Saturday, 16 June 2012

Mechanism of DNA translocation in a replicative hexameric helicase by Enemark and Joshua-Tor

The E1 protein of papillomavirus is a hexameric ring helicase belonging to the AAA + family. The mechanism that couples the ATP cycle to DNA translocation has been unclear. Here we present the crystal structure of the E1 hexamer with single-stranded DNA discretely bound within the hexamer channel and nucleotides at the subunit interfaces. This structure demonstrates that only one strand of DNA passes through the hexamer channel and that the DNA-binding hairpins of each subunit form a spiral ‘staircase’ that sequentially tracks the oligonucleotide backbone. Consecutively grouped ATP, ADP and apo configurations correlate with the height of the hairpin, suggesting a straightforward DNA translocation mechanism. Each subunit sequentially progresses through ATP, ADP and apo states while the associated DNA-binding hairpin travels from the top staircase position to the bottom, escorting one nucleotide of single-stranded DNA through the channel. These events permute sequentially around the ring from one subunit to the next.
  • viral initiator proteins E1, large T-antigen and Rep belong to helicase superfamily III
  • members of AAA family
  • form hexameric rings 
  • encircle substrate DNA and unwind it with  3′ → 5′ polarity.
  • T7gp4 and DNaB encircle only one of 2 DNA strands
  • E1 and Tag initiate unwinding from completely dsSNA
  • cause helix melting and entry of DNA helicase onto a ss region


Overall architecture

  • crystal has 2 hexamers
  • each encircles a single strand of DNA
  • 6 oligomerisation domains form a rigid collar with 6fold rotational symmetry
  • most protein-DNA interactions occur at hairpins at interior of channnel
  • subunits have varying modes of Mg2+-ADP coordination
  • each P-loop has an assoc ADP mol except subunit L
DNA binding in E1 hexameric helicase. 
ac, Interactions involving the AAA + hairpins and ssDNA shown as a schematic (a) and as a ribbon diagram in two perpendicular views (bc) for hexamer 1. K506 coordinates a ssDNA phosphate, and the main-chain amide of H507 interacts with the phosphate of an adjacent nucleotide. K506 also mediates interactions with three sites on the adjacent hairpin to form the staircase of hairpins—the side chain of D504 and the main chain carbonyl groups of R505 and K508 (the latter two side chains are omitted for clarity). Hydrogen bonds are drawn as dotted (a) or dashed (bd) lines. Hairpins of the individual subunits are colour-coded as in Fig. 1. In b and c only H507 and K506 side chains are shown. The letters in parentheses identify the subunits. Other components of the protein are coloured grey with transparency. The DNA is in light blue. d, Same as c with superimposed Fo - Fc difference electron density calculated before inclusion of any DNA in the model. The electron density is contoured at 3sigma (red) and at 6sigma (blue).

DNA binding

  • residues observed to interact with ssDNA are at interior of hexmieric ring on AAA+ domains
  • K506 ammonium grp interacts with 1 ssDNA phosphate oxygen
  • while H507 main chain amide protin forms a H bond with ssDNA phosphate of an adj nt
  • aliphatic portion of K 506 and aromatic grps of F646 and H507 form vdA with ssDNA rugar moiety linking 2 phosphates
  • interactions permute sequentially round hexameric ring
  • 5'end of ssDNA directs towards Nterminal oligomersation domains
  • 3' end is directed towards C terminus
  • one hexamer has 5 of subunit hairpins engaged in ssDNA phosphate coordination
  • other hexamer engages all 6 subunit hairpins

Intersubunit interactions and ADP binding

  • most robust intersubunit interactions occur between oligomerisation domains thru interactions at each interface
  • interactions between AAA+ domains mediate nt binding or involved in staircasing the hairpins
  • Aa responsible for nt coordination and hydrolysis are on adj subunits
  • Walker A and B motifs coordinate disphosphate grp of an ADP mol and an assoc Bg2+ ion


