PDBsum entry 3bb7

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protein links
Hydrolase PDB id
Protein chain
314 a.a. *
Waters ×371
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Structure of prevotella intermedia prointerpain a fragment 3 (mutant c154a)
Structure: Interpain a. Chain: a. Fragment: unp residues 84-403. Engineered: yes. Mutation: yes
Source: Prevotella intermedia. Organism_taxid: 28131. Gene: pin0048. Expressed in: escherichia coli. Expression_system_taxid: 562.
1.50Å     R-factor:   0.156     R-free:   0.193
Authors: N.Mallorqui-Fernandez,S.P.Manandhar,G.Mallorqui-Fernandez,I. K.Wawrzonek,T.Kantyka,M.Sola,I.B.Thogersen,J.J.Enghild,J.Po F.X.Gomis-Ruth
Key ref:
N.Mallorquí-Fernández et al. (2008). A New Autocatalytic Activation Mechanism for Cysteine Proteases Revealed by Prevotella intermedia Interpain A. J Biol Chem, 283, 2871-2882. PubMed id: 17993455 DOI: 10.1074/jbc.M708481200
09-Nov-07     Release date:   20-Nov-07    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
A9J7N5  (A9J7N5_PREIN) -  Interpain A
868 a.a.
314 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 8 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     proteolysis   1 term 
  Biochemical function     cysteine-type peptidase activity     1 term  


DOI no: 10.1074/jbc.M708481200 J Biol Chem 283:2871-2882 (2008)
PubMed id: 17993455  
A New Autocatalytic Activation Mechanism for Cysteine Proteases Revealed by Prevotella intermedia Interpain A.
N.Mallorquí-Fernández, S.P.Manandhar, G.Mallorquí-Fernández, I.Usón, K.Wawrzonek, T.Kantyka, M.Solà, I.B.Thøgersen, J.J.Enghild, J.Potempa, F.X.Gomis-Rüth.
Prevotella intermedia is a major periodontopathogen contributing to human gingivitis and periodontitis. Such pathogens release proteases as virulence factors that cause deterrence of host defenses and tissue destruction. A new cysteine protease from the cysteine-histidine-dyad class, interpain A, was studied in its zymogenic and self-processed mature forms. The latter consists of a bivalved moiety made up by two subdomains. In the structure of a catalytic cysteine-to-alanine zymogen variant, the right subdomain interacts with an unusual prodomain, thus contributing to latency. Unlike the catalytic cysteine residue, already in its competent conformation in the zymogen, the catalytic histidine is swung out from its active conformation and trapped in a cage shaped by a backing helix, a zymogenic hairpin, and a latency flap in the zymogen. Dramatic rearrangement of up to 20A of these elements triggered by a tryptophan switch occurs during activation and accounts for a new activation mechanism for proteolytic enzymes. These findings can be extrapolated to related potentially pathogenic cysteine proteases such as Streprococcus pyogenes SpeB and Porphyromonas gingivalis periodontain.
  Selected figure(s)  
Figure 2.
FIGURE 2. Expression and activity of InpA. A, expression and purification of pro-cd-InpA C154A. Lanes 1 and 2, E. colihomogenate before and 3 h after protein expression induction, respectively.Lane3, recombinant protein after affinity chromatography purification. Molecular masses of the distinct protein species (40 and 27 kDa) are shown on the left. B, same for wt pro-cd-InpA. C, time-course analysis of autocatalytic processing and activation of wt pro-cd-InpA (final concentration, 10 µM) incubated with 1 mM HgCl[2]. The reaction was initiated by adding EDTA (5 mM final concentration) as an Hg^2+-chelator, i.e. by releasing metal-mediated inhibition. Samples were withdrawn at the time intervals specified (O/N, overnight incubation; lane C, pro-cd-InpA alone). D, same as C but after addition of active InpA (10 nM final concentration) to the reaction mixture. In this case, the reaction proceeded much faster. E, a subset volume of the withdrawn aliquots from C and D was used to quantify the activity released from wt pro-cd-InpA in the absence ( ) and presence ( ) of catalytic amounts of wt cd-InpA. As a control, pro-cd-InpA spiked with InpA but without EDTA was incubated in parallel ( ). F, concentration-dependent autoactivation of pro-cd-InpA. The reaction was initiated by releasing Hg^2+-mediated inhibition in mixtures containing 0.04 µM ( ), 2 µM ( ), and 10 µM ( ) zymogen. At indicated time points, 50 µl( ), 10 µl( ), and 2 µl( ) were withdrawn from each reaction mixture and directly assayed for activity. G, SDS-PAGE of the digestion of pro-cd-InpA C154A by wt cd-InpA. The zymogen (final concentration of 10 µM) was incubated with cd-InpA (0.1 µM) for time intervals as specified (lane C, control pro-cd-InpA C154 incubated alone). N-terminal amino acid sequences of pro-cd-InpA derived fragments are indicated on the right. H, Western blot analysis of culture supernatant of P. intermedia using InpA-specific rabbit antiserum (lane 3). Wt cd-InpA and pro-cd-InpA (C154A) were loaded on lanes 1 and 2, respectively, for comparison.
