title: Atypical OmpR/PhoB subfamily response regulator GlnR of actinomycetes functions as a homodimer, stabilized by the unphosphorylated conserved Asp-focused charge interactions.
abstract: The OmpR/PhoB subfamily protein GlnR of actinomycetes is an orphan response regulator that globally coordinates the expression of genes related to nitrogen metabolism. Biochemical and genetic analyses reveal that the functional GlnR from Amycolatopsis mediterranei is unphosphorylated at the potential phosphorylation Asp(50) residue in the N-terminal receiver domain. The crystal structure of this receiver domain demonstrates that it forms a homodimer through the α4-β5-α5 dimer interface highly similar to the phosphorylated typical response regulator, whereas the so-called "phosphorylation pocket" is not conserved, with its space being occupied by an Arg(52) from the β3-α3 loop. Both in vitro and in vivo experiments confirm that GlnR forms a functional homodimer via its receiver domain and suggest that the charge interactions of Asp(50) with the highly conserved Arg(52) and Thr(9) in the receiver domain may be crucial in maintaining the proper conformation for homodimerization, as also supported by molecular dynamics simulations of the wild type GlnR versus the deficient mutant GlnR(D50A). This model is backed by the distinct phenotypes of the total deficient GlnR(R52A/T9A) double mutant versus the single mutants of GlnR (i.e. D50N, D50E, R52A and T9A), which have only minor effects upon both dimerization and physiological function of GlnR in vivo, albeit their DNA binding ability is weakened compared with that of the wild type. By integrating the supportive data of GlnRs from the model Streptomyces coelicolor and the pathogenic Mycobacterium tuberculosis, we conclude that the actinomycete GlnR is atypical with respect to its unphosphorylated conserved Asp residue being involved in the critical Arg/Asp/Thr charge interactions, which is essential for maintaining the biologically active homodimer conformation.
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology.
authors.forename: Wei
authors.initials: W
authors.lastname: Lin
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology, the State Key Laboratory of Genetic Engineering, Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology.
authors.forename: Xiaobiao
authors.initials: X
authors.lastname: Han
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology.
authors.forename: Zilong
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Chengyuan
authors.initials: C
authors.lastname: Wang
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology.
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.affiliation: the Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Huaiyu
authors.initials: H
authors.lastname: Yang
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology.
authors.forename: Yinhua
authors.initials: Y
authors.lastname: Lu
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology.
authors.forename: Weihong
authors.initials: W
authors.lastname: Jiang
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology, the State Key Laboratory of Genetic Engineering, Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China, the Shanghai-MOST Key Laboratory of Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, Shanghai 201203, China, the Department of Microbiology and Li Ka Shing Institute of Health Sciences, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China, and gpzhao@sibs.ac.cn.
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Zhao
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology, State Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China, pengzhang01@sibs.ac.cn.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhang
mesh_terms: D000192:Actinomycetales / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D048372:Streptomyces coelicolor / Q000378:metabolism*; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C082451:GlnR protein, Streptomyces coelicolor; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; C061278:PhoB protein, Bacteria; D001224:Aspartic Acid
keywords: Actinobacteria; Crystal Structure; Gene Regulation; Homodimer; OmpR/PhoB Subfamily; Phosphorylation; Protein Conformation; Receiver Domain; Unphosphorylated Aspartate
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