A coordinated escort mechanism for DNA translocation

  • hexameric strs display varying nt coordination modes 
  • ssDNA nt's are arranged with a 1 nt per subunit increment
  • in DNA translocation mech, each DNA-binding region maintains continuous contact with one unique nt of ssDNA
  • migrates downwards by ATP hydrolysis
  • ADP released at subunit interfaces
  • ATP hydrolysis occurs between subunits located towards top of staircase
  • ADP release occurs between subunits located towards bottom
  • downward movements of hairpins are coordinated by staircasing interactions
  • hairpin at bottom of staircase releases its assoc ssDNA phosphate
  • initiate its escorted journey thru channel and repeat process
  • for one full cycle of hexamer, each subunit hydrolyses 1 ATP, releases 1 ADP and translocates 1 nt thru interior channel
  • A full cycle translocates 6 nt with assoc hydrolysis of 6 ATP and release 6 ADP

fig 4. cartoon depiction of a coordinated escort mech for E1 hexameric helicase. Each subunit is depicted as a wagon (or boxcar), colour-coded as in Fig. 1. Each wagon transports one DNA nucleotide from the right side to the left. Staircasing interactions are depicted by the wagon couplers. ATP hydrolysis and ADP release occur along the path as depicted by the nucleotide coordination type between the wagons. The red, leftmost wagon ejects its associated DNA nucleotide and returns to the right side upon binding a new ATP molecule. This wagon then picks up the next DNA nucleotide of the series, couples to the purple wagon, and carries its cargo towards the left. Figure prepared by J. Duffy.    
  • both hexamers display a gap between AAA+ domains at top and bottom positions of staircase
  • this interface is where most sizeable movements occur in mech
  • on ATP binding, subunit at bottom moves to top and closes this gap
  • a new gap opens between thissubunit and previous subunit (now bottom)
  • for hexamer 1, an ATP-type configuration observed between subunits A and B
  • hairpin of subunit A is not engaged in ssDNA coordination
  • Subunit A is consistent with a subunit that has recently bound an ATP, but not yet bound DNA
  • in hexamer 3, subunit at top has an ATP state and binds ssDNA
  • hexamer has 2 empty nt states whereas hexamer 1 has only 1
  • state that directly precedes ATP binding
  • only subunit in str where no ADP is observed at P loop
  • similarities to T7gp4 operation
  • In T7gp4 proposal, successive loops bind successive nt's but pass them thru channel
  • by handing them off from one loop to next in a bucket brigade manner
  • loops would maintian a nearly fixed height as nt's are passed down from one loop to next
  • in escort mech, each hairpin maintains a continuous set in interactions with one nt
  • entire unit collectively migrates downwards
  • for T7gp4, subunits engaged in ATP coordination have higher affinity for DNA than ADP and empty states
  • same relative order of affinities in E1 hexamer leads to add driving force for coordinated escort mech of cyclic DNA translocation
  • subunits at top of staircase have higher affinity for DNA (more prone to bind) than those at bottom (more prone to release)
  • binding new ATP mol by a subunit in empty state at bottom would return it to top and increase its affinity for DNA

Translocation direction

  • coupled ATP hydrolysis to DNA translocation
  • ATP hydrolysis drives sequential movement of hairpins and pump ssDNA in that direction
  • consistent with  3′ → 5′ polarity for SF3 helicases and observed polarity of DNA in str with 5' end located towards top (oligomerisation domain side) of complex

Oligomerization domain as processivity factor

  • rigid collar formed by oligomerisation domains is a ss equivalent of ds processivity factors eg polymerase sliding clamps like PCNA
  • Static ring formed by 6 oligomerisation domains in E1 helicase keeps 2 strands topologically apart
  • prevents ring from falling off substrate DNa
  • AAA+ domain have weak interactions between subunits
  • intersubunit interactions of ring ensure hexamer continues to surround single strand

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