skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: combined crystallographic and NMR studies of the GroEL Apical Domain with implications for substrate interactions

Abstract

A homologue of the Escherichia coli GroEL apical domain was obtained from thermophilic eubacterium Thermus thermophilus. The domains share 70 % sequence identity (101 out of 145 residues). The thermal stability of the T. thermophilus apical domain (T{sub m}>100{sup o}C as evaluated by circular dichroism) is at least 35{sup o}C greater than that of the E. coli apical domain (T{sub m}=65{sup o}C). The crystal structure of a selenomethione-substituted apical domain from T. thermophilus was determined to a resolution of 1.78 {angstrom} using multiwavelength-anomalous-diffraction phasing. The structure is similar to that of the E. coli apical domain (root-mean-square deviation 0.45 {angstrom} based on main-chain atoms). The thermophilic structure contains seven additional salt bridges of which four contain charge-stabilized hydrogen bonds. Only one of the additional salt bridges would face the 'Anfinsen cage' in GroEL. High temperatures were exploited to map sites of interactions between the apical domain and molten globules. NMR footprints of apical domain-protein complexes were obtained at elevated temperature using {sup 15}N-{sup 1}H correlation spectra of {sup 15}N-labeled apical domain. Footprints employing two polypeptides unrelated in sequence or structure (an insulin monomer and the SRY high-mobility-group box, each partially unfolded at 50{sup o}C) are essentially the same and consistentmore » with the peptide-binding surface previously defined in E. coli GroEL and its apical domain-peptide complexes. An additional part of this surface comprising a short N-terminal {alpha}-helix is observed. The extended footprint rationalizes mutagenesis studies of intact GroEL in which point mutations affecting substrate binding were found outside the 'classical' peptide-binding site. Our results demonstrate structural conservation of the apical domain among GroEL homologues and conservation of an extended non-polar surface recognizing diverse polypeptides.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
943090
Report Number(s):
ANL/BIO/JA-37420
Journal ID: ISSN 0022-2836; JMOBAK; TRN: US201002%%587
DOE Contract Number:  
DE-AC02-06CH11357
Resource Type:
Journal Article
Journal Name:
J. Mol. Biol.
Additional Journal Information:
Journal Volume: 306; Journal Issue: 3 ; Feb. 23, 2001; Journal ID: ISSN 0022-2836
Country of Publication:
United States
Language:
ENGLISH
Subject:
08 HYDROGEN; 36 MATERIALS SCIENCE; BACTERIA; COMPLEXES; CORRELATIONS; CRYSTAL STRUCTURE; DICHROISM; ESCHERICHIA COLI; GENE MUTATIONS; HYDROGEN; INSULIN; INTERACTIONS; MONOMERS; MUTAGENESIS; NUCLEAR MAGNETIC RESONANCE; POLYPEPTIDES; RESIDUES; SALTS; SPECTRA; STABILITY; SUBSTRATES; SURFACES; TEMPERATURE RANGE 0400-1000 K

Citation Formats

Hua, Q X. H., Dementieva, I S. D., Walsh, M A. W., Hallenga, K H, Weiss, M A. W., Joachimiak, A J, Biosciences Division, Case Western Reserve Univ., Angeletti, IRBM P, and Purdue Univ. A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: combined crystallographic and NMR studies of the GroEL Apical Domain with implications for substrate interactions. United States: N. p., 2001. Web. doi:10.1006/jmbi.2000.4405.
Hua, Q X. H., Dementieva, I S. D., Walsh, M A. W., Hallenga, K H, Weiss, M A. W., Joachimiak, A J, Biosciences Division, Case Western Reserve Univ., Angeletti, IRBM P, & Purdue Univ. A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: combined crystallographic and NMR studies of the GroEL Apical Domain with implications for substrate interactions. United States. https://doi.org/10.1006/jmbi.2000.4405
Hua, Q X. H., Dementieva, I S. D., Walsh, M A. W., Hallenga, K H, Weiss, M A. W., Joachimiak, A J, Biosciences Division, Case Western Reserve Univ., Angeletti, IRBM P, and Purdue Univ. 2001. "A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: combined crystallographic and NMR studies of the GroEL Apical Domain with implications for substrate interactions". United States. https://doi.org/10.1006/jmbi.2000.4405.
@article{osti_943090,
title = {A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: combined crystallographic and NMR studies of the GroEL Apical Domain with implications for substrate interactions},
author = {Hua, Q X. H. and Dementieva, I S. D. and Walsh, M A. W. and Hallenga, K H and Weiss, M A. W. and Joachimiak, A J and Biosciences Division and Case Western Reserve Univ. and Angeletti, IRBM P and Purdue Univ.},
abstractNote = {A homologue of the Escherichia coli GroEL apical domain was obtained from thermophilic eubacterium Thermus thermophilus. The domains share 70 % sequence identity (101 out of 145 residues). The thermal stability of the T. thermophilus apical domain (T{sub m}>100{sup o}C as evaluated by circular dichroism) is at least 35{sup o}C greater than that of the E. coli apical domain (T{sub m}=65{sup o}C). The crystal structure of a selenomethione-substituted apical domain from T. thermophilus was determined to a resolution of 1.78 {angstrom} using multiwavelength-anomalous-diffraction phasing. The structure is similar to that of the E. coli apical domain (root-mean-square deviation 0.45 {angstrom} based on main-chain atoms). The thermophilic structure contains seven additional salt bridges of which four contain charge-stabilized hydrogen bonds. Only one of the additional salt bridges would face the 'Anfinsen cage' in GroEL. High temperatures were exploited to map sites of interactions between the apical domain and molten globules. NMR footprints of apical domain-protein complexes were obtained at elevated temperature using {sup 15}N-{sup 1}H correlation spectra of {sup 15}N-labeled apical domain. Footprints employing two polypeptides unrelated in sequence or structure (an insulin monomer and the SRY high-mobility-group box, each partially unfolded at 50{sup o}C) are essentially the same and consistent with the peptide-binding surface previously defined in E. coli GroEL and its apical domain-peptide complexes. An additional part of this surface comprising a short N-terminal {alpha}-helix is observed. The extended footprint rationalizes mutagenesis studies of intact GroEL in which point mutations affecting substrate binding were found outside the 'classical' peptide-binding site. Our results demonstrate structural conservation of the apical domain among GroEL homologues and conservation of an extended non-polar surface recognizing diverse polypeptides.},
doi = {10.1006/jmbi.2000.4405},
url = {https://www.osti.gov/biblio/943090}, journal = {J. Mol. Biol.},
issn = {0022-2836},
number = 3 ; Feb. 23, 2001,
volume = 306,
place = {United States},
year = {Fri Feb 23 00:00:00 EST 2001},
month = {Fri Feb 23 00:00:00 EST 2001}
}