human ddx19 cdna was obtained from the mammalian gene collection ( mgc ; 
  accession number bc003626 ) provided by the national institute of health . the 
  sequences encoding ddx19 ( full - length protein ) , 
  ddx19 , and ddx19 were amplified 
  by pcr and inserted into pnic28-bsa4 by ligation - independent cloning . the 
  expression constructs included a tobacco etch virus protease - cleavable 
  n - terminal hexahistidine tag . protein expression in escherichia coli 
  strain bl21(de3 ) ( ddx19 ) , bl21(de3 ) gold prare2 
  ( ddx19 ) , or bl21(de3 ) r3 prare 
  ( ddx19 ) was done in terrific broth medium supplemented 
  with 8 g / liter glycerol and 50 g / ml kanamycin , induction with 0.5 
  mm isopropyl-1-thio--d - galactopyranoside , and 
  culture at 18 c for 20 h. cell pellets were resuspended in 50 
  mm hepes , ph 7.8 , 500 mm nacl , 10 mm 
  imidazole , 10% glycerol , 0.5 mm tcep , and complete edta - free 
  protease inhibitor ( roche applied science ) . cells were lysed by a freeze / thaw 
  cycle followed by the addition of benzonase ( novagen ) and sonication ( sonics 
  vibracell ) . cleared and filtered lysates were loaded onto hitrap chelating hp columns 
  ( ge healthcare ) pre - equilibrated with buffer 1 ( 30 mm hepes , ph 
  7.5 , 500 mm nacl , 10 mm imidazole , 10% glycerol , and 0.5 
  mm tcep ) . bound protein was eluted with buffer 1 containing 500 
  mm imidazole and loaded onto a hiload 16/60 superdex-200 column ( ge 
  healthcare ) pre - equilibrated with buffer 2 ( 30 mm hepes , ph 7.5 , 
  500 mm nacl , 10% glycerol , and 0.5 mm tcep ) . fractions 
  containing ddx19 protein were pooled , and the concentration of tcep was 
  adjusted to 2 mm . the n - terminal hexahistidine tag was removed by 
  incubation of ddx19 proteins with his - tagged tobacco etch virus protease at a 
  molar ratio of 30:1 overnight at room temperature and subsequent passage over 
  a 1-ml histrap hp column in buffer 1 . the adp - bound ddx19 apoenzyme structure 
  ( pdb entry 3ews ) was obtained with uncleaved protein . the purified proteins 
  were concentrated into buffer 2 using vivaspin ( sartorius ) centrifugal 
  concentrators . all 
  proteins were verified by time - of - flight mass spectrometry analysis . for the adp - bound ddx19 enzyme ( pdb entry 3ews ) , 
  needle - like crystals of ddx19 were grown using vapor diffusion at 4 c by 
  mixing 0.1 l of protein solution ( 18.3 mg / ml ) including 20 mm 
  adp , 10 mm mgcl2 , and 0.1 l of reservoir solution 
  containing 17% polyethylene glycol 10000 , 0.1 m ammonium acetate , 
  0.1 m bis - tris , ph 5.5 . reservoir solution supplemented with 20% 
  glycerol was used as cryo - solution and added directly to the drop . diffraction data to 2.7 
   resolution were collected at berliner 
  elektronenspeicherring - gesellschaft fr synchrotronstrahlung ( bessy ) , 
  berlin , germany ( beamline bl14 - 2 ) . the structure was solved 
  by phaser ( 20 ) using pdb entry 
  1fuu as a search model . domains were searched for separately and were edited 
  according to sequence alignment with chainsaw 
  ( 21 ) before molecular 
  replacement . model building was done 
  using coot ( 23 ) . for further 
  details , see supplemental table s1 . crystals of ddx19 in complex with rna and mg - adpnp 
  were obtained by vapor diffusion in sitting drops incubated at 4 c by 
  mixing 0.1 l of protein solution ( 20 mg / ml ) including 10-molar excess of 
  decauracil ssrna , adpnp , and mgcl2 and 0.2 l of reservoir 
  solution containing 14% polyethylene glycol monomethyl ether 2000 , 0.25 
  m trimethylamine n - oxide , 0.1 m tris , ph 8 . reservoir solution supplemented with 25% 
  glycerol was used as cryosolution and added directly to the drop . diffraction data to 2.7 
   resolution were collected at the european synchrotron radiation 
  facility ( esrf ) grenoble , france ( beamline id-29 ) . the 
  structure was solved by phaser using the previous ddx19 structure ( pdb entry 
  3ews ) as a search model . geometry of the models was analyzed with molprobity 
  ( 25 ) sequence alignments were 
  obtained using espript ( 26 ) . 
