Close proximity of phosphorylation sites to ligand in the phosphoproteome of the extreme thermophile Thermus thermophilusHB8

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.

Phosphoproteomic Analysis Reveals the Effects of PilF Phosphorylation on Type IV Pilus and Biofilm Formation in Thermus thermophilus HB27

“…Among the identified phosphopeptides, the locations of 67 nonredundant phosphorylation sites were determined with the highest level of confidence (class I sites: 99% peptide identification confidence (48)): 38 (56.7%) on serine, 24 (35.8%) on threonine, and 5 (7.4%) on tyrosine. The relative Ser/Thr/Tyr phosphorylation ratio was similar to that of other bacterial phosphoproteomes (28,29,31,33,35). Annotated MS/MS spectra of all identified phosphopeptides and a detailed list of the identified phosphoproteins are provided in supplemental Fig.…”

“…In a comparison of phosphoproteomes within the same species (i.e. T. thermophilus strains HB27 (this study) and HB8 (35)), ϳ45% of the phosphoproteins were found to be identical (supplemental Table S8), and, with the exception of valyl-tRNA synthetase (TTC0805) and chaperonin GroEL (TTC1714), the phosphorylation sites of these proteins were the same. The remaining 55% of the phosphoproteins of these two closely related thermophiles were distinct, perhaps accounting for strain-specific differences in regulatory mechanisms and biological functions.…”

“…These observations have been followed by more detailed, species-specific phosphoproteomics investigations, including in Bacillus subtilis (28), Escherichia coli (29), Lactococcus lactis (30), Halobacterium salinarum (31), Klebsiella pneumonia (32), Pseudomonas spp. (33), Rhodopseudomonas palustris (34), and T. thermophilus HB8 (35). In this study, the role played by the global phosphorylation network of the thermophile T. thermophilus HB27 in the physiological processes that mediate the stress responses and thermotolerance of this bacterium was examined.…”

Lysine Propionylation Is a Prevalent Post-translational Modification in Thermus thermophilus

“…Recently, we reported the results of phosphoproteome and acetylome analyses performed during a proteomic characterization of Thermus thermophilus HB8, an extremely thermophilic Gram-negative eubacterium (22,33). The 2.2-Mb genome of T. thermophilus HB8 contains 2238 open reading frames, about half the number in Escherichia coli.…”

“…The simplicity of this genome is greatly advantageous in attempts to achieve a comprehensive understanding of the bacterium's physiology. We have studied T. thermophilus as a model organism with the goal of understanding biological phenomena in bacteria, using "-omics" approaches such as structural genomics (34 -36), transcriptomics (37), metabolomics (38), whole-cell proteomics (39), and PTM proteomics (22,33).…”