Saturday, 16 June 2012

Crystal Structures of Complexes of PcrA DNA Helicase with a DNA Substrate Indicate an Inchworm Mechanism by Velankar


  • helicases couple free energy of ATP hydrolysis to separation of DNA or RNA duplex into component strands
  • Eg PCrA helicase in Bacillus subtilis and S. aureus
  • involved in repair and rolling circle replication
  • enz has 4 domains, 1A, 1B, 2A and 2B
  • domains 1A and 2A have similar folds
  • ATP-binding site is in cleft between comains 1A and 2A
  • lined with conserved sequence motifs characteristic of helicases
  • PcrA is member of helicase family with 3' ---> 5' directionality
  • Other members are Rep and Uvr helicases
  • crystal structure of Rep helicase complexes with ss DNA crystalised in 2 diff conforms
  • open and closed
  • 2 mols sit adj to each other on ss oligont
  • Conform diff between mols comprised large rigid body rotation of 2B domain by 130 degrees
  • 2 models for helicase mech is inchworm and active rolling models
  • Distinct features for each mech
  1. Rolling model requires a dimeric protein at least. Inchworm model is consistent with any oligomeric model inc monomeric.
  2. DNA binding. In rolling model. each dimer subunit can bind either ssDNA or duplex DNA but never both at the same time. For inchworm model, each proteinm monomer has to bind smulatenously both ssDNA and duplex DNA at least one point in reaction cycle.
  3. step size for each reaction cycle. Inchworm mech could involve progression as little as 1 bp at a time. A rolling model implies a larger step size, at least as large as individual binding site of each monomer

Figure 1. Active Rolling and Inchworm ModelsThe two most popular mechanisms for unwinding of nucleic acid duplexes by helicases ( [26] and [23]). Both mechanisms require the hydrolysis of ATP, but it is not certain at which step this hydrolysis takes place, although ATP binding appears to be associated with an increased affinity of the enzyme for duplex DNA or RNA. In the scheme for the active rolling model, the two subunits of the dimer are colored differently in order to distinguish between them. Initially, the subunits of the dimer are both bound to ssDNA. As a consequence of binding ATP, one of the subunits releases the ssDNA and binds to the duplex region at the fork. This is followed by helix destabilization and the release of one of the DNA strands in a process that accompanies the hydrolysis of ATP. For the inchworm model, the enzyme monomer is bound to ssDNA and then translocates along the DNA strand to the fork region, probably upon binding ATP. Helix destabilization and release of one of the ssDNA strands takes place as ATP is hydrolyzed.
  • Determine 2 diff crystal str of PcrA helicase complexed with a 10bp DNA duplex with a 7 base ss 3' tail. 
  • One complex also incl a bound nonhydrolysable ATP analog
  • traps a substrate complex
  • other complex has a bound sulphate ion in position normally occupied by a phosphate after ATP hydrolysis
  • represents  product complex
  • Large conform changes assoc with binding of DNA and nt support inchworm rather than rolling model

Figure 2. Domain Orientations of the Protein in the ComplexesDomain structure of (A) the product complex and (B) the substrate complex, with domain 1A colored green, domain 1B in yellow, domain 2A in red, and domain 2B in blue. The bound DNA is colored magenta, with ADPNP and sulphate in gold. These figures were produced using RIBBONS (Carson 1991). (C) Stereo diagram of an overlay of the Ca backbone of each of the two complexes illustrating the domain movements. The molecules are superimposed on domain 2A. The substrate complex is colored white, and the product complex is red. DNA and ADPNP have been omitted for clarity.
  • 5 bases in single stranded tail bind in a groove along top of domain 1A across onto domain 2A
  • many contacts between protein and ss tail of duplex
  • few contacts with duplex DNA itself

Structure of a Complex with DNA and ADPNP—A Substrate Complex
  • Positions of domains 1B and 2B altered due to cleft closing between domains 1A and 2A

  • conform changes trap bound ADPNP(analog) in site previously occupied by ADP in PcrA
  • conserved residues in domain 2A comes into closer contact with bound nt than in open str
  • closed form of protein is stabilisd by closer intercations mediated by bound ATP
  • no add protein-protein contacts made between domains 1A and 2A across the cleft
  • important add contacts involve either gamma-phosphate with domain 2A or bound Mg2+ with domain 1A
  • Q254, R487 and R610 make direct contacts with gamma-phosphate of ADPNP
  • replacement each residue with Ala ---> mutatn PcrA proteins with impaired ATPAse and helicase activities
  • Small shift in position of glycin-rich loop that contacts triphosphate tail

Comparison of the Substrate and Product Complexes

  • Ligand-induced conform changes 
  • cleft between domains 1A and 2A close around bound ADPNP
  • domains 1B and 2B move 
  • alter positions relative to each other
  • conform changes that occur on binding ATP set up protein surface to bind duplex DNA
  • move domains 1B and 2B into positions that form a surface comp to shape and charge of duplex DNA
  • incorrectly formed prior to conform changes
  • before ATP binding, appropriate conform changes in mains 1B and 2B are prevented from taking place
  • low affinity of this surface for duplex DNA
  • one function of  coupling ATP binding and hydrolysis to enz activtiy can modulate affinity of complex for duplex DNA
  • change in str of ssDNA tail of substrate
  • In substrate complex, region of 4 bases extends across protein centre
  • in product complex there are 5 bases across equivalent region
  • add base in product complex occupies a pocket on domain 1A not accesible in substrate complex
  • in substrate complex side chain of a consreved F64 in motif rotated around   Cα–Cβ bond and is now filling the pocket

Evidence for an Inchworm Mechanism

  •  The model we propose can be divided into two processes, DNA translocation and duplex destabilization, and it is the coupling of these properties that gives rise to helicase activity.