  the figures were created using pymol 
  ( 27 ) . atpase activities of the ddx19 protein constructs ( 0.5 m ; in 
  30 mm hepes , ph 7.5 , 2 mm atp , 5 mm 
  mgcl2 , 75 mm nacl , 2 mm tcep ) were measured 
  using the enzchek phosphate assay kit ( invitrogen ) at 22 c . the assay was 
  performed in either the presence or the absence of rna ( poly(u ) rna or 
  poly(i)-poly(c ) ( sigma - aldrich ) ) and in the presence and absence of 250 units 
  of benzonase ( merck ) . in an effort to determine the mechanism of ddx19 action and of its 
  regulation , we determined the crystal structures of human ddx19 ( residues 
  glu  glu , encompassing the two conserved 
  domains ) as a complex with adp and as a ternary complex with adpnp and rna 
  ( fig . 1 ; see supplemental table 
  s1 for details of data collection and refinement statistics ) . both structures 
  revealed the canonical dexd / h - box rna helicase n - terminal atpase and 
  c - terminal helicase domains in the typical arrangement , with the two domains 
  coming together to form the nucleotide binding cleft . the structure of rna - free ddx19 bound to adp revealed a surprising feature 
  ( fig . the section 
  asp  ser of the n - terminal extension , which was 
  partly disordered in the rna complex , folded into an -helix that 
  occupied the central cleft between the two lobes . the helix extended from the 
  rna binding site at its n terminus to the nucleotide binding site , where it 
  contacted the phosphate groups of adp ( fig . 
  1 , b and e ) . the helix is predicted to disrupt 
  the atp binding site as it displaces the arginine finger ( arg ) 
  that is essential for the atpase reaction to occur 
  ( fig . 1 , a , c , e , and 
  f ) ( 6 , 
  28 ) . a similar placement of a 
  secondary structural element has never been observed in a dexd / h - box 
  protein . ser -helix makes a 
  significant number of side chain interactions with both conserved domains 
  ( fig . 1b ) . the loop 
  leading out of the c terminus of the -helix hydrogen bonds with two 
  hydroxyls of the nucleotide ribose ( the backbone amide of asn 
  with the 3 hydroxyl and the backbone amide of leu with the 
  ring oxygen ) , suggesting selective binding to nucleotides at that site 
  ( fig . 1 , b and 
  e ) . the significance of these interactions is supported 
  by the conservation of the sequence of this n - terminal flanking segment , as 
  well as the side chains it makes contacts with , in the dexd / h - box rna 
  helicase , ddx25/grth ( fig . the structure of the ternary complex with a decauracil ( u10 ) 
  mrna mimic and the non - hydrolyzable nucleotide , adpnp , shows that both lobes 
  contribute to nucleotide and rna binding 
  ( fig . six bases of the rna 
  molecule are visible in the electron density , positioned across the top of the 
  cleft ; this mode of binding is consistent with the mechanism of rna binding 
  predicted for other dexd / h - box rna helicases , as determined by the 
  structures of the complexes of rna with ddx48/elf4aiii 
  ( 12 , 
  13 ) and vasa 
  ( 14 ) . also common to the 
  mechanisms of the dexd / h - box rna helicase interactions with rna is a 
  kink that the proteins introduce in the rna backbone between u4 and u5 . figure 1.structure of human ddx19 . a , overview of ddx19 with adp 
  bound and the n - terminal flanking helix in the central cleft . the 
  arg side chain that acts as an arginine finger is presented as 
  sticks . b , schematic representation of the cleft - inserted helix with 
  the two conserved domains of the protein , shown in the same view as in 
  panel a. residues that are conserved in ddx25 are shown in blue . c , overview of the ddx19-rna complex , with mg - adpnp bound in the central 
  cleft . e , detail of 
  the nucleotide binding site in the open conformation , with the electron 
  density ( 2fobs  fcalc ) for adp 
  rendered at 1.5 . f , detail of the nucleotide binding site in 
  the rna complex , with the electron density ( 2fobs  
  fcalc ) for mg - adpnp rendered at 1.5 . in all 
  panels , the conserved domain-1 ( yellow ) , the conserved domain-2 
  ( red ) , and the n - terminal flanking sequence ( green ) are 
  indicated . a , overview of ddx19 with adp 
  bound and the n - terminal flanking helix in the central cleft . the 
  arg side chain that acts as an arginine finger is presented as 
  sticks . b , schematic representation of the cleft - inserted helix with 
  the two conserved domains of the protein , shown in the same view as in 
  panel a. residues that are conserved in ddx25 are shown in blue . c , overview of the ddx19-rna complex , with mg - adpnp bound in the central 
  cleft . e , detail of 
  the nucleotide binding site in the open conformation , with the electron 
  density ( 2fobs  fcalc ) for adp 
  rendered at 1.5 . f , detail of the nucleotide binding site in 
  the rna complex , with the electron density ( 2fobs  
  fcalc ) for mg - adpnp rendered at 1.5 . in all 