Figure 4.
FIGURE 4. Structures of N1pro-cd-InpA C154A and wt cd-InpA. A, Richardson diagram of N1pro-cd-InpA C154A in standard orientation. The pro-domain is displayed in blue/cyan and the mature protein moiety (subdivided into a right and a left subdomain) in yellow/brown. The subdomains, the regular secondary structure elements (see Fig. 1), the N- and the C terminus, the primary activation point (at Asn^111-Val^112), and the structure regions responsible for latency maintenance are marked and labeled. B, superimposition of the C -carbon traces of N1pro-cd-InpA C154A (yellow) and wt mature cd-InpA (red) in standard orientation. Some residues of N1pro-cd-InpA C154A are labeled for reference. C, close-up view of the active site of N1pro-cd-InpA C154A. Orientation as in B after a horizontal rotation of 45°. D, same as in C but for wt active cd-InpA. E, C -trace of the structure of N1pro-cd-InpA C154A (yellow) and wt mature cd-InpA (red) around the active site, including the catalytic cysteine residue (Cys^154; mutated to alanine in N1pro-cd-InpA C154A), imbedded in active-site helix 2(circled 1), the zymogenic hairpin (circled 3) encompassing the catalytic histidine (His^305) (undefined from Ser^295 to Gln^301 in N1pro-cd-InpA C154A) (circled 4), the backing helix 1 (absent in cd-InpA) (circled 1), and the latency-flap, displayed from Tyr^332 to Met^351 for either structure (circled 2). The gray arrows indicate the displacements of the keynote structural elements upon zymogen activation as explained in the text.
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 2871-2882) copyright 2008.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21166898 N.Cerdà-Costa, T.Guevara, A.Y.Karim, M.Ksiazek, K.A.Nguyen, J.L.Arolas, J.Potempa, and F.X.Gomis-Rüth (2011).
The structure of the catalytic domain of Tannerella forsythia karilysin reveals it is a bacterial xenologue of animal matrix metalloproteinases.
  Mol Microbiol, 79, 119-132.
PDB codes: 2xs3 2xs4
19814715 D.P.Byrne, K.Wawrzonek, A.Jaworska, A.J.Birss, J.Potempa, and J.W.Smalley (2010).
Role of the cysteine protease interpain A of Prevotella intermedia in breakdown and release of haem from haemoglobin.
  Biochem J, 425, 257-264.  
20635859 K.Ishihara, K.Wawrzonek, L.N.Shaw, S.Inagaki, M.Miyamoto, and J.Potempa (2010).
Dentipain, a Streptococcus pyogenes IdeS protease homolog, is a novel virulence factor of Treponema denticola.
  Biol Chem, 391, 1047-1055.  
20416045 R.F.Thornton, T.F.Kagawa, P.W.O'Toole, and J.C.Cooney (2010).
The dissemination of C10 cysteine protease genes in Bacteroides fragilis by mobile genetic elements.
  BMC Microbiol, 10, 122.  
19237546 C.C.Wang, H.C.Houng, C.L.Chen, P.J.Wang, C.F.Kuo, Y.S.Lin, J.J.Wu, M.T.Lin, C.C.Liu, W.Huang, and W.J.Chuang (2009).
Solution structure and backbone dynamics of streptopain: insight into diverse substrate specificity.
  J Biol Chem, 284, 10957-10967.
PDB code: 2jtc
19247445 M.Potempa, J.Potempa, T.Kantyka, K.A.Nguyen, K.Wawrzonek, S.P.Manandhar, K.Popadiak, K.Riesbeck, S.Eick, and A.M.Blom (2009).
Interpain A, a cysteine proteinase from Prevotella intermedia, inhibits complement by degrading complement factor C3.
  PLoS Pathog, 5, e1000316.  
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