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Title: Short strong hydrogen bonds in proteins: a case study of rhamnogalacturonan acetylesterase

Abstract

The short hydrogen bonds in rhamnogalacturonan acetylesterase have been investigated by structure determination of an active-site mutant, {sup 1}H NMR spectra and computational methods. Comparisons are made to database statistics. A very short carboxylic acid carboxylate hydrogen bond, buried in the protein, could explain the low-field (18 p.p.m.) {sup 1}H NMR signal. An extremely low-field signal (at approximately 18 p.p.m.) in the {sup 1}H NMR spectrum of rhamnogalacturonan acetylesterase (RGAE) shows the presence of a short strong hydrogen bond in the structure. This signal was also present in the mutant RGAE D192N, in which Asp192, which is part of the catalytic triad, has been replaced with Asn. A careful analysis of wild-type RGAE and RGAE D192N was conducted with the purpose of identifying possible candidates for the short hydrogen bond with the 18 p.p.m. deshielded proton. Theoretical calculations of chemical shift values were used in the interpretation of the experimental {sup 1}H NMR spectra. The crystal structure of RGAE D192N was determined to 1.33 Å resolution and refined to an R value of 11.6% for all data. The structure is virtually identical to the high-resolution (1.12 Å) structure of the wild-type enzyme except for the interactions involving the mutation andmore » a disordered loop. Searches of the Cambridge Structural Database were conducted to obtain information on the donor–acceptor distances of different types of hydrogen bonds. The short hydrogen-bond interactions found in RGAE have equivalents in small-molecule structures. An examination of the short hydrogen bonds in RGAE, the calculated pK{sub a} values and solvent-accessibilities identified a buried carboxylic acid carboxylate hydrogen bond between Asp75 and Asp87 as the likely origin of the 18 p.p.m. signal. Similar hydrogen-bond interactions between two Asp or Glu carboxy groups were found in 16% of a homology-reduced set of high-quality structures extracted from the PDB. The shortest hydrogen bonds in RGAE are all located close to the active site and short interactions between Ser and Thr side-chain OH groups and backbone carbonyl O atoms seem to play an important role in the stability of the protein structure. These results illustrate the significance of short strong hydrogen bonds in proteins.« less

Authors:
; ; ;  [1];  [1];  [2];  [1];  [3];  [1]
  1. Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen (Denmark)
  2. Department of Chemistry, University of Iowa, IA 52242 (United States)
  3. Novozymes, Novo Allé, DK-2880 Bagsvaerd (Denmark)
Publication Date:
OSTI Identifier:
22348015
Resource Type:
Journal Article
Journal Name:
Acta Crystallographica. Section D: Biological Crystallography
Additional Journal Information:
Journal Volume: 64; Journal Issue: Pt 8; Other Information: PMCID: PMC2483496; PUBLISHER-ID: gx5126; PMID: 18645234; OAI: oai:pubmedcentral.nih.gov:2483496; Copyright (c) Langkilde et al. 2008; This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0907-4449
Country of Publication:
Denmark
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; ATOMS; CARBONYLS; CHEMICAL SHIFT; CRYSTAL STRUCTURE; CRYSTALS; HYDROGEN; INTERACTIONS; MOLECULES; NMR SPECTRA; NUCLEAR MAGNETIC RESONANCE; ORIGIN; PROTEIN STRUCTURE; SIGNALS; STABILITY

