protein production  production and purification of 
  recombinant human tpp1 from a chinese hamster ovary ( cho )
expression system 
  were conducted using modifications of methods described previously 
  ( 9 ) .
cells were cultured in 
  1050-cm roller bottles for 9 days with serum - free dulbecco 's 
  modified eagle's / f-12 medium containing 1 g / ml kifunensin .
conditioned 
  media were harvested and adjusted to contain 2.5 m nacl as well as 
  20 mm tris , ph 8.0 , and 20 m cacl2 ( tc 
  buffer ) .
the solution was clarified by centrifugation and filtration and 
  then applied to a butyl - sepharose 4 fast - flow column ( ge healthcare ) .
after 
  washing with 2.5 m nacl in tc buffer , the column was eluted with a 
  7.5-column volume linear gradient of 2.5 to 0 m nacl in tc buffer . 
 
fractions containing most of the pro - tpp1 were concentrated and equilibrated 
  with 20 mm nacl / tc buffer by diafiltration .
the solution was 
  filtered , loaded onto a mono q12 column ( bio - rad ) , and washed with 4 column 
  volumes of 20 mm nacl in tc buffer .
the column was eluted with 10 
  column volumes of a linear gradient of 20 to 515 mm nacl in tc 
  buffer .
peak fractions containing pro - tpp1 were adjusted to contain 100 
  mm sodium citrate , ph 5.5 .
protein was deglycosylated by incubation 
  with endo hf ( new england biolabs , 26,500 units per mg of pro - tpp1 ) 
  at 25 c for 15 h followed by 1 h at 37 c .
the solution was buffer 
  exchanged and repurified by mono q12 chromatography as described above . 
 
fractions containing pro - tpp1 were concentrated and applied to a superdex 75 
  column equilibrated in 150 mm nacl in tc buffer to separate 
  pro - tpp1 monomer from a minor amount of aggregated material .
a similar procedure was used to isolate glycosylated pro - tpp1 , omitting 
  kifunensin from the cell culture media and the endo h digestion and second 
  mono q12 chromatography steps .
hexahistidine - tagged human pro - tpp1 was 
  produced using an insect cell system and purified as described previously 
  ( 14 ) except that all column 
  buffers contained 20 m cacl2 , and a final superdex 
  75 gel filtration step was performed as described above . tpp1 activation kinetics 
proenzyme was diluted to the 
  desired concentration using 0.1% triton x-100 , 150 mm nacl , 20 
  mm tris , ph 8.0 . for experiments using hexahistidine tpp1 ,
the 
  protein was preincubated with 1 mm e-64 to inhibit an insect cell 
  thiol protease contaminant that was present in trace amounts in some 
  preparations .
proenzyme ( 20 l ) was mixed with 180 l of 0.1% triton 
  x-100 , 150 mm nacl , and 100 mm sodium formate adjusted 
  with hcl to the indicated ph .
the reaction was incubated at 25 c for the 
  indicated time , and the activation process was terminated by diluting 1 part 
  sample into 9 parts ice - cold 0.1% triton x-100 , 150 mm nacl , 500 
  mm sodium acetate , ph 5.0 , frozen on dry ice , and stored at -80 
  c .
the amount of enzymatically active tpp1 ( total final tpp1 
  concentration typically 1.5 nm ) was measured using the previously 
  described kinetic fluorescence assay 
  ( 35 ) using 200 
  m ala - ala - phe - amc substrate in 0.1% triton x-100 , 150 
  mm nacl , 500 mm sodium acetate , ph 5.0 .
data for 
  activation conducted at ph 4.0 were fit to a one - phase exponential association 
  model using built in functions in prism version 5.01 ( graphpad software ) .
the 
  activation reactions at ph 2.5 to 3.5 were fit to an irreversible consecutive 
  reaction model where the inactive tpp1 zymogen ( z ) is converted to an 
  intermediate ( i ) with partial activity , which is then converted into a fully 
  active mature form ( m ) as shown in reaction 1 , using this model , one can calculate the activity at any given time from 
  equation 1 , 
  ( eq.1)\documentclass[10pt]{article } 
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 \begin{equation*}\;\;v_{t}\hspace{1em}=\hspace{1em}v_{max}({\alpha}_{1}[{\mathrm{z}}]_{t}\hspace{1em}+\hspace{1em}{\alpha}_{2}[{\mathrm{i}}]_{t}\hspace{1em}+\hspace{1em}{\alpha}_{3}[{\mathrm{m}}]_{t})\;\;\end{equation*}\end{document } 
  here vt denotes the observed tpp1 enzymatic activity at 
  any given time ; vmax denotes the enzymatic activity of the 
  preparation when existing as the fully activated form ;  denotes the 
  relative enzymatic activity of the different forms , and for any given time 
  [ z]t + [ i]t + [ m]t 
  is a constant that corresponds to the total added tpp1 , with all existing as z 
  at time = 0 .
36 using prism version 5.01 , 
  with the following constraints : no activity for the zymogen 
  ( 1 = 0 ) , partial activity for the intermediate form ( 0  
  2  1 ) , and maximal activity for the mature form 
  ( 3 = 1 ) .
pro - tpp1 was activated as described above . 
  for matrix - assisted laser desorption ionization time - of - flight mass 
  spectrometry ( maldi - tof ms ) ,
reactions were terminated by mixing with an equal 
  volume of ice - cold solution containing 0.5% trifluoroacetic acid , 50% 
  acetonitrile , mixed with sinapinic acid matrix , and analyzed using an applied 
  biosystems / mds - sciex 4800 mass spectrometer operated in the linear mode . for 
  sds - page analysis , reactions were terminated using reducing sample buffer and 
  fractionated on 10% nupage bistris gels using mops running buffer 
  ( invitrogen ) .
gels were either stained using sypro ruby ( molecular probes ) or 
  transferred to membranes , stained with coomassie blue , and relevant regions of 
  the membrane excised and sequenced as described previously 
  ( 9 , 
  14 ) .
crystallization of pro - tpp1purified endo h - deglycosylated 
  pro - tpp1 was dialyzed into tc buffer containing 100 mm nacl , 
  concentrated to 7 mg / ml using a 30,000 centrifugal device ( millipore ) , and 
  filtered through 0.22-m pore size cellulose acetate filters ( corning 
  costar ) .
initial crystallization conditions were identified by high throughput 
  screening at the hauptman woodward medical research institute , buffalo , ny . in 
  the optimized condition , crystals of pro - tpp1 ranging in size between 50 
   100  100 m and 50  100  300 m appeared in 
  3 days when set up and grown at 4 c using hanging drop diffusion by 
  mixing 1 l of pro - tpp1 solution at 5 mg / ml and 1 l of reservoir 
  solution containing 0.1 m citrate , 4 - 7% polyethylene glycol 6000 , 
  ph 5.0 .
data for two native , one mercury - derivatized , and one 
  lead - derivatized crystals were collected to 1.85  at x4c at the 
  national synchrotron light source , brookhaven national laboratory , upton , ny . 
  the lead and mercury derivatives were obtained by soaking the crystals 
  overnight at 4 c in reservoir solutions containing heavy atom salts at 
  concentrations of 2.5 mm pb(no3)2 and 0.5 
  mm c2h5hgcl .
cryoprotection was achieved with 
  passage of the crystals held in cryoloops through 1-l drops of 50% 
  ethylene glycol for 1 s and flash - cooling in liquid n2 stream . for 
  all crystals ,
all four data sets were processed and scaled in the primitive orthorhombic 
  p212121 space group using denzo and scalepack 
  ( 37 ) 
  ( table 1 and supplemental table 
  s1 ) .
initial attempts for structure solution with molecular replacement using 
  a homology model of tpp1 ( 33 ) 
  or homologous structures ( 24 , 
  27 , 
  28 ) did not yield reliable 
  solutions .
furthermore , attempts for solving the structure by multiple 
  isomorphous replacement using multiple heavy atom derivatives were 
  unsuccessful because of lack of isomorphism between the datasets ( supplemental 
  table s1 ) .
subsequently , the crystal structure was solved by single wavelength 
  anomalous dispersion ( sad ) technique using the mercury dataset .
four mercury 
  sites were obtained from the anomalous differences using shelx programs 
  ( 38 ) with the hkl2map software 
  ( 39 ) .
the sites were used for 
  sad phasing at 2.5  resolution through solve / resolve 
  ( 40 , 
  41 ) as implemented in the 
  software package phenix ( 42 ) . 
  taking advantage of the anisomorphism among the data sets ,
the sad phases were 
  improved and extended to 1.9  resolution through multiple crystal form 
  averaging four data sets ( two native , mercury - soaked and lead - soaked ) using 
  dmmulti ( 43 ) integrated into 
  ccp4 version 6.0 package ( 44 ) . 
 
