Structural basis for action of the external chaperone for a propeptide-deficient serine protease from Aeromonas sobria

Hidetomo Kobayashi, Toru Yoshida, Takuya Miyakawa, Mitsuru Tashiro, Keinosuke Okamoto, Hiroyasu Yamanaka, Masaru Tanokura, Hideaki Tsuge

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Subtilisin-like proteases are broadly expressed in organisms ranging from bacteria to mammals. During maturation of these enzymes, N-terminal propeptides function as intramolecular chaperones, assisting the folding of their catalytic domains. However, we have identified an exceptional case, the serine protease from Aeromonas sobria (ASP), that lacks a propeptide. Instead, ORF2, a protein encoded just downstream of asp, appears essential for proper ASP folding. The mechanism by which ORF2 functions remains an open question, because it shares no sequence homology with any known intramolecular propeptide or other protein. Here we report the crystal structure of the ORF2-ASP complex and the solution structure of free ORF2. ORF2 consists of three regions: an N-terminal extension, a central body, and a C-terminal tail. Together, the structure of the central body and the C-terminal tail is similar to that of the intramolecular propeptide. The N-terminal extension, which is not seen in other subtilisin-like enzymes, is intrinsically disordered but forms some degree of secondary structure upon binding ASP. We also show that C-terminal (ΔC1 and ΔC5) or N-terminal (ΔN43 and ΔN64) deletion eliminates the ability of ORF2 to function as a chaperone. Characterization of the maturation of ASP with ORF2 showed that folding occurs in the periplasmic space and is followed by translocation into extracellular space and dissociation from ORF2, generating active ASP. Finally, a PSI-BLAST search revealed that operons encoding subtilases and their external chaperones are widely distributed among Gram-negative bacteria, suggesting that ASP and its homologs form a novel family of subtilases having an external chaperone.

Original languageEnglish
Pages (from-to)11130-11143
Number of pages14
JournalJournal of Biological Chemistry
Volume290
Issue number17
DOIs
Publication statusPublished - Apr 24 2015

Fingerprint

Aeromonas
Subtilisin
Serine Proteases
Tail
Periplasm
Bacteria
Extracellular Space
Enzymes
Operon
Sequence Homology
Gram-Negative Bacteria
Mammals
Catalytic Domain
Proteins
Peptide Hydrolases
Crystal structure

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Structural basis for action of the external chaperone for a propeptide-deficient serine protease from Aeromonas sobria. / Kobayashi, Hidetomo; Yoshida, Toru; Miyakawa, Takuya; Tashiro, Mitsuru; Okamoto, Keinosuke; Yamanaka, Hiroyasu; Tanokura, Masaru; Tsuge, Hideaki.

In: Journal of Biological Chemistry, Vol. 290, No. 17, 24.04.2015, p. 11130-11143.

Research output: Contribution to journalArticle

Kobayashi, H, Yoshida, T, Miyakawa, T, Tashiro, M, Okamoto, K, Yamanaka, H, Tanokura, M & Tsuge, H 2015, 'Structural basis for action of the external chaperone for a propeptide-deficient serine protease from Aeromonas sobria', Journal of Biological Chemistry, vol. 290, no. 17, pp. 11130-11143. https://doi.org/10.1074/jbc.M114.622852
Kobayashi, Hidetomo ; Yoshida, Toru ; Miyakawa, Takuya ; Tashiro, Mitsuru ; Okamoto, Keinosuke ; Yamanaka, Hiroyasu ; Tanokura, Masaru ; Tsuge, Hideaki. / Structural basis for action of the external chaperone for a propeptide-deficient serine protease from Aeromonas sobria. In: Journal of Biological Chemistry. 2015 ; Vol. 290, No. 17. pp. 11130-11143.
@article{d26f70b53fb64cd38956a08a45a3c090,
title = "Structural basis for action of the external chaperone for a propeptide-deficient serine protease from Aeromonas sobria",
abstract = "Subtilisin-like proteases are broadly expressed in organisms ranging from bacteria to mammals. During maturation of these enzymes, N-terminal propeptides function as intramolecular chaperones, assisting the folding of their catalytic domains. However, we have identified an exceptional case, the serine protease from Aeromonas sobria (ASP), that lacks a propeptide. Instead, ORF2, a protein encoded just downstream of asp, appears essential for proper ASP folding. The mechanism by which ORF2 functions remains an open question, because it shares no sequence homology with any known intramolecular propeptide or other protein. Here we report the crystal structure of the ORF2-ASP complex and the solution structure of free ORF2. ORF2 consists of three regions: an N-terminal extension, a central body, and a C-terminal tail. Together, the structure of the central body and the C-terminal tail is similar to that of the intramolecular propeptide. The N-terminal extension, which is not seen in other subtilisin-like enzymes, is intrinsically disordered but forms some degree of secondary structure upon binding ASP. We also show that C-terminal (ΔC1 and ΔC5) or N-terminal (ΔN43 and ΔN64) deletion eliminates the ability of ORF2 to function as a chaperone. Characterization of the maturation of ASP with ORF2 showed that folding occurs in the periplasmic space and is followed by translocation into extracellular space and dissociation from ORF2, generating active ASP. Finally, a PSI-BLAST search revealed that operons encoding subtilases and their external chaperones are widely distributed among Gram-negative bacteria, suggesting that ASP and its homologs form a novel family of subtilases having an external chaperone.",
author = "Hidetomo Kobayashi and Toru Yoshida and Takuya Miyakawa and Mitsuru Tashiro and Keinosuke Okamoto and Hiroyasu Yamanaka and Masaru Tanokura and Hideaki Tsuge",
year = "2015",
month = "4",
day = "24",
doi = "10.1074/jbc.M114.622852",
language = "English",
volume = "290",
pages = "11130--11143",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "17",