Citation Formats

Langkilde, Annette, Kristensen, Søren M., Lo Leggio, Leila, Mølgaard, Anne, Jensen, Jan H., Department of Chemistry, University of Iowa, IA 52242, Houk, Andrew R., Navarro Poulsen, Jens-Christian, Kauppinen, Sakari, Larsen, Sine, and European Synchrotron Radiation Facility. Short strong hydrogen bonds in proteins: a case study of rhamnogalacturonan acetylesterase. Denmark: N. p., 2008. Web. doi:10.1107/S0907444908017083.
Langkilde, Annette, Kristensen, Søren M., Lo Leggio, Leila, Mølgaard, Anne, Jensen, Jan H., Department of Chemistry, University of Iowa, IA 52242, Houk, Andrew R., Navarro Poulsen, Jens-Christian, Kauppinen, Sakari, Larsen, Sine, & European Synchrotron Radiation Facility. Short strong hydrogen bonds in proteins: a case study of rhamnogalacturonan acetylesterase. Denmark. doi:10.1107/S0907444908017083.
Langkilde, Annette, Kristensen, Søren M., Lo Leggio, Leila, Mølgaard, Anne, Jensen, Jan H., Department of Chemistry, University of Iowa, IA 52242, Houk, Andrew R., Navarro Poulsen, Jens-Christian, Kauppinen, Sakari, Larsen, Sine, and European Synchrotron Radiation Facility. Fri . "Short strong hydrogen bonds in proteins: a case study of rhamnogalacturonan acetylesterase". Denmark. doi:10.1107/S0907444908017083.
@article{osti_22348015,
title = {Short strong hydrogen bonds in proteins: a case study of rhamnogalacturonan acetylesterase},
author = {Langkilde, Annette and Kristensen, Søren M. and Lo Leggio, Leila and Mølgaard, Anne and Jensen, Jan H. and Department of Chemistry, University of Iowa, IA 52242 and Houk, Andrew R. and Navarro Poulsen, Jens-Christian and Kauppinen, Sakari and Larsen, Sine and European Synchrotron Radiation Facility},
abstractNote = {The short hydrogen bonds in rhamnogalacturonan acetylesterase have been investigated by structure determination of an active-site mutant, {sup 1}H NMR spectra and computational methods. Comparisons are made to database statistics. A very short carboxylic acid carboxylate hydrogen bond, buried in the protein, could explain the low-field (18 p.p.m.) {sup 1}H NMR signal. An extremely low-field signal (at approximately 18 p.p.m.) in the {sup 1}H NMR spectrum of rhamnogalacturonan acetylesterase (RGAE) shows the presence of a short strong hydrogen bond in the structure. This signal was also present in the mutant RGAE D192N, in which Asp192, which is part of the catalytic triad, has been replaced with Asn. A careful analysis of wild-type RGAE and RGAE D192N was conducted with the purpose of identifying possible candidates for the short hydrogen bond with the 18 p.p.m. deshielded proton. Theoretical calculations of chemical shift values were used in the interpretation of the experimental {sup 1}H NMR spectra. The crystal structure of RGAE D192N was determined to 1.33 Å resolution and refined to an R value of 11.6% for all data. The structure is virtually identical to the high-resolution (1.12 Å) structure of the wild-type enzyme except for the interactions involving the mutation and a disordered loop. Searches of the Cambridge Structural Database were conducted to obtain information on the donor–acceptor distances of different types of hydrogen bonds. The short hydrogen-bond interactions found in RGAE have equivalents in small-molecule structures. An examination of the short hydrogen bonds in RGAE, the calculated pK{sub a} values and solvent-accessibilities identified a buried carboxylic acid carboxylate hydrogen bond between Asp75 and Asp87 as the likely origin of the 18 p.p.m. signal. Similar hydrogen-bond interactions between two Asp or Glu carboxy groups were found in 16% of a homology-reduced set of high-quality structures extracted from the PDB. The shortest hydrogen bonds in RGAE are all located close to the active site and short interactions between Ser and Thr side-chain OH groups and backbone carbonyl O atoms seem to play an important role in the stability of the protein structure. These results illustrate the significance of short strong hydrogen bonds in proteins.},
doi = {10.1107/S0907444908017083},
journal = {Acta Crystallographica. Section D: Biological Crystallography},
issn = {0907-4449},
number = Pt 8,
volume = 64,
place = {Denmark},
year = {2008},
month = {8}
}