the initial model consisting of 511 of the 544 residues was obtained by 
  automated chain tracing with the arp / warp routine 
  ( 45 ) using the averaged 
  phases .
the remaining residues were modeled following iterative cycles of 
  maximum likelihood and isotropic temperature refinement with refmac 5.1.2.4 
  ( 46 ) , and manual model 
  building using coot ( 47 ) .
the 
  translation / libration / screw ( tls ) motion determination server 
  ( 48 - 50 ) 
  was used to determine the optimal number of tls groups ( five groups used ) and 
  to generate parameters that were used in the final stages of refinement in 
  refmac 5.1.2.4 ( 46 ) .
initial 
  models for solvent molecules were obtained from the hic - up server 
  ( 51 ) .
the final model , 
  refined to 1.85  with rwork and 
  rfree of 0.179 and 0.205 , respectively , includes all 544 
  residues , 1 bound ca ion , 4 
  n - acetyl - d - glucosamine ( glcnac ) groups , 4 ethylene glycol 
  molecules , and 237 water oxygen atoms 
  ( table 1 ) .
the structure was 
  validated using the molprobity server 
  ( 52 ) , which reported 97.05% of 
  the residues in the ramachandran favored regions .
loop regions 
  thr - leu and glu - thr 
  were modeled using a combination of composite omit and difference fourier maps 
  calculated with map algorithms within cns version 1.2 
  ( 53 ) , and side chain 
  conformations for residues in the aforesaid loops as well as for residues 
  lys , glu , gln , arg , 
  and arg were modeled as the most common rotamer , because of 
  disordered electron density corresponding to these regions .
table 1data collection and refinement statisticsvalues corresponding to highest resolution shells are shown in 
  parentheses .
data collection and refinement statistics values corresponding to highest resolution shells are shown in 
  parentheses .
structural analyses  superposition of structural models was 
  accomplished using lsqkab ( 54 ) 
  from the ccp4 program suite version 6.0 
  ( 44 ) .
structural comparisons 
  were made using the secondary structure matching server 
  ( 55 ) , and overall root mean 
  square deviation ( r.m.s.d . )
the protein - protein 
  interaction server ( 56 , 
  57 ) , the imoltalk server 
  ( 58 ) , and the whatif web 
  interface ( 59 ) were used for 
  analyses of interdomain and lattice contacts .
buried surface areas were 
  calculated using cns version 1.2 
  ( 53 ) , and all structural 
  figures were generated using pymol version 1.0 
  ( 60 ) .
electrostatic potential calculations  analysis of ph 
  dependence on interdomain binding energy in pro - tpp1 was accomplished using 
  the adaptive poisson - boltzmann solver version 0.5.1 ( apbs ) 
  ( 61 ) .
the pdb2pqr server 
  ( 62 , 
  63 ) implementing the parse 
  forcefield and the propka program 
  ( 64 ) to assign specific 
  protonation states at different ph conditions ( ph 2.5 - 8.0 ) was used to convert 
  the pro - tpp1 protein data bank ( pdb ) coordinates to three sets of pqr files , 
  one for the intact zymogen ( residues 20 - 563 ) ( zph ) , one for the 
  propiece ( residues 20 - 195 ) ( pph ) , and the third for the mature 
  domain ( residues 196 - 563 ) ( mph ) for a given ph condition .
these 
  ph - specific pqr files were used as input for electrostatic binding energy 
  calculations at that ph using a linearized poisson - boltzmann equation ( pb ) 
  using cubic b - spline discretization of the charge distributions .
pb 
  calculations were performed at 298 k with a dielectric constant of 78.0 for 
  water and 2.0 for the protein interior .
the ion concentrations were set to 
  0.15 m with ionic radius of 2.0  .
care was taken to remove 
  self - interaction energies by using exactly the same grid spacing , length and 
  center , determined by the psize.py script provided in the apbs distribution . 
 
the electrostatic binding energy at a given ph ( beph ) is 
  calculated by apbs as shown in equation 2 , 
  ( eq.2)\documentclass[10pt]{article } 
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 \begin{equation*}\;\;be_{{\mathrm{ph}}}\hspace{1em}=\hspace{1em}z_{{\mathrm{ph}}}\hspace{1em}-\hspace{1em}(p_{{\mathrm{ph}}}\hspace{1em}+\hspace{1em}m_{{\mathrm{ph}}})\;\;\end{equation*}\end{document } 

production of deglycosylated tpp1initial attempts to obtain 
  suitable crystals from previously characterized glycosylated recombinant human 
  pro - tpp1 produced in insect cells 
  ( 14 ) and in cho cells 
  ( 9 ) were unsuccessful ( data not 
  shown).5 given that 
  heterogeneity in glycosylation may have been a complicating factor , we tried 
  different methods to deglycosylate the protein produced from cho cells .
in our 
  hands , peptide : n - glycosidase f was not completely effective in 
  removing oligosaccharides unless the protein was denatured , suggesting that 
  steric hindrance prevented cleavage of the glcnac - asn bond .
endo h , which 
  cleaves between the first two asn - linked glc - nac residues , was partially 
  effective in deglycosylating the native molecule , but some n - linked 
  glycans were retained , reflecting the presence of oligosaccharides that had 
  been processed to endo h - resistant forms 
  ( 15 , 
  65 ) .
we therefore cultured the 
  cho cells in the presence of the endoplasmic reticulum mannosidase i inhibitor 
  kifunensin , which prevents further processing of n - linked glycans to 
  endo h - resistant forms ( 66 ) . 
  when treated with endo h ,
pro - tpp1 produced in this manner yielded a single 
  species by sds - page analysis , corresponding to a deglycosylated form in which 
  four single asn - linked glc - nac monosaccharides remain , as confirmed by the 
  crystal structure ( supplemental fig .
properties of deglycosylated tpp1glycosylation has been 
  reported to be critical for the folding , processing , and/or stability of tpp1 
  in cultured cells ( 17 , 
  67 ) . in this study , it was 
  therefore important to compare the functional properties of deglycosylated 
  tpp1 prepared for structural studies and the previously well characterized 
  glycosylated native protein .
purified pro - tpp1 is a zymogen that undergoes low ph - triggered 
  autoactivation and processing 
  ( 9 , 
  13 , 
  14 , 
  16 , 
  68 ) .
the kinetics for 
  autoactivation of deglycosylated pro - tpp1 were essentially identical to those 
  for glycosylated protein produced from untreated cho cells or a 
  hexahistidine - tagged protein produced from insect cells 
  ( fig .
rates were greatly 
  accelerated at lower ph values and showed biphasic kinetics at ph 3.5 and 
  below ( fig .
1 ) . we also measured the activity of preactivated glycosylated and 
  deglycosylated tpp1 using two different fluorogenic substrates , 
  ala - ala - phe - amc and arg - nle - nle - amc .
the kcat 
  values ( vmax / protein concentration ) for the deglycosylated 
  and glycosylated proteins were similar 
  ( fig .
although it appeared that the deglycosylated protein 
  had slightly higher activity , the difference fell within the variation that we 
  typically observe with independent preparations of glycosylated tpp1 .
2 , a and 
  b ) and substrate specificity constant 
  ( kcat / km ) 
  ( fig .
our results do not rule out a role for glycosylation in folding or trafficking 
  of pro - tpp1 , nor do they address the role of the terminal asn - linked glcnac 
  that remains after endo h - deglycosylation .
however , they do demonstrate that 
  endo h - deglycosylated tpp1 is equivalent in terms of autocatalytic and 
  enzymatic function to fully glycosylated tpp1 .
highly purified human recombinant tpp1 
  preparations ( filled circles , hexahistidine - tagged pro - tpp1 from an 
  insect cell expression system ; open circles , fully glycosylated 
  pro - tpp1 from cho cells ; and open squares , endo h - deglycosylated 
  pro - tpp1 from kifunensin - treated cho cells ) were incubated at the indicated ph 
  for the specified time before stopping the reaction and measuring tpp1 
  activity as described under  experimental procedures .
 data for 
  each experiment were normalized to plateau values , all of which were 
  comparable .
the curves shown are fit to the reaction curves for the 
  hexahistidine - tagged pro - tpp1 preparation as described under 
   experimental procedures . 
highly purified human recombinant tpp1 
  preparations ( filled circles , hexahistidine - tagged pro - tpp1 from an 
  insect cell expression system ; open circles , fully glycosylated 
  pro - tpp1 from cho cells ; and open squares , endo h - deglycosylated 
  pro - tpp1 from kifunensin - treated cho cells ) were incubated at the indicated ph 
  for the specified time before stopping the reaction and measuring tpp1 
  activity as described under  experimental procedures . 
data for 
  each experiment were normalized to plateau values , all of which were 
  comparable .
the curves shown are fit to the reaction curves for the 
  hexahistidine - tagged pro - tpp1 preparation as described under 
   experimental procedures .
 figure 2.enzymatic properties of glycosylated and endo h - deglycosylated tpp1 . 
  cho cell - produced enzyme preparations described in 
  fig .
enzyme activity measurements were conducted using eight 
  different concentrations of either ala - ala - phe - amc ( 800 to 6.25 
  m ) or arg - nle - nle - amc ( 400 to 3.12 m ) substrate . 
 
