AAA+ domain against large AAA+ domain of clockwise subunit
  • adj small and large AAA +domains can be viewed as a single rigid-body unit 
  • hexamer comprises 6 such units
  • change in rotation between large and small AAA+ domains of a single monomer propagate via rigid-body interfaces
  • affect orientation of adj large AAA+ domain
  • Less conserved subunit interfaces occur between adj large AAA+ domain


Closing the Hexameric Ring

  • topologically closed rings of both hexamers showed a 112112 pattern of type 1 and 2 subunits

A Mechanism for Substoichiometric Nucleotide Binding


  • ClpX hexamers bind a max of 4 ATP mols in solution
  • In AAA+ enz, ATP/ADP binds interface between large and small AAA+ domains of one subunit and large AAA+ domain of an adj subunit
  • create 6 potential bindings sites
  • IN ClpX, only 4 sites can bind ATP/ADP
  • small domain in type 2 ClpX subunits occupies space where nt's would normally bind
  • in type 1 subunits, adenine base of a nt can interact with short linker between large and small domains
  • mech to link binding to str changes in hexamer conform

Motions Driven by Nucleotide Binding

  • Nt binding has 2 results
  • 1st, orientation of large and small domains of type 1 subunits change
  • rotaton between large and small domains of subunit A increased by 15 degrees after nt binding
  • domains in subnit B rotated 14 degrees in opposite direction
  • 2nd.rotations cause complex set of motions of assoc rigid body units
  • in units with a type1 small domain, this domain moves dowanwards and inward towards bottom face of hexamer
  • its partner large domain moves upwards and inwards, closing the pore
  • in units with a type 2 small domain. entire rigid body element moves up and away from pore
  • cause ClpX hexamer to flex in plane of ring
  • nt bound str is taller, narrower and more constricted pore than nt-free str


Axial Staggering of Pore Loops

  • GYVG and pore 2 loops project into lumen of axial pore
  • RKH loops surround upper entry to pore
  • All ClpX loops play roles in binding of ssrA degradation tag of substrates
  • subsets mediate translocation, unfolding and dynamic contacts with ClpP

DISCUSSION

  • 6 subunits of hexamer have same seq and identical folds for large and small AAA+ subunits
  • but different orientation between large and small AAA+ domains of individual subunits define 2 general classes of subunits
  • type 1 subunits bind nt
  • type 2 subunits do no bind nt
  • major interface between subunits is formed by packing a small AAA+ domain against neighbouring large AAA+ domain
  • unit moves as a rigid body around restricted swivel points defined by linker conforms in adj subunits
  • closed hexameric ring is formed by a 112112 pattern of type 1 and 2 subunits
  • cause staggering of domains and structural elements with respect to central pore axis
  • nt binding type 1 subunits affect conform of linker between large and small AAA+ domains
  • change rotation between these domains
  • movements propagate around the ring
  • cause flexing motion
  • some str els move down
  • others move up relative to pore axis
  • ADP binding , hydrolysis to ADP.Pi, re;ease of Pi and / or ADP during ATPase cycle will alter roation between large and small AAA+ domains of subunit
  • cause rigid body conform changes thruout ClpX hexamer
  • coupled to mechanical work on protein and pp substrates
  • in intact Clpx, a 15 residue linker connects each N domain to the large AAA+domain
  • And IGF seq in ClpX makes important docking contacts with hydrophobic clefts on surface on ClpP ring

Functional Consequences of Pore-Loop Staggering

  • loops forming axial pore of ClpX are involved in recognising ssrA degradation tag, binding and communicating with ClpP,. substrate unfolding and translocation
  • basic RKH loops surround pore entry
  • interact favourably with -vely charged a-carboxylate of sswA tag
  • stabilise an encounter complex
  • before tag moves deeper into pore to interact with GYVG and pore 2 loops
  • position from which RKH loops project from hexamer are axially staggered
  • provdes a path to guide ssrA tag into pore lumen
  • where GYVG and pore 2 loops engage substrate
  • faciliate unfolindg and treanslocation
  • pore 4 loops crosslink N temrinal loops of ClpP which contact ClpX near bottom of axial pore
  • ClpP loops contact SspB adaptor protein
  • interactions with ClpP mediated by pore 2 loops regulate ATPase rate of wt ClpX
  • poor 2 loops of type 1 subunits may alter rate of ATP hydrolysis in these aubunits
  • Walker B motif contacts ATP and takes part in catalysis
  • GYVG loops and pore 2 loops required for strong binding t ssrA tag and protein unfolding
  • only GYVG loops are highly conserved in other AAA+ proteases
  • play major role in substrate translocation and protein unfolding by ClpX
  • GYVG loops help grip pp substrate
  • ATP binding or hydroylysis may cause a GYVG loop to move downwards in pore
  • due to rigid body movement of large AAA+ domain where it resides
  • could pull or drag a bound pp substate along
  • tyr 153 in GYVG loop of chain E moves down thru pore on nt binding
  • mech for translocation and force application
Figure 6
Models for Protein Unfolding and Pore Expansion
(A) Cartoon showing how ATP hydrolysis might change rotations between the large and small AAA+ domains of two ClpX subunits. These domain-domain rotations, in turn, could drive rigid-body movements that result in unfolding and translocation of a bound native substrate.
(B) The cartoon on the left shows that the ClpX hexamer can be viewed as consisting of two jaw-like elements. The main contacts between these jaws are formed by the interfaces between the large and small AAA+ domains of the type 2 subunits (red/dark red). Opening of these interfaces, as shown in the exaggerated right cartoon, provides a potential mechanism for pore expansion to accommodate large substrates, including those with multiple chains.

A Mechanism for Pore Elasticity

  • ClpX must translocate 2 pp together when it degrades a disulphide bonded proteins
  • If axial pore is like a mouth
  • hexameric ring has 2 jaws connected by hinge like linker between large and small AA+ domains of each type 2 subunit
  • hinge interfaces may open in elastic fashion
  • allow passage if substrates too large to transit undistorted pore
  • refolding could close pore








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