Sunday, 17 June 2012

The 'glutamate switch' provides a link between ATPase activity and ligand binding in AAA+ proteins by Zhang and Wigley


AAA+ proteins carry out diverse functions in cells. In most cases, their ATPase activity is tightly regulated by protein partners and target ligands, but the mechanism for this control has remained unclear. We have identified a conserved link between the ligand binding and ATPase sites in AAA+ proteins. This link, which we call the 'glutamate switch', regulates ATPase activity directly in response to the binding of target ligands by controlling the orientation of the conserved glutamate residue in the DExx motif, switching it between active and inactive conformations. The reasons for this level of control of the ATPase activity are discussed in the context of the biological processes catalyzed by AAA+ proteins.

  • AAA+ are distributed across all kingdoms
  • classified in 7 clades
  • involved in protein degradation, chaperone activities, membrane fusion, regulating transcription. DNA repl and repair
  • Many systems are cargo delivery
  • ligand delivered to a site
  •  released before protein returns to pick up another ligand
  • eg DNAP procsesivity clamp loading
  • some involved in remodelling macromolecular ligands
  • convert them from one state to another
  • eg protein chaperones and transcriptional regulators
  • AAA+ enzyme systems need ATP in reactions that are energetically neutral and favourable
  • initiating transcription in some bacterial genes requires AAA+ trasncriptional a ctivators eg PspF and NtrC
  • Protease activity of AAA+ proteases is ATP dependent
  • other proteases do not require ATP
  • cleavage of peptide bond is energetically favourable
  • total protein digestion possible without ATP
  • rate of ATP turnover of AA+ enz is poor compared to other ATPases
  • ATPase activity is regulated by ligand binding
  • binding DNA to replication factor C (RFC) stimulates ATPase activity of protein
  • DNA binding origin recognition complex (ORC) proteins represses ATPase activity
  • ATPase active site of AAA+ proteins contains Walker A and B
  • AAA+ enz have conserved residues in active site
  • promote ATP hydrolysis
  • key residues in Walker A and B motifs coordinate Mg2+, promotes catalysis
  • Arginie finger reaches in ATPase site
  • polarise gamma-phosphate ---> facilitate hydrolysis
  • a conserved glu is within Walker B motif in AAA+ but lies at end of anj beta-strand in other ATPases
  • glu polarises a water mol for inline attack of gamma-phosphate during hydrolysis
  • replace with ala ---> proteins can bind ATP
  • but hydrolysis activity is impraired.
fig 1 essential residues in active site of ATPases. ATP hydrolysis is promoted by several residues in this generalized active site of ATPases. Only residues with an established role in catalysis rather than binding are shown. When present, the 'arginine finger' is usually provided in trans from an adjacent subunit or domain. The magnesium ion can be coordinated in different ways, but that shown is one of the most common, involving interactions with a threonine (or serine) residue from the Walker A motif, an aspartate residue from the Walker B motif and oxygens from the beta- and gamma-phosphates of the ATP. The role of the glutamate residue (in the DExx motif of AAA+ proteins) is to activate a water molecule by making the oxygen more electronegative and, hence, a better nucleophile for attack of the gamma-phosphorus.   

Results

  • An AAA+ protein for wh many nt complexes are available at high resolution is bacterial transcription activator, PspF
  • PspF activates   sigma54-dependent RNA polymerase (RNAP) 
  • by converting it from a closed (inactive) state to the open (active) conformation, which initiates transcription.
  • Compare ADP and ATP complexes: str superimpose well except a few regions that respond to presence of gamma-phosphate
  • one change is in ATPase site
  • gluatmate residue of DExx motif moves a lot
  • In ADP complex, glutamate side chain is in a position similar to that seen in most other ATPase active sites
  • in ATP complex, glutamate is rotated 100 degrees from conform in ADP complex
  • forms a H bond with an asparagine on an adj beta-strand
  • rotation moves the Glu from a position in wh it could activate incoming attacking water mol to one in wh it would not.
  • ATP binding inactivates ATPase activity
  • switch active site from active config to inactive
  • glutamate switch converts enz from low-energy ground state to higher energ form
  • now trapped in inactive state
  • protein conform stabilised by water-mediated interactions
  • involve gamma-phosphate of bound ATP, glu and Asn side chain and Mg2+
  • Glu-Asn is present in ATP-noung complex from  related sigma54 activator ZraR15 but is not formed in the ADP-bound complex of another regulator, NtrC1
  • 2nd area of movement in PspF complexes is remote from active site
  • involves 2 loops, L1 and L2 that interact with   sigma54-RNAP
  • Direct peptide linkage between E-M pair and these loops
  • provide communication between ATPase and ligand-binding sites
  • complex between PspF and  sigma54-RNAP is most stable when formed with TS analog ADP-A;Fx rather than ADP or ATP
  • interaction between sigma54-RNAP and PspF must stabilize the transition state and hence stimulate ATPase activity, 
  • probably by releasing the glutamate in the ATPase active site from the inactive to the active configuration.
  • gluatmate switch operates similarly to other AAA+ protein clades
  • eg RFc, ORC, p97
  • conserved glu switch pair found in members of 6 of 7 AA+ clades
  • glu is almost invariant across all AAA+ proteins
          --- key role in promoting ATP hydrolysis
  • active conform (ADP state of PspF) shows tigether clustering of chi2 angles compared to that of the inactive conformation represented by the ATP state of PspF   
  • geometric constraints for positioning glu correctly
  • to polarise water mol