final reactions contained 150 mm nacl , 0.1% triton x-100 , and 
  either 100 mm sodium formate , ph 2.5 , or sodium acetate ( all other 
  ph values ) adjusted to the indicated ph using hcl .
kinetic constants were 
  calculated using the built - in michaelis - menten curve fitting functions in 
  prism version 5.01 .
c , circles , 
  ala - ala - phe - amc substrate ; squares , arg - nle - nle - amc substrate .
enzyme activity measurements were conducted using eight 
  different concentrations of either ala - ala - phe - amc ( 800 to 6.25 
  m ) or arg - nle - nle - amc ( 400 to 3.12 m ) substrate . 
 
final reactions contained 150 mm nacl , 0.1% triton x-100 , and 
  either 100 mm sodium formate , ph 2.5 , or sodium acetate ( all other 
  ph values ) adjusted to the indicated ph using hcl .
kinetic constants were 
  calculated using the built - in michaelis - menten curve fitting functions in 
  prism version 5.01 .
c , circles , 
  ala - ala - phe - amc substrate ; squares , arg - nle - nle - amc substrate .
crystal structure of pro - tpp1 reveals a conserved subtilisinlike 
  fold  we have determined the crystal structure of the inactive 
  precursor of endo h - deglycosylated form of tpp1 at 1.85  resolution 
  ( supplemental fig . s1 and fig . 
 
the data processed in the 
  p212121 space group with one molecule in the 
  asymmetric unit with a mathhews coefficient and solvent content of 2.37 
   / da and 48.0% , respectively .
the structure is markedly 
  similar to the glycosylated form of pro - tpp1 determined at 2.35  
  resolution in the p21212 space group with two molecules 
  in the asymmetric unit ( pdb i d 3ee6 ) .
superposition of c 
  coordinates of deglycosylated pro - tpp1 with those of the two chains of 
  glycosylated pro - tpp1 yield r.m.s.d .
values of 0.61 and 0.66  , 
  respectively , with differences in positioning of a few solvent exposed loops 
  and conformations of a couple of active site residues ( as discussed below ) . 
 
pro - tpp1 exhibits a characteristic bi - domain precursor structure with a 
  c - shaped prodomain ( residues ser - phe ) tightly 
  packed against a more globular catalytic domain ( residues 
  leu - pro ) connected by a 24-residue linker 
  ( gly - gly ) .
the propiece has an 
  antiparallel-/antiparallel- fold and the catalytic domain has a 
  typical subtilisin - like fold . the n - terminal portion ( residues 172 - 179 ) of the propiece linker is well 
  ordered and threads through the substrate - binding groove making strong short 
  distance contacts ( one hydrogen bond , one salt bridge , and three hydrophobic 
  interactions ) with the catalytic domain .
in contrast , the remaining portion of 
  the linker ( residues 180 - 195 ) loops out of the interface making only minimal 
  long range van der waal contacts .
the conformation of this portion of the 
  linker is stabilized by a few strong lattice contacts involving residues 
  180 - 190 as discussed below .
interestingly , the well structured nature of the 
  n - terminal portion of the linker is supported by identical conformations and 
  its involvement in interdomain contacts in both glycosylated and 
  deglycosylated structures .
the flexible nature of the c - terminal portion of 
  the linker is emphasized by its involvement in lattice contacts in the 
  deglycosylated structure and by its absence in the glycosylated structure , 
  possibly because of disorder .
furthermore , a tls motion analysis 
  ( 48 - 50 ) 
  for evidence of thermal motion detects significant flexibility in the linker 
  region as compared with the rest of the protein ( supplemental fig .
such 
  flexibility may be important in positioning the linker for autocatalytic 
  cleavage during zymogen activation ( see below ) .
stereo view of pro - tpp1 backbone 
  depicted in coil representation with the propiece shown in cyan and 
  blue and the catalytic domain in gold and red , for 
   helices and  strands , respectively , and the linker loop shown in 
  magenta .
side chains of active site catalytic triad 
  ( ser , glu , and asp ) and oxyanion 
  hole ( asp ) residues are shown in cpk mode .
glycosylation sites 
  with residual glcnac moieties ( circles , asn , 
  1 ; asn , 2 ; asn , 3 ; and 
  asn , 4 ) and disulfide bridges ( squares , 
  cys - cys , 1 ; 
  cys - cys , 2 ; and 
  cys - cys , 3 ) are depicted in stick 
  mode .
stereo view of pro - tpp1 backbone 
  depicted in coil representation with the propiece shown in cyan and 
  blue and the catalytic domain in gold and red , for 
   helices and  strands , respectively , and the linker loop shown in 
  magenta .
side chains of active site catalytic triad 
  ( ser , glu , and asp ) and oxyanion 
  hole ( asp ) residues are shown in cpk mode .
glycosylation sites 
  with residual glcnac moieties ( circles , asn , 
  1 ; asn , 2 ; asn , 3 ; and 
  asn , 4 ) and disulfide bridges ( squares , 
  cys - cys , 1 ; 
  cys - cys , 2 ; and 
  cys - cys , 3 ) are depicted in stick 
  mode .
the structure has marked similarities with those of other s53 family 
  members ( 24 , 
  27 , 
  28 , 
  30 ) in terms of the overall 
  fold , barring minor differences in the presence or absence of a few secondary 
  structural elements in both the propiece and mature domain and orientation of 
  the linker ( supplemental figs .
superpositions of 
  c coordinates of corresponding regions of pro - tpp1 with 
  propiece of prokumamolisin ( pdb i d 1t1e ) 
  ( 28 ) , and mature enzymes of 
  sedolisin ( pdb i d 1ga4 ) ( 24 ) , 
  kumamolisin ( pdb i d 1gtg ) 
  ( 27 ) , and kumamolisin - as ( pdb 
  i d 1sn7 ) ( 30 ) yield r.m.s.d . 
 
values of 2.17 , 2.07 , 1.81 , and 1.92  , respectively . compared with 
  prokumamolisin , the propiece and mature domain in pro - tpp1 are more compact 
  with 550  more buried surface area than the former , whereas 
  the respective linkers are oriented at an angle of 30 ( supplemental 
  fig .
the total buried surface area between the propiece and mature domain 
  in pro - tpp1 is 2200 a with 22 direct and 10 water - mediated 
  hydrogen bonds , 3 salt bridges , and 6 hydrophobic interactions ( supplemental 
  fig . s5 and supplemental table s2 ) .
key features of the s53 family members , including c 
  positions of catalytic residues forming the charge transfer complex 
  ( viz .
ser , glu , asp , and 
  asp ) , two cis - peptide bonds , and the calcium - binding 
  site are conserved .
three cis - pro residues are found in pro - tpp1 
  ( pro , pro , and pro ) , of which the 
  former two are completely conserved in the bacterial homologs ( supplemental 
  fig .
s3 ) . the structural role and binding partners of the octahedrally 
  coordinated calcium ion , including the side chain carboxyl groups of 
  asp and asp , backbone carbonyl groups of 
  val , gly , and gly , and a bound water 
  molecule , are conserved within the s53 family .
in addition , three disulfide 
  linkages unique to pro - tpp1 ( viz . 
 
