Limited proteolysis of mycobacterial DNA-binding protein 1 with an extended, lysine-rich, intrinsically disordered region to unveil posttranslational modifications

Yutaka Yoshida; Akihito Nishiyama; Desak Nyoman Surya Suameitria Dewi; Tomoya Yamazaki; Akira Yokoyama; Daiki Kobayashi; Hitoshi Kondo; Yuriko Ozeki; Sohkichi Matsumoto

doi:10.1016/j.bbrc.2023.09.028

Limited proteolysis of mycobacterial DNA-binding protein 1 with an extended, lysine-rich, intrinsically disordered region to unveil posttranslational modifications

Biochem Biophys Res Commun. 2023 Nov 12:681:111-119. doi: 10.1016/j.bbrc.2023.09.028. Epub 2023 Sep 20.

Authors

Yutaka Yoshida¹, Akihito Nishiyama², Desak Nyoman Surya Suameitria Dewi², Tomoya Yamazaki², Akira Yokoyama², Daiki Kobayashi³, Hitoshi Kondo², Yuriko Ozeki², Sohkichi Matsumoto²

Affiliations

¹ Department of Bacteriology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-Dori, Chuo-Ku, Niigata, 951-8510, Japan. Electronic address: yoshida.yutaka@med.niigata-u.ac.jp.
² Department of Bacteriology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-Dori, Chuo-Ku, Niigata, 951-8510, Japan.
³ Omics Unit, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-Dori, Chuo-Ku, Niigata, 951-8510, Japan.

PMID: 37774568
DOI: 10.1016/j.bbrc.2023.09.028

Abstract

The basic, intrinsically disordered regions of eukaryotic histones and their bacterial counterparts are presumed to act as signaling hubs to regulate the compaction of chromosomes or nucleoids and various DNA processes such as gene expression, recombination, and DNA replication. Posttranslational modifications (PTMs) on these regions are pivotal in regulating chromosomal or nucleoid compaction and DNA processes. However, the low sequence complexity and the presence of short lysine-rich repeats in the regions have hindered the accurate determination of types and locations of PTMs using conventional proteomic procedures. We described a limited proteolysis protocol using trypsin to analyze PTMs on mycobacterial DNA-binding protein 1 (MDP1), a nucleoid-associated protein in mycobacterial species that possesses an extended, lysine-rich, intrinsically disordered region in its C-terminal domain. This limited proteolysis approach successfully revealed significant methylation on many lysine residues in the C-terminal domain of MDP1 purified from Mycobacterium tuberculosis, which was lacking in the corresponding region of recombinant MDP1 expressed in Escherichia coli.

Keywords: Bottom-up proteomics; HupB; Intrinsically disordered region; Limited proteolysis; Mycobacterial DNA-binding protein 1; Posttranslational modification; Protein methylation.