title: Close proximity of phosphorylation sites to ligand in the phosphoproteome of the extreme thermophile Thermus thermophilus HB8.
abstract: We performed phosphoproteome analysis of proteins from the extremely thermophilic Gram-negative eubacterium Thermus thermophilus HB8 using gel-free mass spectrometric method. We identified 52 phosphopeptides from 48 proteins and determined 46 phosphorylation sites: 30 on serine, 12 on threonine, and 4 on tyrosine. The identified phosphoproteins are known to be involved in a wide variety of cellular processes. To help elucidate the functional roles of these phosphorylation events, we mapped the phosphorylation sites on the known tertiary structures of the respective proteins. In all, we succeeded in mapping 46 sites (approximately 88%) on the corresponding structures. Most of the phosphorylation sites were found to be located on loops and terminal regions of the secondary structures. Surprisingly, 28 of these sites were situated at or near the active site of the enzyme. In particular, 18 sites were within 4 Å of the ligand, including substrate or cofactor. Such structural locations suggest direct effects of the phosphorylation on the binding of ligand in addition to inducing a conformational change. Interestingly, 19 of these 28 phosphorylation sites were situated near the phosphate moiety of a substrate or cofactor. In oligomeric proteins, 5 phosphorylation sites were found at the subunit interface. Based on these results, we propose a regulatory mechanism that involves Ser/Thr/Tyr phosphorylation in T. thermophilus HB8.
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Takahata
authors.forename: Masao
authors.initials: M
authors.lastname: Inoue
authors.forename: Kwang
authors.initials: K
authors.lastname: Kim
authors.forename: Yota
authors.initials: Y
authors.lastname: Iio
authors.forename: Masaaki
authors.initials: M
authors.lastname: Miyamoto
authors.forename: Ryoji
authors.initials: R
authors.lastname: Masui
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ishihama
authors.forename: Seiki
authors.initials: S
authors.lastname: Kuramitsu
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D002952:Citric Acid Cycle; D008024:Ligands; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D021122:Protein Subunits; D020543:Proteome / Q000032:analysis / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D016963:Thermus thermophilus / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D010750:Phosphoproteins; D021122:Protein Subunits; D020543:Proteome