cys - cys , cys - cys , and 
  cys - cys ) stabilize the fold .
the lone 
  cys is completely buried within the core of the mature domain 
  and is within hydrogen bonding distance of the side chain carboxyl of 
  asp ( see below ) .
endo h treatment leaves one glcnac moiety at each n - linked 
  glycosylation site .
asn , 
  asn , asn , asn , and 
  asn ) are utilized 
  ( 67 ) .
we find clear evidence 
  for single glcnac moieties at all sites except for asn , a 
  surface residue , which is clearly not modified in our structure .
the structure 
  of the glycosylated form of pro - tpp1 also shows that asn is not 
  utilized ( pdb i d 3ee6 ) .
it is formally possible that this site is 
  heterogeneously glycosylated and that crystallization conditions have selected 
  out the four - sugar glycosylated form from a population of four- and five - sugar 
  forms . in our structure ,
the glcnac moiety linked to asn 
  participates in lattice contacts with a symmetry - related molecule 
  ( supplemental fig . s6 and supplemental table s3 ) .
active site of pro - tpp1 and maintenance of the zymogen in the inactive 
  state  the active site in pro - tpp1 contains an sed 
  ( ser - glu - asp ) catalytic triad , 
  similar to all other members of the s53 family of serine peptidases and 
  contrasting with the shd catalytic triad of canonical serine peptidases .
the 
  replacement of the his residue with the acidic residue glu in the s53 family 
  allows the active site serine to function in nucleophilic attack of substrate 
  at acidic ph ( 14 ) , with the 
  glu abstracting the hydroxyl proton from the serine 
  ( 14 , 
  23 - 26 , 
  34 ) . in addition
, there is a 
  second conserved asp residue ( asp ) , designated here as d , 
  in place of a conserved asn in the subtilisin family .
the role of the latter 
  is to bind and stabilize the oxyanion of the tetrahedral intermediate of the 
  peptidase reaction by hydrogen bond interactions , as described by nmr analyses 
  and high resolution crystal structures of members of the subtilisin family 
  bound to inhibitors acting as transition - state analogs that mimic the 
  tetrahedral intermediate ( reviewed in ref . 
 
the catalytic efficiency 
  in proteolytic enzymes is significantly increased by transition - state 
  stabilization of the tetrahedral intermediate in the reaction .
computational 
  studies on kumamolisin - as suggest that the d residue has multiple roles 
  in catalyzing peptide bond hydrolysis , serving as a general acid to protonate 
  the substrate during formation of the tetrahedral intermediate and 
  subsequently functioning as a general base , extracting a proton from the 
  tetrahedral intermediate during conversion to the acyl enzyme 
  ( 73 , 
  74 ) . in tpp1 , these roles are 
  likely to be performed by asp . the d residue is conserved 
  in the s53 family , suggesting a common proton transfer mechanism that 
  distinguishes these enzymes from canonical serine proteases . figure 4.comparison of the active site of pro - tpp1 zymogen with that of mature 
  kumamolisin .
backbones are shown as ribbon representations with 
  featured side chains in ball - and - stick mode , bound water molecules 
  depicted as red spheres , and hydrogen bonds shown as dashed 
  lines with distances in  . a , stereo view of tpp1 showing 
  catalytic residues in a conformation that is recalcitrant to activity . in 
  tpp1 ,
glu ( e ) , asp ( d ) , and 
  ser ( s ) form the catalytic triad , and asp 
  ( d ) forms part of the stabilizing oxyanion hole . 
 
side chain oxygen atoms are shown in red and backbone 
  carbonyl oxygens in yellow .
a bound water molecule ( 1 ) occupies the position 
  expected for one of the carboxyl oxygens of glu , if the active 
  site were poised for activity .
one of the carboxyl oxygens of 
  asp forms a hydrogen bond to a water molecule ( 2 ) , and 
  the other is within weak hydrogen bond distance to the sulfhydryl of unpaired 
  cys .
the side chain hydroxyls of ser and 
  ser participate in a strong hydrogen bond .
a third water 
  molecule ( 3 ) makes a strong hydrogen bond with the ser 
  hydroxyl and is also within hydrogen bond distance of the asp 
  carboxyl constituting the oxyanion hole , close to where the carbonyl oxygen of 
  the scissile peptide bond is expected to be positioned .
b , comparison 
  of catalytic machinery of pro - tpp1 with that in mature kumamolisin .
least 
  square superpositions of c coordinates of the pro - tpp1 
  catalytic domain with those of all available structures of s53 family members 
  were performed and yielded similar results .
superposition of tpp1 with mature 
  kumamolisin ( pdb i d 1gtl ) ( 27 ) 
  is shown with ribbon depictions in gray and green , 
  respectively .
although the corresponding oxyanion d residues in tpp1 
  and kumamolisin are in similar conformations , the corresponding catalytic glu 
  and asp residues are both in different conformations .
furthermore , the 
  s residue in tpp1 ( ser ) is in a different rotameric 
  conformation , and its c position is displaced by 1.5  
  from the corresponding s ( ser ) in kumamolisin , resulting 
  in different hydrogen bond schemes with the active site ser .
comparison of the active site of pro - tpp1 zymogen with that of mature 
  kumamolisin .
backbones are shown as ribbon representations with 
  featured side chains in ball - and - stick mode , bound water molecules 
  depicted as red spheres , and hydrogen bonds shown as dashed 
  lines with distances in  .
a , stereo view of tpp1 showing 
  catalytic residues in a conformation that is recalcitrant to activity . in 
  tpp1 ,
glu ( e ) , asp ( d ) , and 
  ser ( s ) form the catalytic triad , and asp 
  ( d ) forms part of the stabilizing oxyanion hole . 
  ser ( s ) is completely conserved in the s53 
  family .
side chain oxygen atoms are shown in red and backbone 
  carbonyl oxygens in yellow .
a bound water molecule ( 1 ) occupies the position 
  expected for one of the carboxyl oxygens of glu , if the active 
  site were poised for activity .
one of the carboxyl oxygens of 
  asp forms a hydrogen bond to a water molecule ( 2 ) , and 
  the other is within weak hydrogen bond distance to the sulfhydryl of unpaired 
  cys .
the side chain hydroxyls of ser and 
  ser participate in a strong hydrogen bond .
a third water 
  molecule ( 3 ) makes a strong hydrogen bond with the ser 
  hydroxyl and is also within hydrogen bond distance of the asp 
  carboxyl constituting the oxyanion hole , close to where the carbonyl oxygen of 
  the scissile peptide bond is expected to be positioned .
b , comparison 
  of catalytic machinery of pro - tpp1 with that in mature kumamolisin .
least 
  square superpositions of c coordinates of the pro - tpp1 
  catalytic domain with those of all available structures of s53 family members 
  were performed and yielded similar results .
superposition of tpp1 with mature 
  kumamolisin ( pdb i d 1gtl ) ( 27 ) 
  is shown with ribbon depictions in gray and green , 
  respectively . although the corresponding oxyanion d residues in tpp1 
  and kumamolisin are in similar conformations , the corresponding catalytic glu 
  and asp residues are both in different conformations .
furthermore , the 
  s residue in tpp1 ( ser ) is in a different rotameric 
  conformation , and its c position is displaced by 1.5  
  from the corresponding s ( ser ) in kumamolisin , resulting 
  in different hydrogen bond schemes with the active site ser .
the crystal structure of pro - tpp1 reveals a number of features that help 
  maintain the zymogen in an inactive state , with the active site assuming a 
  geometry that is suboptimal for catalysis .
low ph - mediated pro - tpp1 zymogen 
  maturation is likely to be associated with different positioning of key 
  elements at the active site as well as different protonation states of active 
  site carboxylates that might favor conformations poised for catalysis .
although the c positions of key catalytic residues , 
  including sedd are superimposable with those of corresponding residues 
  in all previously determined structures of bacterial homologs 
  ( 24 , 
  27 , 
  28 , 
  30 ) , the side chain 
  conformations and hydrogen bond network at the active site in pro - tpp1 are 
  different ( fig .
as revealed 
  by several high resolution crystal structures of free and inhibitor - bound 
  mature enzymes of sedolisin , kumamolisin , and kumamolisin - as , the geometry of 
  an active site poised for catalysis is characterized by a charge transfer 
  complex involving the key sed residues with hydrogen bonds between one of the 
  side chain carboxyl oxygens of the glu with the hydroxyl of the ser and the 
  other with one of the carboxyl oxygens of the asp .
in contrast , in pro - tpp1 , 
  side chains of both glu and asp are directed away 
  from each other and from the active site ser .
such alternate 
  rotameric conformations of these catalytic residues allow hydrogen bonds 
  distinct from those characteristic of a charge transfer complex 
  ( fig .
propka predictions 
  ( 64 ) yield 
  pka values of 3.6 and 6.4 for glu and 
  asp in the mature enzyme , respectively ( supplemental table s4 ) . 
  from this
it follows that such rotameric conformations for the glu and the 
  asp , as observed in pro - tpp1 , might arise from electrostatic charge repulsions 
  and could likely be favored at neutral ph , at which both of these residues are 
  predicted to be deprotonated and negatively charged .
however , at the 
  crystallization ph condition , ph 5.0 , only glu and not 
  asp is predicted to be deprotonated .
the active site glu is also 
  found in dual conformations in some inhibitor complexes in structures of 
  homologs crystallized at higher ph 
  ( 25 ) .
in addition , the 
  conformations of glu and asp in the glycosylated 
  form of pro - tpp1 are similar to those found in mature enzymes of bacterial 
  homologs .
these results suggest that the acidic residues at the active site 
  most likely exist in equilibrium between alternate rotameric states and that 
  different conditions favoring different ph - driven protonation states increase 
  the probability of one state over another .
the model was 
  constructed by docking a hexapeptide substrate , aaffaa , with a fixed position 
  of the carbonyl oxygen of the p1 residue ( phe ) relative to the catalytic 
  domain of tpp1 , based on the prokumamolisin structure ( pdb i d 1t1e ) 
  ( 28 ) , onto the catalytic 
  domain of tpp1 in which the side chains of active site residues 
  glu and asp were reoriented in the presumed active 
  conformation as found in mature bacterial homologs . a ribbon diagram and a 
  transparent surface rendition of the catalytic domain
are shown in 
  gray , a ball - and - stick depiction of the modeled hexapeptide 
  substrate in yellow , and the superimposed linker with side chains of 
  featured residues shown in ball - and - stick mode in cyan . 
 