fig 3 conservation of glutamate switch in AAA+ proteins. (a) Sequence alignments of selected members of each clade for which the crystal structures are known. The color scheme follows that used in Figure 4. RFCs, replication factor C small subunit from Archaeoglobus fulgidus; Orc1, Orc1 protein from Aeropyrum pernix; p97D1, p97 D1 AAA+ domain from Mus musculus; SV40, SV40 large T antigen; PspF, PspF from Escherichia coli; HslU, HslU from E. coli; RuvBL1, RuvB-like 1 (TIP49a, Pontin) from Homo sapiens. (b) Plot of side chain torsion angles for 50 active site glutamate (DExx box) residues from AAA+ protein structures in the Protein Data Bank (http://www.rcsb.org/) with a resolution better than 3.5 Å. Only one copy from the asymmetric unit was used if angles were similar, to reduce redundancy. The appropriate glutamate residue was selected from individual PDB files and the side chain torsion angles were calculated using the CCP4 program ANGLES40, then normalized to 0–360° for display. The values for the PspF–ADP complex (cyan diamond) and PspF–ATP complex (magenta diamond) are overlaid as examples of residues in the two conformations.   

  • binding site for target ligands on AAA+ domain involves same region of fold in all clades
  • possible conservation of linkage between ATPase and ligand binding sites
  • for type 2 AAA+ protein ef p07. 2nd AAA+ domain (D2) is  not directly involved in substrate binding
  • interacts with D1 via similar regions in D2

fig 4 link between glutamate switch motif and ligand binding site. (a) Location of the glutamate switch pair within a typical AAA+ domain and the linkage to the ligand binding site. The example shown is theA. pernix ORC1-DNA complex (clade 2, PDB 2V1U). The Walker A motif is shown in red, the glutamate switch region in orange (with the glutamate-asparagine pair in magenta), the ligand binding site between the glutamate switch and the DExx box in cyan, the DExx box in green and the bound ADP in blue. A portion of the bound DNA is shown in wheat. (b) D1 (wheat) and D2 (gray) domains of p97 (PDB 3CF3) showing the interaction between the two ATPase domains in a type II AAA+ protein. The coloring of the D2 domain follows the scheme used in a   
  • formation of switch pair when ATP is bound is means to suppress ATPase
  • can be alleviated on ligand binding
  • linkage can be reversed
  • in ORC and DnaA, glu residue (Asp in DnaA_ remains in active config in both ADP and ATP analog complexes in absence of DNA
  • This family of proteins is unusual
  • ligand binding (DNA) inhinits ATPase activity rather than stimulating it
  • in archaeal ORC proteins bound to DNA targets, glu-asn pair is formed
  • ligand binding inhibits ATPase activity
  • glu switch can function in either direction
          --- stimulate or inhibit ATP hydrolysis at diff stages
  • ATPase activtiy of HSlU is stimulate by binding HsIV and substrate
  • glu residue is locked in inactive config in ADP complex
  • free in ATP-bound form
  • In SV40 large T antigen (LTag) catalyse unwinding long DNA stretches during virus repl
  • glu remains in active config in ADP and ATP analog complexes
  • Full length LTAg contains a domain that binds SV40 replication origin
  • helicase activity must be restrained before origin firing
  • glu-asn pair may be swtich on repl origin binding
  • after origin firing, enz must finction as processive helicase
  • switch no longer required
  • ATPase activity of human papilloma virus E1 helicease (closely related to LTag and has conserved switch pair) is inhibited by E2 origin binding protein
  • mutations at glutamate residue reduces ATPase activity
  • for RFC, glu ---> ala mutant has reudced ATPase activity and reduced basal activity is no longer stimulated by DNA
  • Mutate asn in PspF ---> abolish inhibitory effects of its negative regulator, PspA
  • Glu-Asn pair communicates ligand binding to ATPase activity
  • PspF hexamers are stabilished by ATP binding
  • mutatns in wh switch pair can no longer form are defective in hexamerisation
  • hexamerisation required for  max ATPase activity