}

TY - JOUR

T1 - Structural basis for action of the external chaperone for a propeptide-deficient serine protease from Aeromonas sobria

AU - Kobayashi, Hidetomo

AU - Yoshida, Toru

AU - Miyakawa, Takuya

AU - Tashiro, Mitsuru

AU - Okamoto, Keinosuke

AU - Yamanaka, Hiroyasu

AU - Tanokura, Masaru

AU - Tsuge, Hideaki

PY - 2015/4/24

Y1 - 2015/4/24

N2 - Subtilisin-like proteases are broadly expressed in organisms ranging from bacteria to mammals. During maturation of these enzymes, N-terminal propeptides function as intramolecular chaperones, assisting the folding of their catalytic domains. However, we have identified an exceptional case, the serine protease from Aeromonas sobria (ASP), that lacks a propeptide. Instead, ORF2, a protein encoded just downstream of asp, appears essential for proper ASP folding. The mechanism by which ORF2 functions remains an open question, because it shares no sequence homology with any known intramolecular propeptide or other protein. Here we report the crystal structure of the ORF2-ASP complex and the solution structure of free ORF2. ORF2 consists of three regions: an N-terminal extension, a central body, and a C-terminal tail. Together, the structure of the central body and the C-terminal tail is similar to that of the intramolecular propeptide. The N-terminal extension, which is not seen in other subtilisin-like enzymes, is intrinsically disordered but forms some degree of secondary structure upon binding ASP. We also show that C-terminal (ΔC1 and ΔC5) or N-terminal (ΔN43 and ΔN64) deletion eliminates the ability of ORF2 to function as a chaperone. Characterization of the maturation of ASP with ORF2 showed that folding occurs in the periplasmic space and is followed by translocation into extracellular space and dissociation from ORF2, generating active ASP. Finally, a PSI-BLAST search revealed that operons encoding subtilases and their external chaperones are widely distributed among Gram-negative bacteria, suggesting that ASP and its homologs form a novel family of subtilases having an external chaperone.

AB - Subtilisin-like proteases are broadly expressed in organisms ranging from bacteria to mammals. During maturation of these enzymes, N-terminal propeptides function as intramolecular chaperones, assisting the folding of their catalytic domains. However, we have identified an exceptional case, the serine protease from Aeromonas sobria (ASP), that lacks a propeptide. Instead, ORF2, a protein encoded just downstream of asp, appears essential for proper ASP folding. The mechanism by which ORF2 functions remains an open question, because it shares no sequence homology with any known intramolecular propeptide or other protein. Here we report the crystal structure of the ORF2-ASP complex and the solution structure of free ORF2. ORF2 consists of three regions: an N-terminal extension, a central body, and a C-terminal tail. Together, the structure of the central body and the C-terminal tail is similar to that of the intramolecular propeptide. The N-terminal extension, which is not seen in other subtilisin-like enzymes, is intrinsically disordered but forms some degree of secondary structure upon binding ASP. We also show that C-terminal (ΔC1 and ΔC5) or N-terminal (ΔN43 and ΔN64) deletion eliminates the ability of ORF2 to function as a chaperone. Characterization of the maturation of ASP with ORF2 showed that folding occurs in the periplasmic space and is followed by translocation into extracellular space and dissociation from ORF2, generating active ASP. Finally, a PSI-BLAST search revealed that operons encoding subtilases and their external chaperones are widely distributed among Gram-negative bacteria, suggesting that ASP and its homologs form a novel family of subtilases having an external chaperone.

UR - http://www.scopus.com/inward/record.url?scp=84928399839&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84928399839&partnerID=8YFLogxK

U2 - 10.1074/jbc.M114.622852

DO - 10.1074/jbc.M114.622852

M3 - Article

C2 - 25784551

AN - SCOPUS:84928399839

VL - 290

SP - 11130

EP - 11143

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 17

ER -