this model is an 
  approximation because the positions of the m3 helix with 
  glu and asp as well as m4 with 
  ser together with the m4-m5 loop 
  with asp are likely to differ in the mature enzyme 
  ( fig .
the n terminus of the 
  tripeptide substrate is likely to interact with the asp side 
  chain carboxyl groups .
the predicted protonation state of asp 
  ( see text ) is consistent with a role in binding to an unsubstituted positively 
  charged n terminus of the p3 residue of a putative substrate , thus providing a 
  possible structural basis for the tripeptidyl peptidase activity .
in addition , 
  in the absence of crystal lattice contacts , positively charged 
  arg of the linker is likely to participate in electrostatic 
  interactions with negatively charged asp of the oxyanion hole , 
  potentially restricting its role in general acid and base catalysis .
the model was 
  constructed by docking a hexapeptide substrate , aaffaa , with a fixed position 
  of the carbonyl oxygen of the p1 residue ( phe ) relative to the catalytic 
  domain of tpp1 , based on the prokumamolisin structure ( pdb i d 1t1e ) 
  ( 28 ) , onto the catalytic 
  domain of tpp1 in which the side chains of active site residues 
  glu and asp were reoriented in the presumed active 
  conformation as found in mature bacterial homologs . a ribbon diagram and a 
  transparent surface rendition of the catalytic domain
are shown in 
  gray , a ball - and - stick depiction of the modeled hexapeptide 
  substrate in yellow , and the superimposed linker with side chains of 
  featured residues shown in ball - and - stick mode in cyan . 
 
this model is an 
  approximation because the positions of the m3 helix with 
  glu and asp as well as m4 with 
  ser together with the m4-m5 loop 
  with asp are likely to differ in the mature enzyme 
  ( fig .
the n terminus of the 
  tripeptide substrate is likely to interact with the asp side 
  chain carboxyl groups .
the predicted protonation state of asp 
  ( see text ) is consistent with a role in binding to an unsubstituted positively 
  charged n terminus of the p3 residue of a putative substrate , thus providing a 
  possible structural basis for the tripeptidyl peptidase activity .
in addition , 
  in the absence of crystal lattice contacts , positively charged 
  arg of the linker is likely to participate in electrostatic 
  interactions with negatively charged asp of the oxyanion hole , 
  potentially restricting its role in general acid and base catalysis .
furthermore , in both structures of the glycosylated and endo 
  h - deglycosylated forms of pro - tpp1 , the side chain hydroxyl group of the 
  active site ser is also involved in a strong hydrogen bond with 
  the side chain hydroxyl of ser at 2.6  .
interestingly , 
  this ser ( designated here as s ) is completely conserved within the s53 
  family ( supplemental fig .
it is noteworthy that in the crystal structures 
  of mature enzymes of the bacterial homologs , the region of the protein 
  containing the conserved s residue exhibits a different conformation 
  and consequently a distinct hydrogen bond geometry with the active site ser 
  ( fig .
, the s residue ser is in a different 
  rotameric conformation , and its c position is displaced by 
  1.5  away from that of the pro - tpp1 s ser .
this 
  geometry supports a hydrogen bond between the backbone carbonyl of the active 
  site ser and the side chain hydroxyl of ser at 2.8 
   ( backbone - side chain bond ) and another between the side chain hydroxyl 
  of the active site ser and the backbone carbonyl of 
  ser at 3.5  ( side chain - backbone bond ) , in place of a 
  strong side chain - side chain hydrogen bond in pro - tpp1 . with lack of 
  mutational data on this residue ,
its contribution to the catalytic machinery 
  is not certain but is worth consideration because of its complete 
  conservation . in pro - tpp1
the differences in positioning of structural 
  elements of this region compared with those in mature enzymes are likely 
  propagated as a result of interdomain contacts involving residues c - terminal 
  to ser , viz . within the 
  m4-m5 loop and the m5 strand 
  ( region iii in supplemental fig .
the side chain conformation of the d residue asp 
  constituting the oxyanion hole is indistinguishable from the corresponding 
  residue in homologous structures except in prokumamolisin where the 
  corresponding asp is in a different rotameric conformation . in 
  addition , a bound water molecule ( water 3 ) occupies a similar position as the 
  backbone carbonyl of the scissile peptide bond of a substrate as observed upon 
  superposition of the pro - tpp1 catalytic domain with an inhibitor bound 
  homologous structure ( 27 ) . 
 
this water makes strong hydrogen bonds with the side chain oxygens of active 
  site ser and asp 
  ( fig .
zymogens are commonly kept in their inactive states by occlusion of their 
  active sites by propeptides that function as competitive inhibitors , occupying 
  the substrate - binding site in a nonhydrolyzable configuration ( reviewed in 
  ref .
75 ) . to test whether the 
  pro - tpp1 linker can function in this role
, we docked a model hexapeptide 
  substrate to the catalytic domain in which the side chain conformations of 
  glu and asp were reoriented to assumed active 
  conformations , based on the structures of mature enzymes of bacterial 
  homologs .
however , when the linker was superimposed on the docked 
  model , the p3 and p3 residues overlap with linker residues 
  pro and arg , respectively , suggesting steric 
  occlusion .
the docking study further suggests that the linker residue 
  arg would hinder approach of the substrate to the s2 and 
  s3 subsites ( fig . 
  5 ) .
in addition , a putative salt bridge between the positively charged 
  arg and negatively charged asp is also likely to 
  render a nonfunctional oxyanion hole .
this is consistent with the 
  pka of 4.7 predicted for asp in the zymogen 
  form ( supplemental table s4 ) .
a corresponding salt bridge between 
  arg and the oxyanion hole asp observed in 
  prokumamolisin has been implicated in substrate - mediated inactivation 
  ( 28 ) . however , in pro - tpp1 
  this salt bridge is absent ( > 5.0  ) , presumably because of crystal 
  lattice interactions involving the linker region c - terminal to 
  arg ( supplemental fig .
s6 ) . structural basis for tripeptidyl peptidase activity  the 
  substrate - binding subsite and the active site pocket are likely to undergo 
  subtle changes in the mature enzyme with different rotameric conformations of 
  catalytic triad and neighboring residues ( such as ser , see 
  above ) . nevertheless ,
a docked model of a hexapeptide substrate aaffaa 
  revealed that the asp side chain is a likely candidate for 
  binding to the positively charged unsubstituted n termini of p3 residues of 
  substrates , thus facilitating release of tripeptides after catalysis 
  ( fig . 5 ) .
asp is 
  completely conserved in all mammalian tpp1 proteins but not among the 
  bacterial homologs ( supplemental fig .
s3 ) . interestingly , in the latter , a 
  neighboring trp side chain blocks this region . in pro - tpp1 ,
the 
  asp carboxyl is 5.3  away from the n terminus of the 
  modeled hexapeptide , although this is likely to change in the mature domain . 
 