Figure 6. A Model for the Mechanism of Helicases(a) A model for helicase activity with regard to the large conformational changes in the protein and the DNA (or RNA) substrate. The intermediates are based upon our structures, but the DNA has been extended at both ends to illustrate how a larger substrate might bind. The colors of the protein are the same as in Figure 2, but to assist in following the translocation process the base pairs have been colored alternately magenta and white. At the initial step in the reaction (A), the protein is bound to the ssDNA tail but does not bind the duplex region of the DNA. Upon binding ATP (B), there is a conformational change in the protein, and the duplex region binds to domains 1B and 2B with a concomitant unwinding of several base pairs at the junction. Finally (C), following the hydrolysis of ATP, the protein conformation returns to that in (A) as the protein translocates along the ssDNA tail by one base and releases the DNA duplex.(b) Cartoon demonstrating the alternation in affinity for ssDNA of domains 1A and 2A during translocation. An open hand represents a loose grip on the DNA, and a closed hand is a tighter grip. (A–C) correspond to those in (a).(c) Cartoon of the ssDNA-binding region at each stage of the reaction to illustrate the conformational changes that occur in this site as bases flip between binding pockets during translocation along single-stranded DNA. Again, (A–C) correspond to those in (a). The bases are numbered arbitrarily in the 3′ to 5′ direction.
  • protein binds ssDNA ---> initiate DNA translocation
  • induce domain swivelling
          ---set up potential for binding DNA duplex
  • at initial stage, ssDNA is bound to 1A and 2A
  • when  ATP binds complex, cleft between domains 1A and 2A closes
  • For this to happen while retaining hold on ssDNA, one of the domains must reslease its grip on DNA and slide along it
  • Cleft closure ---> substrate complex structure
  • bound ATP is hydrolysed
  • release protein from this conform state
  • hydrolysis destabilises cleft closure
  • contacts mediated thr gamma phorphate are broken
  • as cleft opens, domain 2A weakens its hold on ssDNA
  • translocation along DNA effected across domain 2A as cleft springs open again 
  • while domain 1A retains tight grip on DNA and pulls ssDNA across surface of domain 2A
  • ATP-dependent DNA translocation occurs until helicase meets a duplex
  • when protein bind ATP and cleft closes, protein has increased affinity for dsDNA
  • binds duplex region adj to fork
  • duplex region of DNA pulled onto surface presesnted by domains 1B and 2B
  • create strain in base pairing at fork as DNA substrate is bent across surface of protein
  • exposed F636 stacks with DNA at the fork
  • stabilises unwound form of DNA
  • duplex is regular B-form DNA substrate complex to begin with but becomes distorted when closer to junction
  • 4-5 bp destabilised 
  • strands beginning to separate
  • binding energy creates add ssDNA along which helicase can move
  • helix destabilisation is coupled to DNA translocation
  • free energy of ATP hydrolysis is used for unidirectional translocation and strand separation, energetically unfavourable processes.

Molecular Details of the DNA Translocation Process

  • ssDNa must slide across surface of domains 1A and 2A separately at diff steps of cycle
  • At intial step, domain 1A has firm grip on DNA with bases in all of the acceptor pockets in this domain
  • complex binds ATP
  • conform changes as cleft between domains closes
  • hold of domain 1A on ssDNA has to be released
          --- allow translocation across this domain
  • accomplished as side chain of F64 moves into pocket B
         --- displace base in it to pocket A
         --- while base formerly in pocket A is pushed outside protein
         ---- as DNA slides across surface of domain 1A
         --- cause structure represented by substrate complex
  • ssDNA translocation has taken place but only across domain 1A
  • ATP hydrolysis
  • cleft reopens between domains 1A and 2A
  • ATP hydrolysis releases F64 side chain from pocket B
  • base in pocket C can flip into pocket B
  • domain 1A now has tighter grip on ssDNA
  • allow ssDNA to be pulled over surface of domain 2A as the cleft opens
  • movement forces a base to flip from stacked pair in pocket D on domain 2A as cleft opens.
  • other base of stacked pair in pocket D moves along one position as next base alond flips from based stacked with F626 and moves into pocket
  • translocation results from a wave of base flipping moving along bound ssDNA tail
  • power stroke  is relaxation of protein as cleft opens
  • proposed mech implies a step size of 1 base for translocation process as bases flip between adj pockets along ssDNA-binding site

Implications of the Model

  • helicase activity is active rather than passive
  • all components reuqired for DNAi transloction are in domains 1A and 2A
  • provides mech for ATP-depdent translocation along DNA in helicases and other enz with conserved helicase seq motifs
  • domain swivelling creates binding site for duplex DNA once protein bound ssDNA
  • protein is prevented from interacting with dsSNA until it is activated by presence of ssDNA
  • cannot initiate strand separation from within a sealed duplex
  • undesirable in a cell
  • helicase monomer can bind both ssDNA and dsDNA at the same time

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