  panels , the conserved domain-1 ( yellow ) , the conserved domain-2 
  ( red ) , and the n - terminal flanking sequence ( green ) are 
  indicated . figure 2.role of the n - terminal flanking sequence in the regulation of ddx19 
  atpase activity . a , schematic diagram of the ddx19 protein 
  constructs used in this study ( not drawn to scale ) . b , relative atpase activities of ddx19 protein 
  constructs in the presence of between 0 and 0.5 mg / ml ssrna . role of the n - terminal flanking sequence in the regulation of ddx19 
  atpase activity . a , schematic diagram of the ddx19 protein 
  constructs used in this study ( not drawn to scale ) . b , relative atpase activities of ddx19 protein 
  constructs in the presence of between 0 and 0.5 mg / ml ssrna . ddx19 differs from ddx48 and vasa in the binding to rna downstream of the 
  kink . in the ddx48 and vasa structures , u5 and u6 remain stacked with each 
  other on the 3 side of the kink , whereas in ddx19 , the u6 base is 
  rotated through 150 degrees around the sugar - phosphate backbone 
  ( fig . this 
  conformation is stabilized by hydrogen - bonding interactions between the 
  2-ribose hydroxyl of u6 and the -amide of lys and 
  between the uracil base and the lys and asp side 
  chains . structure overlays suggest that a similar flipped conformation of u6 
  could not be accommodated by either ddx48 or vasa . the interactions of the cleft - inserted helix with the conserved domains and 
  with the nucleotide attest to the specificity and selectivity of the 
  positioning of the n - terminal extension and point to a potential regulatory 
  mechanism for the intrinsic atpase activity of ddx19 
  ( fig . 1 , b and 
  e ) . to investigate this possibility , we measured the 
  atpase activities of three different ddx19 protein constructs 
  ( fig . 2a ) . in the 
  absence of ssrna , full - length ddx19 ( met  asn ) did not hydrolyze appreciable amounts of atp , but atpase activity was 
  stimulated to a rate of 0.77  0.2 min ( s.d . ) 
  by 0.5 mg / ml ssrna ( fig . the shorter protein construct 
  glu  glu , lacking the native n terminus but 
  containing the cleft insertion helix , hydrolyzed atp at an intermediate rate 
  of 25% of the ssrna - stimulated full - length protein . the construct 
  lys  glu , which also lacked the cleft 
  insertion -helix , displayed the highest turnover of atp ( 200% of 
  the ssrna - stimulated full - length protein ) , again independent of the presence 
  of ssrna . further tests showed that the atpase activities of the ddx19 
  proteins were not stimulated nor inhibited by the presence of double - stranded 
  rna ( supplemental fig . glu ) represses the atpase activity of the 
  core protein but does not confer rna dependence to it . further addition of the 
  native n terminus ( i.e. resulting in full - length ddx19 ) completely 
  abolishes the atpase activity of the protein in the absence of ssrna . the 
  presence of ssrna overcomes the complete inhibition of atpase activity in the 
  full - length protein . the presence of the native n terminus is also required 
  for ssrna concentration - dependent stimulation of atpase activity , indicating 
  that ssrna binding is sufficient to displace the n terminus , release the helix 
  from the cleft , and allow formation of the catalytic triad in the atp binding 
  site . rna- and atp - induced cleft closure has been shown to occur in solution 
  ( 29 ) , and the crystal 
  structures of human ddx19 presented here demonstrate how cleft closure can be 
  utilized in the regulation of atpase activity . our results suggest a model 
  where an -helical segment of the n - terminal flanking region is inserted 
  between the core domains in the rna - free protein . this helix prevents cleft 
  closure and positions the n - terminal flanking sequence in the rna binding 
  groove or in its proximity . rna binding then displaces the native n terminus , 
  releasing the helix from the cleft to allow formation of the closed state and 
  subsequent nucleotide hydrolysis . we predict that this mechanism of 
  autoregulation also applies to ddx25 , the other dexd / h - helicase in 
  which the sequence of the cleft insertion helix is highly conserved , and 
  possibly to other family members .
dexd / h - box rna helicases couple atp hydrolysis to rna remodeling 
  by an unknown mechanism . 
 we used x - ray crystallography and biochemical 
  analysis of the human dexd / h - box protein ddx19 to investigate its 
  regulatory mechanism . 
 the crystal structures of ddx19 , in its rna - bound 
  prehydrolysis and free posthydrolysis state , reveal an -helix that 
  inserts between the conserved domains of the free protein to negatively 
  regulate atpase activity . 
 this finding was corroborated by biochemical data 
  that confirm an autoregulatory function of the n - terminal region of the 
  protein . 
 this is the first study describing crystal structures of a 
  dexd / h - box protein in its open and closed cleft conformations .