in addition , pka predictions using the propka program 
  ( 64 ) indicate that the 
  pka of asp is 6.2 when it is buried as part 
  of pro - tpp1 , whereas it is 3.9 when exposed in the mature enzyme . from this
it 
  follows that at ph < 3.9 it is likely to be protonated when its binding to 
  the positively charged n terminus of a substrate is suboptimal .
this 
  correlates with the km profile of the enzyme , which 
  reveals a substantial increase in km below ph 4.0 
  ( fig .
such a role for 
  asp was also suggested from the previously predicted homology 
  model for tpp1 ( 33 ) , supported 
  by site - directed mutagenesis 
  ( 34 ) .
figure 6.model for autoproteolytic endopeptidase cleavage during ph - dependent 
  pro - tpp1 zymogen maturation . a , ms analysis of autoproteolytic 
  cleavage products .
endo h - deglycosylated pro - tpp1 was incubated at either 25 
  c for 0.5 min ( ph 2.5 and 3.0 ) , 6.3 min ( ph 3.5 ) , 13 min ( ph 4.0 ) , or at 
  37 c for 18 h ( ph 4.5 ) .
indicated m / z are those of the 
  predicted tpp1 fragments spanning residues 20 - 168 ( 16640 ) , 20 - 181 
  ( 18024 ) , and 20 - 189 ( 19029 ) .
b , endopeptidase cleavage sites and 
  positions of ionizable residues at the interdomain interface .
the pro - tpp1 
  backbone is shown with the catalytic domain in orange , the prodomain 
  in cyan , and the propiece linker in magenta .
the 
  endopeptidase cleavage sites ( 1 , ser - leu ; 
  2 , gln - val ; and 3 , 
  asp - phe ) and active site residues 
  ( ser , glu , asp , and 
  asp ) are depicted in stick mode .
the 
  c positions of ionizable interface residues with 
  pka values predicted to be within the relevant 
  physiological ph range for tpp1 ( supplemental table s4 ) are shown as spheres : 
  1 , glu ; 2 , glu ; 3 , 
  glu ; 4 , glu ; 5 , asp ; 
  6 , asp ; 7 ; asp ; 8 , 
  glu ; and 9 , asp .
details of these 
  interactions are shown in supplemental fig . s4 and supplemental table s2 . 
 
c , titration curve of binding energies of the propiece and mature 
  domain of pro - tpp1 .
a schematic depiction of pro - tpp1 is shown with colors , and 
  endopeptidase cleavage sites and interdomain contacts involving ionizable 
  residues are indicated as in b. the active site cleft is depicted as 
  a triangle within the catalytic domain .
the geometry of one side of 
  the cleft ( top half of triangle ) defined by the acidic residues is 
  subject to ph - driven protonation states , whereas the structure of the other 
  side ( lower half of triangle ) defined by ser and 
  ser is subject to restructuring by propagation of long range 
  prodomain interactions . at higher ph
, the prodomain and mature domains are 
  tightly bound , and the active site catalytic machinery is preformed , and the 
  active site exists in equilibrium between two states , one suboptimal for 
  activity ( hatched triangle with mouth closed ) and one poised 
  for activity ( white triangle with open mouth ) .
as the ph is 
  lowered , changes in protonation states progressively disrupt interdomain 
  interactions and affect the rotameric conformations of the active site 
  residues favoring an active site geometry poised for catalysis .
as the ph is 
  lowered to ph 4.5 , electrostatic interactions holding the 3-strand leaf 
  of the prodomain -sandwich weaken and peel away from the interface .
this 
  is likely to result in repositioning of the linker loop bringing cleavage site 
  1 close to the active site . at progressively lower ph , interactions involving 
  the 4-strand leaf of the propiece
this allows sufficient kinking in the linker 
  conformation so that site 2 can be brought close to the active site and 
  targeted for endopeptidase action . at ph
< 3.0 , the interdomain binding 
  interface is completely disrupted , allowing access to site 3 .
model for autoproteolytic endopeptidase cleavage during ph - dependent 
  pro - tpp1 zymogen maturation . a , ms analysis of autoproteolytic 
  cleavage products .
endo h - deglycosylated pro - tpp1 was incubated at either 25 
  c for 0.5 min ( ph 2.5 and 3.0 ) , 6.3 min ( ph 3.5 ) , 13 min ( ph 4.0 ) , or at 
  37 c for 18 h ( ph 4.5 ) . indicated m / z are those of the 
  predicted tpp1 fragments spanning residues 20 - 168 ( 16640 ) , 20 - 181 
  ( 18024 ) , and 20 - 189 ( 19029 ) .
b , endopeptidase cleavage sites and 
  positions of ionizable residues at the interdomain interface .
the pro - tpp1 
  backbone is shown with the catalytic domain in orange , the prodomain 
  in cyan , and the propiece linker in magenta .
the 
  endopeptidase cleavage sites ( 1 , ser - leu ; 
  2 , gln - val ; and 3 , 
  asp - phe ) and active site residues 
  ( ser , glu , asp , and 
  asp ) are depicted in stick mode .
the 
  c positions of ionizable interface residues with 
  pka values predicted to be within the relevant 
  physiological ph range for tpp1 ( supplemental table s4 ) are shown as spheres : 
  1 , glu ; 2 , glu ; 3 , 
  glu ; 4 , glu ; 5 , asp ; 
  6 , asp ; 7 ; asp ; 8 , 
  glu ; and 9 , asp .
details of these 
  interactions are shown in supplemental fig . s4 and supplemental table s2 . 
 
c , titration curve of binding energies of the propiece and mature 
  domain of pro - tpp1 .
a schematic depiction of pro - tpp1 is shown with colors , and 
  endopeptidase cleavage sites and interdomain contacts involving ionizable 
  residues are indicated as in b. the active site cleft is depicted as 
  a triangle within the catalytic domain .
the geometry of one side of 
  the cleft ( top half of triangle ) defined by the acidic residues is 
  subject to ph - driven protonation states , whereas the structure of the other 
  side ( lower half of triangle ) defined by ser and 
  ser is subject to restructuring by propagation of long range 
  prodomain interactions . at higher ph
, the prodomain and mature domains are 
  tightly bound , and the active site catalytic machinery is preformed , and the 
  active site exists in equilibrium between two states , one suboptimal for 
  activity ( hatched triangle with mouth closed ) and one poised 
  for activity ( white triangle with open mouth ) . as
the ph is 
  lowered , changes in protonation states progressively disrupt interdomain 
  interactions and affect the rotameric conformations of the active site 
  residues favoring an active site geometry poised for catalysis .
as the ph is 
  lowered to ph 4.5 , electrostatic interactions holding the 3-strand leaf 
  of the prodomain -sandwich weaken and peel away from the interface .
this 
  is likely to result in repositioning of the linker loop bringing cleavage site 
  1 close to the active site . at progressively lower ph , interactions involving 
  the 4-strand leaf of the propiece
this allows sufficient kinking in the linker 
  conformation so that site 2 can be brought close to the active site and 
  targeted for endopeptidase action . at ph
< 3.0 , the interdomain binding 
  interface is completely disrupted , allowing access to site 3 .
model for autoproteolytic endopeptidase cleavage of 
  pro - tpp1maldi - tof - mass spectrometric ( ms ) analysis revealed 
  differences in the size of the liberated propeptide at different ph values 
  ( fig .
this 
  indicates that the zymogen can utilize three different endopeptidase cleavage 
  sites ( fig .
6b ) as 
  follows : 1 ) ser - leu site within ph range 4.0 - 4.5 ; 
  2 ) gln - val site within ph range 3.0 - 4.0 ; and 3 ) 
  asp - phe site within ph range 2.5 - 4.0 .
cleavages at 
  sites 1 and 2 have been reported previously 
  ( 13 ) .
calculations using apbs 
  electrostatic analysis software 
  ( 61 ) with pro - tpp1 domains 
  revealed that the binding energy between the propiece and mature domain is 
  more positive ( i.e. interactions are less stable ) with lower ph 
  ( fig .
a similar 
  profile is also observed for calculations done using prokumamolisin 
  coordinates ( data not shown ) .
key salt bridge interactions as well as hydrogen bonds 
  contributed by ionizable residues may be disrupted at low ph where acidic 
  residues are likely to become protonated .
this conclusion is supported by a 
  recent report that the extent of inhibition of tpp1 activity by the prodomain 
  decreases with lower ph and is negligible at ph 3.5 
  ( 68 ) .
in addition , complex 
  formation between kumamolisin - as e78h / d164n mutant mature domain and wild - type 
  propiece was observed to be less at lower ph 
  ( 29 ) .
these results are 
  consistent with a ph - induced change in binding between the propiece and the 
  mature domain . in pro - tpp1 ,
the binding interface has many electrostatic bonds 
  and ionizable residues with pka values within the 
  physiological ph range ( fig . 
 
