Protein histidine phosphorylation: Increased stability of thiophosphohistidine

Lasker, Michael V.; Bui, Cuong D.; Besant, Paul G.; Sugawara, Kiyoshi; Thai, Phung N.; Medzihradszky, Gergely; Turck, Christoph W.

doi:10.1110/ps.8.10.2177

Cited by 46 publications

(42 citation statements)

References 22 publications

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“…For this experiment we used ␥S-ATP instead of ATP. ␥S-ATP is an ATP analogue, which is stabilized by replacement of the double-bonded oxygen of the ␥-phosphate residue with a sulphur atom resulting in minimized degradation by ATP-hydrolyzing enzymes due to the lower electronegativity of the sulphur (Lasker et al, 1999). Fig.…”

Section: Figmentioning

confidence: 99%

ATP-dependent stabilization and protection of fibroblast growth factor 2

Rose

Gast

Seeger

et al. 2010

Journal of Biotechnology

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Section: Figmentioning

confidence: 99%

ATP-dependent stabilization and protection of fibroblast growth factor 2

Rose

Gast

Seeger

et al. 2010

Journal of Biotechnology

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“…However, with time, the intensity of the ␥-32 P label associated with NSP2 decreased, perhaps due to hydrolysis of the phosphohistidine (8). With ATP-␥-S, the bound thiophosphate is retained longer because of the increased stability of the P-N linkage (between the ␥-phosphate of the thiophosphate contributed by ATP-␥-S and the N-ε of the histidine side chain) due to lower electronegativity of the sulfur atom (25). The longer retention time has been advantageous for crystallographic visualization of phosphohistidine intermediates.…”

Section: Vol 81 2007 Ndp Kinase Activity Of Rotavirus Nsp2 12275mentioning

confidence: 99%

Crystallographic and Biochemical Analysis of Rotavirus NSP2 with Nucleotides Reveals a Nucleoside Diphosphate Kinase-Like Activity

Kumar

Jayaram

Carpió

et al. 2007

J Virol

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Rotavirus, the major pathogen of infantile gastroenteritis, carries a nonstructural protein, NSP2, essential for viroplasm formation and genome replication/packaging. In addition to RNA-binding and helix-destabilizing properties, NSP2 exhibits nucleoside triphosphatase activity. A conserved histidine (H225) functions as the catalytic residue for this enzymatic activity, and mutation of this residue abrogates genomic double-stranded RNA synthesis without affecting viroplasm formation. To understand the structural basis of the phosphatase activity of NSP2, we performed crystallographic analyses of native NSP2 and a functionally defective H225A mutant in the presence of nucleotides. These studies showed that nucleotides bind inside a cleft between the two domains of NSP2 in a region that exhibits structural similarity to ubiquitous cellular HIT (histidine triad) proteins. Only minor conformational alterations were observed in the cleft upon nucleotide binding and hydrolysis. This hydrolysis involved the formation of a stable phosphohistidine intermediate. These observations, reminiscent of cellular nucleoside diphosphate (NDP) kinases, prompted us to investigate whether NSP2 exhibits phosphoryl-transfer activity. Bioluminometric assay showed that NSP2 exhibits an NDP kinase-like activity that transfers the bound phosphate to NDPs. However, NSP2 is distinct from the highly conserved cellular NDP kinases in both its structure and catalytic mechanism, thus making NSP2 a potential target for antiviral drug design. With structural similarities to HIT proteins, which are not known to exhibit NDP kinase activity, NSP2 represents a unique example among structure-activity relationships. The newly observed phosphoryl-transfer activity of NSP2 may be utilized for homeostasis of nucleotide pools in viroplasms during genome replication.Rotaviruses are the major cause of life-threatening diarrhea in children worldwide (13). The genome (ϳ18 kb) of rotavirus consists of 11 segments of double-stranded RNA (dsRNA) encoding six structural and six nonstructural proteins (NSPs) (13). Several studies have shown that endogenous transcription of dsRNA segments, genome replication/packaging, and assembly of subviral particles take place in specialized compartments called viroplasms, which appear early during infection (30). NSP2, along with NSP5, is essential for the formation of these perinuclear, non-membrane-bound, electron-dense inclusions (14). Coexpression of NSP2 and NSP5 alone has been shown to be sufficient for the formation of viroplasm-like structures (14). NSP2 also appears to interact directly or indirectly with other structural proteins present in the viroplasm, such as VP2 (30), which forms the innermost capsid layer of the virion (32), and VP1, which is the viral RNA-dependent RNA polymerase (45).Biochemical studies on recombinant NSP2 have shown that in addition to single-stranded-RNA (ssRNA)-binding and helix-destabilizing activities (42), NSP2 exhibits nucleotide triphosphatase (NTPase) activity (40). The protein cleaves...

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“…The course of the phosphotransfer in the process or the origin and fate of the high energy phosphate that passes through PtsN in vivo is, however, difficult to assess. This is because phospho-histidines are labile in any of the procedures that allow detection of other phospho-amino acids (15,16). To overcome this difficulty, we have resorted to the release of much of the intact proteome of live P. putida cells directly into a native polyacrylamide gel 3 by means of the in situ electrophoresis of whole bacteria.…”

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confidence: 99%

Growth-dependent Phosphorylation of the PtsN (EIINtr) Protein of Pseudomonas putida

Pflüger

2007

Journal of Biological Chemistry

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The nitrogen-related branch of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) of Pseudomonas putida includes the ptsN gene encoding the EII Ntr (PtsN) enzyme. Although the implication of this protein in a variety of cellular functions has been observed in diverse bacteria, the physiological signals that bring about phosphorylation/dephosphorylation of the PtsN protein are not understood. This work documents the phosphorylation status of the EII Ntr enzyme of P. putida at various growth stages in distinct media. Culture conditions were chosen to include fructose (the uptake of which is controlled by the PTS) or glucose (a non-PTS sugar in P. putida) in minimal medium with casamino acids, ammonia, or nitrate as alternative nitrogen sources. To quantify the relative ratio of PtsN/PtsN ϳ P in live cells, we resorted to the in situ electrophoresis of whole bacteria expressing an E-epitope-tagged EII Ntr followed by the fractionation of the thereby released native proteome in a non-denaturing gel. Although the PtsN species phosphorylated in amino acid His 68 was detected under virtually all growth scenarios, the relative levels of the non-phosphorylated form varied dramatically depending on the growth phase and the nutrients available in the medium. The share of phosphorylated PtsN increased along growth in a fashion apparently independent of any trafficking of sugars. The large variations of non-phosphorylated PtsN in different growth conditions, in contrast to the systematic excess of the phosphorylated PtsN form, suggested that the P-free PtsN is the predominant signaling species of the protein.

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Protein histidine phosphorylation: Increased stability of thiophosphohistidine

Cited by 46 publications

References 22 publications

ATP-dependent stabilization and protection of fibroblast growth factor 2

ATP-dependent stabilization and protection of fibroblast growth factor 2

Crystallographic and Biochemical Analysis of Rotavirus NSP2 with Nucleotides Reveals a Nucleoside Diphosphate Kinase-Like Activity

Growth-dependent Phosphorylation of the PtsN (EIINtr) Protein of Pseudomonas putida

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