Discussion


  • why is ATPase activity tightly regulated in AAA+ enz?
  • many ATPases use free energy of ATP hydrolysis to drive reactions
          ---transfer chem energy
          --- or convert to mechanical energy
  • reactions catalysed by AAA+ enz are diff
  • ATPase activity inhibition is found in systems with complex assembly pathways and miltiple intmt steps
  • ATP hydrolysis is suppressed until system is fully assembled
          --- thru atp binding in RFC
          --- or ligand binding in ORC
  • ATP turnover then allows completion of reaction and/or recycling of coponents
  • many reactions involve single events on a spec marcromol substrate
  • require series of steps in a precise pathway
  • in these systems, ATP us used to control directionality of reaction
  • regulate assembly of multicomponent system,
  • ATP ensures correct assembly of components
  • as system begins to assemble, ATP binds 
  • but hydrolysis is suppressed until system is fully competent.
  • eg in synaptic vesicle fusion, complex between NSF,SNAPs and SNAREs is stabilised on ATP binding
  • disassembles when ATP is hydrolysed
  • eg loading of proliferating cell nuclear antigen (PCNA) rings onto DNA primer junctions by RFC
  • ATP binding sifficient to stabilised RFC-DNA complex and support loading of clamp around DNA
  • DNA-stimulated ATP hydrolysis releases PCNA onto DNA
  • recycle RFC components
  • allow them to pick up another PCNA ring
  • another eg is  activation of RNAP-sigma54 by transcriptional activators such as PspF or NtrC39
  • RNAP-sigma54binds to the promoter site 
  • form stable, closex complex
  • cannot inititate transcription without being remodelled by activators
  • activators bind a spec activation seq
  • 80-150 bp upstream of transcriptional start site
  • ATP binding required for stable hexamer formation of activators and their interactions with  RNAP-sigma54 through DNA looping
  • facilitated by DNA-bending protein eg integrative host factor, IHF
  • ATP hydrolysis releases activators from activated   RNAP-sigma54 allowing transcription to proceed.
overall mechanism of 2 well-characterised AAA+ protein-catalysed reactions.  (a) PCNA clamp loading by RFC. Upon ATP binding, RFC forms a stable complex with PCNA (cargo pickup) that is competent to load onto DNA at primer-template junctions (cargo delivery). ATP hydrolysis by the small subunits (blue) releases PCNA (cargo release), which is then bound by DNA polymerase. Finally, ATP hydrolysis at the large subunit (orange) recycles the RFC and allows pick up of the next PCNA (cargo reloading). (b) Bacterial transcription activation by PspF. Upon ATP binding, PspF, which binds to the DNA sequence upstream of the transcription start site, interacts with RNAP-sigma54 through DNA looping (cargo pickup). At the point of ATP hydrolysis, PspF forms a stable complex with RNAP-sigma54 and initial remodeling of RNAP-sigma54/DNA occurs (cargo remodeling). Upon the completion of ATP hydrolysis, transcription proceeds and PspF dissociates from the complex (cargo remodeling and reloading).   

  • ATP hydrolysis is a switch to control process and recycle compoenents after reaction completion
  • coupling to ATP hydrolysis allows control of process
  • explains requirement for ATP in processes energetically favourable of neutral
  • some ring helicases can load themselves onto DNA
  • ATP-dependent helicase loaders (eg ORC and DnaC) provides controlled loading at spec sites and times during cell cycle
         --- rather than random sites over genome
  • althout peptide bond cleavage is favourable
          --- controlled proteolysis by AAA+ proteases eg ClpXP and HsIUV is more discriminating
          --- only digests proteins selectribely delivered to proteases by unfolding them in situ
  • small energetic cost of using ATP as a switch is outweighed by advantages of assembling systems correctly 
  • control reactions that might have drastic consequences for the cell.

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