predictions of pka values of ionizable residues 
  at the interdomain interface reveal that these residues cluster into groups 
  that map to specific regions of the binding interface ( supplemental fig .
these regions are associated with distinct ranges 
  of pka values , suggesting ph - specific disruption of domain 
  contacts .
we propose a model of activation that takes into the account the ph - induced 
  change in binding together with the ph - specific cleavage site preference 
  ( fig .
the cleavage site 
  his - phe is positioned close to the active site ser 
  ( ser ) ( supplemental fig .
s4 ) . this corresponds to cleavage site 
  1 ( ser - leu ) in pro - tpp1 , which is relatively close 
  to the active site ser , with the carbonyl carbon of the scissile 
  peptide being 12.0  away . in the absence of the lattice interactions 
  that alter the position of the linker in our crystals grown at ph 5.0
however , under the conditions 
  that we use in our in vitro activation reactions , autocatalytic 
  cleavage at ph 4.5 and above is extremely slow , with the intact pro - tpp1 
  zymogen persisting for days ( data not shown ) .
it is likely that tight binding 
  between the prodomain and catalytic domain at this ph holds the zymogen in a 
  conformation that favors the nonoptimal active site geometry , and that the 
  small amount of cleavage observed might arise from local breathing motions 
  that allow acquisition of a conformationally active catalytic site .
however , 
  given that the prodomain retains high affinity for the catalytic domain and 
  remains associated even after cleavage 
  ( 68 ) , the equilibrium would 
  favor the inactive conformation . at lower ph ,
the prodomain - catalytic domain 
  interface could become destabilized , allowing assumption of a more 
  conformationally optimal active site in the catalytic domain , as well as 
  reorientation of the prodomain and linker .
after endoproteolytic cleavage , the 
  liberated propeptide as well as portions of the linker region still attached 
  to the catalytic domain could then be further digested by the free catalytic 
  domain ( see below ) .
consistent with this model , rates of pro - tpp1 activation are greatly 
  accelerated in the presence of denaturants such as urea ( supplemental fig .
in addition , the environment of 
  late endosomes and lysosomes may be conducive to destabilization of the 
  prodomain - catalytic domain complex , allowing rapid activation at the 
  physiological ph of these organelles ( ph 6 to 4.5 ) 
  ( 76 , 
  77 ) .
for instance , 
  glycosaminoglycans have been shown to accelerate autoactivation of pro - tpp1 at 
  mildly acidic ph ( 16 ) , as do 
  liposomes containing bis(monoacylglycero)phosphate ( also called 
  lysobisphosphatidic 
  acid),6 an anionic 
  lipid enriched in late endosomes 
  ( 78 ) . nonetheless , decreasing 
  the ph to optimize the protonation states of residues in the catalytic center , 
  combined with reorientation of the catalytic serine and glutamic acid that 
  would be favored by destabilization of the prodomain - catalytic domain 
  interaction , are likely to be key elements in acquisition of activity .
analysis of 
  hexahistidine - tagged glycosylated recombinant tpp1 using sds - page 
  ( fig .
7c ) , 
  and maldi - tof ms ( data not shown ) corroborates the ms analysis of the endo 
  h - deglycosylated untagged tpp1 ( fig . 
 
6a ) , and it also reveals that autocatalytic processing 
  involves a series of ph - dependent endoproteolytic cleavages and tripeptidyl 
  aminopeptidase cleavages . at ph 4.0 ,
the predominant initial cleavage products 
  correspond to an intermediate starting at val 
  ( fig .
7a , band marked f2 and maldi - tof ms ; data not shown ) . 
  over time , the intermediate undergoes two tripeptidyl - peptidase cleavages , 
  yielding an intermediate starting at thr and the mature protein 
  starting at leu ( fig . 
 
, 
  the predominant initial cleavage products correspond to an intermediate 
  starting at phe ( fig . 
  7 , a , band marked , and c ) and 
  residues 20 - 168 of the propeptide ( fig . 
 
7a , band marked f3 and maldi - tof ms ; data not 
  shown ) . with increasing time
, the intermediate undergoes a second 
  endoproteolytic cleavage to liberate the intermediate form starting at 
  val with subsequent tpp1 cleavage as above 
  ( fig .
initial 
  endoproteolytic cleavages of peptide bonds asp - phe 
  and gln - val occur both at ph 3.0 and 2.5 
  ( fig .
in addition to the tripeptidylpeptidase cleavages of 
  the form with an n - terminal val observed at ph 4.0 , there is 
  also processing of the form starting at phe , yielding 
  intermediates with n - terminal gly and arg .
this is 
  entirely consistent with the known exopeptidase specificity of tpp1 , where 
  peptides with prolines at either p1 or p1 are extremely poor substrates 
  ( 20 ) , thus preventing further 
  exopeptidyl digestion past arg .
6a ) ( 13 ) 
  could not be processed by progressive tripeptidyl peptidase cleavages to yield 
  mature tpp1 because of both frame and specificity considerations , and would be 
  predicted to undergo a second endoproteolytic cleavage at 
  gln - val to yield the form with an n - terminal 
  leu .
the liberated propeptide has an n terminus sysp that is 
  unlikely to be efficiently digested by tpp1 exopeptidase activity .
thus , in 
  the absence of any additional proteases , its hydrolysis probably involves tpp1 
  endoproteolytic cleavage .
activation at ph 4.0 can be accelerated by other 
  proteases ( data not shown ) , and given the protease - rich environment of the 
  lysosome , it is likely that downstream hydrolysis of the initial propeptide 
  cleavage product is not rate - limiting in vivo .
it has been recently reported that the propeptide can inhibit the activity 
  of mature tpp1 at neutral and mildly acidic ph , with an ic50 in the 
  micromolar range at ph 4.0 
  ( 68 ) .
our results on comparing 
  zymogen cleavage and acquisition of tpp1 activity are consistent with this . 
 
there was a marked lag between the two events at ph 4.0 
  ( fig .
in contrast , the 
  appearance and disappearance of the free propeptide could be fit with a 
  two - step consecutive irreversible reaction model 
  ( 36 ) , with the second phase 
  having a t of 7 h , paralleling that of 
  acquisition of tpp1 activity .
thus , the lag is likely to reflect the time 
  required for proteolysis of the inhibitory prodomain .
the lag at lower ph 
  values was much less pronounced , with the difference in the half - times between 
  the first phase of the activation reaction and zymogen cleavage differing by 
  at most a factor of 2 and both processes occurring rapidly 
  ( fig .
7b , ph 2.5 , 3.0 , 
  and 3.5 ; t cleavage 3 , 6 , and 20 s ; and 
  t activation 5 , 12 , and 20 s , respectively ) .
this 
  is consistent with rapid dissociation of the cleaved propeptide at more acidic 
  ph values .
activation reactions were conducted 
  using 1 - 1.5 m pro - tpp1 , and aliquots ( typically 0.5 - 1 g ) 
  were analyzed by sds - page as described under  experimental 
  procedures .
 z , position of 66-kda glycosylated pro - tpp1 
  zymogen ; asterisk , 49-kda intermediate ; m , 46-kda 
  glycosylated mature tpp1 ; f1 , f2 , and 
  f3 , fragments cleaved at sites 1 , 2 , and 3 , respectively , 
  with assignment of f1 being based on maldi - tof analysis of 
  deglycosylated tpp1 ( fig . 
 
