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Title: The structure of the Thermococcus gammatolerans McrB N-terminal domain reveals a new mode of substrate recognition and specificity among McrB homologs

Abstract

McrBC is a two-component, modification-dependent restriction system that cleaves foreign DNA-containing methylated cytosines. Previous crystallographic studies have shown that Escherichia coli McrB uses a base-flipping mechanism to recognize these modified substrates with high affinity. The side chains stabilizing both the flipped base and the distorted duplex are poorly conserved among McrB homologs, suggesting that other mechanisms may exist for binding modified DNA. In this paper we present the structures of the Thermococcus gammatolerans McrB DNA-binding domain (TgΔ185) both alone and in complex with a methylated DNA substrate at 1.68 and 2.27 Å resolution, respectively. The structures reveal that TgΔ185 consists of a YT521-B homology (YTH) domain, which is commonly found in eukaryotic proteins that bind methylated RNA and is structurally unrelated to the E. coli McrB DNA-binding domain. Structural superposition and co-crystallization further show that TgΔ185 shares a conserved aromatic cage with other YTH domains, which forms the binding pocket for a flipped-out base. Mutational analysis of this aromatic cage supports its role in conferring specificity for the methylated adenines, whereas an extended basic surface present in TgΔ185 facilitates its preferential binding to duplex DNA rather than RNA. Together, these findings establish a new binding mode and specificity among McrBmore » homologs and expand the biological roles of YTH domains.« less

Authors:
ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [1]
  1. Cornell Univ., Ithaca, NY (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE Office of Science (SC); National Institutes of Health (NIH)
OSTI Identifier:
1593451
Grant/Contract Number:  
AC02-06CH11357; GM120242; P41 GM103403; S10 RR029205
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 295; Journal Issue: 3; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; X-ray crystallography; DNA binding protein; protein-nucleic acid interaction; protein structure; RNA-protein interaction; structural biology; 6-methyladenosine; DNA binding; McrB; restriction system; YTH domain

Citation Formats

Hosford, Christopher J., Bui, Anthony Q., and Chappie, Joshua S. The structure of the Thermococcus gammatolerans McrB N-terminal domain reveals a new mode of substrate recognition and specificity among McrB homologs. United States: N. p., 2019. Web. https://doi.org/10.1074/jbc.RA119.010188.
Hosford, Christopher J., Bui, Anthony Q., & Chappie, Joshua S. The structure of the Thermococcus gammatolerans McrB N-terminal domain reveals a new mode of substrate recognition and specificity among McrB homologs. United States. https://doi.org/10.1074/jbc.RA119.010188
Hosford, Christopher J., Bui, Anthony Q., and Chappie, Joshua S. Tue . "The structure of the Thermococcus gammatolerans McrB N-terminal domain reveals a new mode of substrate recognition and specificity among McrB homologs". United States. https://doi.org/10.1074/jbc.RA119.010188. https://www.osti.gov/servlets/purl/1593451.
@article{osti_1593451,
title = {The structure of the Thermococcus gammatolerans McrB N-terminal domain reveals a new mode of substrate recognition and specificity among McrB homologs},
author = {Hosford, Christopher J. and Bui, Anthony Q. and Chappie, Joshua S.},
abstractNote = {McrBC is a two-component, modification-dependent restriction system that cleaves foreign DNA-containing methylated cytosines. Previous crystallographic studies have shown that Escherichia coli McrB uses a base-flipping mechanism to recognize these modified substrates with high affinity. The side chains stabilizing both the flipped base and the distorted duplex are poorly conserved among McrB homologs, suggesting that other mechanisms may exist for binding modified DNA. In this paper we present the structures of the Thermococcus gammatolerans McrB DNA-binding domain (TgΔ185) both alone and in complex with a methylated DNA substrate at 1.68 and 2.27 Å resolution, respectively. The structures reveal that TgΔ185 consists of a YT521-B homology (YTH) domain, which is commonly found in eukaryotic proteins that bind methylated RNA and is structurally unrelated to the E. coli McrB DNA-binding domain. Structural superposition and co-crystallization further show that TgΔ185 shares a conserved aromatic cage with other YTH domains, which forms the binding pocket for a flipped-out base. Mutational analysis of this aromatic cage supports its role in conferring specificity for the methylated adenines, whereas an extended basic surface present in TgΔ185 facilitates its preferential binding to duplex DNA rather than RNA. Together, these findings establish a new binding mode and specificity among McrB homologs and expand the biological roles of YTH domains.},
doi = {10.1074/jbc.RA119.010188},
journal = {Journal of Biological Chemistry},
number = 3,
volume = 295,
place = {United States},
year = {2019},
month = {12}
}

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