Focus on phosphohistidine

Summary. Phosphohistidine has been identified as an enzymic intermediate in numerous biochemical reactions and plays a functional role in many regulatory pathways. Unlike the phosphoester bond of its cousins (phosphoserine, phosphothreonine and phosphotyrosine), the phosphoramidate (P–N) bond of phosphohistidine has a high ΔG° of hydrolysis and is unstable under acidic conditions. This acid-lability has meant that the study of protein histidine phosphorylation and the associated protein kinases has been slower to progress than other protein phosphorylation studies. Histidine phosphorylation is a crucial component of cell signalling in prokaryotes and lower eukaryotes. It is also now becoming widely reported in mammalian signalling pathways and implicated in certain human disease states. This review covers the chemistry of phosphohistidine in terms of its isomeric forms and chemical derivatives, how they can be synthesized, purified, identified and the relative stabilities of each of these forms. Furthermore, we highlight how this chemistry relates to the role of phosphohistidine in its various biological functions.     

Negatively charged phospholipids influence the activity of the sensor kinase KdpD of Escherichia coli

Synthesis of the high-affinity K⁺-translocating Kdp-ATPase of Escherichia coli, encoded by the kdpFABC operon, is regulated by the membrane-bound sensor kinase KdpD and the soluble response regulator KdpE. K⁺ limitation or a sudden increase in osmolarity induces the expression of kdpFABC. Due to the importance of K⁺ to maintain turgor, it has been proposed that KdpD is a turgor sensor. Although the primary stimulus that KdpD senses is unknown, alterations in membrane strain or the interaction between KdpD and membrane components might be good candidates. Here, we report a study of the influence of the membrane phospholipid composition on the function of KdpD in vivo and in vitro using various E. coli mutants defective in phospholipid biosynthesis. Surprisingly, neither the lack of the major E. coli phospholipid phosphatidylethanolamine nor the drastic reduction of the phosphatidylglycerol/cardiolipin content influenced induction of kdpFABC expression significantly. However, in vitro reconstitution experiments with synthetic phospholipids clearly demonstrated that KdpD kinase activity is dependent on negatively charged phospholipids, whereas the structure of the phospholipids plays a minor role. These results indicate that electrostatic interactions are important for the activity of KdpD. Received: 29 March 1999 / Accepted: 26 July 1999     

Role of Histidine Residues in the Adenosine A2a Receptor Ligand Binding Site

Abstract: The pH dependency of the binding of ligands to adenosine A_2a receptors in rat striatal membranes was examined. For those agonists sensitive to adenosine deaminase a solubilised membrane preparation was used. A two- to fourfold increase in affinity was observed for CGS-21680, 5'- N -ethylcarboxamidoadenosine, adenosine, 3'-deoxyadenosine, 5'-deoxyadenosine, inosine, and N ⁶-methoxypurine riboside on lowering the ambient pH from 7.0 to 5.5. In contrast, no such pH dependency was observed with 2'-deoxyadenosine, although 2'-methoxyadenosine binding was pH dependent. This effect on the affinity of CGS-21680 was reduced by diethylpyrocarbonate and restored by hydroxylamine and implied a pK value of 7.0 for the histidine residue involved. No such dependence was observed with cyclopentyltheophylline or dimethylpropargylxanthine. It is concluded that one of the histidines conserved in the adenosine receptor binding site acts as a hydrogen bond donor to the oxygen of the 2'-hydroxyl group of adenosine agonists.     

[Pt(dien)]2+ migrates intramolecularly from methionine S to imidazole Nz 2 in the peptides H-His-Gly-Met-OH and Ac-His-Ala-Ala-Ala-Met-NHPh

The pH- and time-dependent reaction of [Pt(dien)(H₂O)]²⁺ with the methionine- and histidine-containing peptides H-His-Gly-Met-OH and Ac-His-Ala-Ala-Ala-Met-NHPh at 313 K has been investigated by HPLC and NMR spectroscopy. For both peptides, initial relatively rapid formation of the kinetically favoured methionine S-bound complex is followed by slow intramolecular migration of the [Pt(dien)]²⁺ fragment to imidazole N_{ε 2} (or, in the case of H-His-Gly-Met-OH, to a much lesser extent to the competing imidazole N_{δ 1}) of the histidine side chain over a period of 500 h. Time-dependent studies for the pentapeptide at pH 8.0 demonstrate that this isomerization can take place by either direct S→N_{ε 2} migration or by a two-step mechanism involving initial N_{ε 2} coordination of a second [Pt(dien)]²⁺ fragment and subsequent cleavage of the orginal Pt-S bond in the resulting dinuclear complex. The rate of κS/κN _{ε 2} isomerization is markedly reduced on lowering the pH to 5.1. Received: 26 February 1999 / Accepted: 14 April 1999     