b , comparison of proteolytic processing and 
  acquisition of tpp1 activity . two to four gels for each ph were used for the 
  analysis and background - corrected intensity data normalized for each gel .
the 
  green upward pointing triangles indicate disappearance of zymogen at 
  each time point , normalized to the intensity at t = 0 ( e.g. 
  1 - zt / zo ) .
the red downward pointing triangles 
  indicate levels of liberated propeptide ( f1 + 
  f2 + f3 ) , normalized to intensity of 
  the highest value obtained for the reaction time course .
open circles 
  indicate tpp1 activity measurements on activation reactions initiated using 
  1.5 m zymogen . for comparison , data from 
  fig .
although 
  the time course was similar at both concentrations for ph 4 and 3.5 , there 
  were noticeable differences at ph 2.5 and 3.0 .
for the latter reactions , the 
  initial phase for acquisition of activity was similar , but the second phase 
  was noticeably slower for the high concentration reactions .
the reason for 
  this is not entirely obvious , but it may reflect experimental considerations 
  such as some reassociation of the prodomain after the activation reactions 
  were arrested by dilution into ph 5.0 buffer . similarly , the faster time 
  course and coincidence of the acquisition of enzymatic activity and 
  endoproteolysis at ph 4.0 that we observed previously 
  ( 14 ) may reflect differences 
  in experimental design , where the reactions were terminated by dilution into 
  ph 4.0 buffer and then assayed immediately for tpp1 activity . under these 
  conditions , the propeptide would be expected to be largely dissociated from 
  the mature protein .
protein ( 17 pmol / time point ) was resolved by 
  sds - page and transferred to membranes .
bands of molecular mass ranging between 
  46 and 49 kda were analyzed by chemical sequencing .
the table lists 
  the amount of each form in picomoles as estimated from the sequencing data 
  ( see  experimental procedures  for details ) .
activation reactions were conducted 
  using 1 - 1.5 m pro - tpp1 , and aliquots ( typically 0.5 - 1 g ) 
  were analyzed by sds - page as described under  experimental 
  procedures .
 z , position of 66-kda glycosylated pro - tpp1 
  zymogen ; asterisk , 49-kda intermediate ; m , 46-kda 
  glycosylated mature tpp1 ; f1 , f2 , and 
  f3 , fragments cleaved at sites 1 , 2 , and 3 , respectively , 
  with assignment of f1 being based on maldi - tof analysis of 
  deglycosylated tpp1 ( fig . 
 
b , comparison of proteolytic processing and 
  acquisition of tpp1 activity . two to four gels for each ph were used for the 
  analysis and background - corrected intensity data normalized for each gel .
the 
  green upward pointing triangles indicate disappearance of zymogen at 
  each time point , normalized to the intensity at t = 0 ( e.g. 
  1 - zt / zo ) .
the red downward pointing triangles 
  indicate levels of liberated propeptide ( f1 + 
  f2 + f3 ) , normalized to intensity of 
  the highest value obtained for the reaction time course .
open circles 
  indicate tpp1 activity measurements on activation reactions initiated using 
  1.5 m zymogen . for comparison , data from 
  fig .
although 
  the time course was similar at both concentrations for ph 4 and 3.5 , there 
  were noticeable differences at ph 2.5 and 3.0 .
for the latter reactions , the 
  initial phase for acquisition of activity was similar , but the second phase 
  was noticeably slower for the high concentration reactions .
the reason for 
  this is not entirely obvious , but it may reflect experimental considerations 
  such as some reassociation of the prodomain after the activation reactions 
  were arrested by dilution into ph 5.0 buffer . similarly , the faster time 
  course and coincidence of the acquisition of enzymatic activity and 
  endoproteolysis at ph 4.0 that we observed previously 
  ( 14 ) may reflect differences 
  in experimental design , where the reactions were terminated by dilution into 
  ph 4.0 buffer and then assayed immediately for tpp1 activity . under these 
  conditions , the propeptide would be expected to be largely dissociated from 
  the mature protein .
protein ( 17 pmol / time point ) was resolved by 
  sds - page and transferred to membranes .
bands of molecular mass ranging between 
  46 and 49 kda were analyzed by chemical sequencing .
the table lists 
  the amount of each form in picomoles as estimated from the sequencing data 
  ( see  experimental procedures  for details ) .
the structure of pro - tpp1 provides some 
  insights into disease - causing mutations in lincl ( discussed in the 
  accompanying paper by pal et al . 
 
s8 ) . 
  there are currently 57 mutations listed in the ncl mutation data base . roughly 
  half ( 29 mutations )
are missense mutations , some of which could respond to 
  small molecule chaperone therapy directed at stabilizing the aberrant protein 
  and thus rescuing function . for these ,
the structure of pro - tpp1 may help in 
  identifying such small molecule therapeutics .
however , most individuals with 
  lincl would not benefit from this approach as two mutations that encode 
  totally nonfunctional proteins represent the majority of disease alleles 
  ( 79 ) .
one is an invariant 
  3 splice acceptor mutation in intron five that introduces a frameshift 
  upstream of the phe codon ; the other is a nonsense mutation at 
  codon arg that does not appear to efficiently respond to 
  potential therapeutics that promote translation readthrough 
  ( 80 ) .
based on these and other considerations , any widely applicable therapy will 
  need to target sufficient amounts of tpp1 to the brain to digest both newly 
  arriving substrates and pre - existing lysosomal storage material . for enzyme 
  replacement therapy using peripherally administered protein
, it will be 
  necessary to modify pro - tpp1 so that it can cross the blood - brain barrier 
  ( e.g. creation of a chimeric protein containing elements that allow 
  transcytosis ) .
in addition , for both enzyme and gene therapy approaches , it 
  would be extremely useful to engineer functional tpp1 derivatives that have a 
  longer half - life in the lysosome , thus increasing the effectiveness of 
  properly targeted protein .
 late infantile neuronal ceroid lipofuscinosis is a fatal childhood 
  neurological disorder caused by a deficiency in the lysosomal protease 
  tripeptidyl - peptidase 1 ( tpp1 ) . 
 tpp1 represents the only known mammalian 
  member of the s53 family of serine proteases , a group characterized by a 
  subtilisin - like fold , a ser - glu - asp catalytic triad , and an acidic ph optimum . 
  
 tpp1 is synthesized as an inactive proenzyme ( pro - tpp1 ) that is 
  proteolytically processed into the active enzyme after exposure to low ph 
  in vitro or targeting to the lysosome in vivo . in this 
  study , we describe an endoglycosidase h - deglycosylated form of tpp1 containing 
  four asn - linked n - acetylglucosamines that is indistinguishable from 
  fully glycosylated tpp1 in terms of autocatalytic processing of the proform 
  and enzymatic properties of the mature protease . 
 the crystal structure of 
  deglycosylated pro - tpp1 was determined at 1.85  resolution . 
 a large 
  151-residue c - shaped prodomain makes extensive contacts as it wraps around the 
  surface of the catalytic domain with the two domains connected by a 24-residue 
  flexible linker that passes through the substrate - binding groove . 
 the 
  proenzyme structure reveals suboptimal catalytic triad geometry with its 
  propiece linker partially blocking the substrate - binding site , which together 
  serve to prevent premature activation of the protease . 
 finally , we have 
  identified numerous processing intermediates and propose a structural model 
  that explains the pathway for tpp1 activation in vitro . 
 these data 
  provide new insights into tpp1 function and represent a valuable resource for 
  constructing improved tpp1 variants for treatment of late infantile neuronal 
  ceroid lipofuscinosis . 
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
Supplementary Material