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Navigation inside a protease: substrate selection and product exit in the tricorn protease from Thermoplasma acidophilum.

Kim JS, Groll M, Musiol HJ, Behrendt R, Kaiser M, Moroder L, Huber R, Brandstetter H
J Mol Biol 324 1041-50 (2002)
Related entries: 1n6d, 1n6e

Cited: 15 times
EuropePMC logo PMID: 12470958

Abstract

The proposed pathway and mechanism of substrate entry and product egress in the hexameric D3 symmetric tricorn protease from Thermoplasma acidophilum were explored by crystallographic studies of ligand complexes and by structure-based mutagenesis. Obstruction of the pore within the 7-bladed beta-propeller (beta7) domain by alkylation or oxidation of an engineered double cysteine mutant strongly decreased enzymatic activities. In line herewith, the crystal structure of the tricorn protease in complex with a trideca-peptide inhibitor modifying the catalytic Ser965 revealed part of the peptide trapped inside the channel of the beta7 domain. The cysteine mutation widening the lumen of the 6-bladed beta-propeller (beta6) domain enhanced catalytic activity, which was restored to normal values after its alkylation. A charge reversal mutant at the putative anchor site of the substrate C terminus, R131E-R132E, drastically reduced the proteolytic activity. The complex crystal structure of a peptide inhibitor with a diketo group at the cleavage site mapped the substrate recognition site and confirmed the role of Arg131-Arg132 as an anchor site. Our results strongly suggest the wider beta7 domain to serve as a selective filter and guide of the substrate to the sequestered active site, while the narrower beta6 domain routes the product to the surface. Moreover, we identified the role of Arg131-Arg132 in anchoring the substrate C terminus.

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Reviews citing this publication (2)

  1. Molecular machines for protein degradation. Groll M, Bochtler M, Brandstetter H, Clausen T, Huber R. Chembiochem 6 222-256 (2005)
  2. Protein Defense Systems against the Lantibiotic Nisin: Function of the Immunity Protein NisI and the Resistance Protein NSR. Khosa S, Lagedroste M, Smits SH. Front Microbiol 7 504 (2016)

Articles citing this publication (12)

  1. The crystal structure of dipeptidyl peptidase IV (CD26) reveals its functional regulation and enzymatic mechanism. Engel M, Hoffmann T, Wagner L, Wermann M, Heiser U, Kiefersauer R, Huber R, Bode W, Demuth HU, Brandstetter H. Proc Natl Acad Sci U S A 100 5063-5068 (2003)
  2. Intrinsic evolutionary constraints on protease structure, enzyme acylation, and the identity of the catalytic triad. Buller AR, Townsend CA. Proc Natl Acad Sci U S A 110 E653-61 (2013)
  3. Chlamydial CT441 is a PDZ domain-containing tail-specific protease that interferes with the NF-kappaB pathway of immune response. Lad SP, Yang G, Scott DA, Wang G, Nair P, Mathison J, Reddy VS, Li E. J Bacteriol 189 6619-6625 (2007)
  4. Crystal structures of the tricorn interacting factor F3 from Thermoplasma acidophilum, a zinc aminopeptidase in three different conformations. Kyrieleis OJ, Goettig P, Kiefersauer R, Huber R, Brandstetter H. J Mol Biol 349 787-800 (2005)
  5. Concerted structural changes in the peptidase and the propeller domains of prolyl oligopeptidase are required for substrate binding. Szeltner Z, Rea D, Juhász T, Renner V, Fülöp V, Polgár L. J Mol Biol 340 627-637 (2004)
  6. Structural basis of lantibiotic recognition by the nisin resistance protein from Streptococcus agalactiae. Khosa S, Frieg B, Mulnaes D, Kleinschrodt D, Hoeppner A, Gohlke H, Smits SH. Sci Rep 6 18679 (2016)
  7. High-resolution structure of ybfF from Escherichia coli K12: a unique substrate-binding crevice generated by domain arrangement. Park SY, Lee SH, Lee J, Nishi K, Kim YS, Jung CH, Kim JS. J Mol Biol 376 1426-1437 (2008)
  8. X-ray snapshots of peptide processing in mutants of tricorn-interacting factor F1 from Thermoplasma acidophilum. Goettig P, Brandstetter H, Groll M, Göhring W, Konarev PV, Svergun DI, Huber R, Kim JS. J Biol Chem 280 33387-33396 (2005)
  9. Crystal structure of the major Malassezia sympodialis allergen Mala s 1 reveals a beta-propeller fold: a novel fold among allergens. Vilhelmsson M, Zargari A, Crameri R, Rasool O, Achour A, Scheynius A, Hallberg BM. J Mol Biol 369 1079-1086 (2007)
  10. A self-compartmentalizing hexamer serine protease from Pyrococcus horikoshii: substrate selection achieved through multimerization. Menyhárd DK, Kiss-Szemán A, Tichy-Rács É, Hornung B, Rádi K, Szeltner Z, Domokos K, Szamosi I, Náray-Szabó G, Polgár L, Harmat V. J Biol Chem 288 17884-17894 (2013)
  11. Functional characterization of hypothetical proteins of Mycobacterium tuberculosis with possible esterase/lipase signature: a cumulative in silico and in vitro approach. Kumar A, Sharma A, Kaur G, Makkar P, Kaur J. J Biomol Struct Dyn 35 1226-1243 (2017)
  12. Molecular dynamics simulation of the last step of a catalytic cycle: product release from the active site of the enzyme chorismate mutase from Mycobacterium tuberculosis. Choutko A, van Gunsteren WF. Protein Sci 21 1672-1681 (2012)


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