1H-N.m.r. and c.d. investigation of the histidine side chain conformation in small peptides

Three series of model peptides containing histidine have been examined by ¹H-n.m.r. and c.d. spectroscopy: X-His peptides with X = Gly, Ala, Leu; His-X peptides with X = Gly, Ala, Leu, Ser, Lys, Phe, Tyr; and Pro-His-X peptides with X = Gly; Ala; Leu; Val; Phe; Tyr, C.d. spectra were obtained for pH values between 1 and 11 to give titration curves [θ] vs. pH; ¹H-n.m.r. spectra were recorded at four selected pH values corresponding to defined ionic species. ¹H-n.m.r. spectra in Me₂SO of the NH₃⁺, Imid⁺, COO^? ionic state (pH 4.5) were also obtained. The histidine side chain conformation in the various peptides and the changing ionic states is reflected in the ³J_αβ,β′ coupling constants, the Δδ ββ′ anisochrony values and the c.d. histidine chromophore contribution at 215 nm, and qualitative and semiquantitative correlations can be established between these parameters. Whereas the histidine side chain conformation is quite different in each of the three series, and varies with the ionic state and environment, it is practically identical for each peptide within a series: the nature of the X-residue does not exert any influence on the histidine side chain conformational behaviour. Thus, the classical rotamer distribution R I > R II > R III which is due to steric factors is usually observed unless specific intramolecular interactions such as hydrogen or ionic bonds override these.     

Carbamoyl phosphate synthetase 1 deficiency in Italy: clinical and genetic findings in a heterogeneous cohort

Carbamoyl Phosphate Synthetase 1 deficiency (CPS1D) is a rare autosomal recessive urea cycle disorder, potentially leading to lethal hyperammonemia. Based on the age of onset, there are two distinct phenotypes: neonatal and late form. The CPS1 enzyme, located in the mitochondrial matrix of hepatocytes and epithelial cells of intestinal mucosa, is encoded by the CPS1 gene. At present more than 220 clear-cut genetic lesions leading to CPS1D have been reported. As most of them are private mutations diagnosis is complicated.Here we report an overview of the main clinical findings and biochemical and molecular data of 13 CPS1D Italian patients. In two of them, one with the neonatal form and one with the late form, cadaveric auxiliary liver transplant was performed. Mutation analysis in these patients identified 17 genetic lesions, 9 of which were new confirming their “private” nature. Seven of the newly identified mutations were missense/nonsense changes. In order to study their protein level effects, we performed an in silico analysis whose results indicate that the amino acid substitutions occur at evolutionary conserved positions and affect residues necessary for enzyme stability or function.     

Tautomerism,acid-base equilibria,and H-bonding of the six histidines in subtilisin BPN' by NMR

We have determined by (15)N, (1)H, and (13)C NMR, the chemical behavior of the six histidines in subtilisin BPN' and their PMSF and peptide boronic acid complexes in aqueous solution as a function of pH in the range of from 5 to 11, and have assigned every (15)N, (1)H, C(epsilon 1), and C(delta2) resonance of all His side chains in resting enzyme. Four of the six histidine residues (17, 39, 67, and 226) are neutrally charged and do not titrate. One histidine (238), located on the protein surface, titrates with pK(a) = 7.30 +/- 0.03 at 25 degrees C, having rapid proton exchange, but restricted mobility. The active site histidine (64) in mutant N155A titrates with a pK(a) value of 7.9 +/- 0.3 and sluggish proton exchange behavior, as shown by two-site exchange computer lineshape simulation. His 64 in resting enzyme contains an extremely high C(epsilon 1)-H proton chemical shift of 9.30 parts per million (ppm) owing to a conserved C(epsilon 1)-H(.)O=C H-bond from the active site imidazole to a backbone carbonyl group, which is found in all known serine proteases representing all four superfamilies. Only His 226, and His 64 at high pH, exist as the rare N(delta1)-H tautomer, exhibiting (13)C(delta1) chemical shifts approximately 9 ppm higher than those for N(epsilon 2)-H tautomers. His 64 in the PMSF complex, unlike that in the resting enzyme, is highly mobile in its low pH form, as shown by (15)N-(1)H NOE effects, and titrates with rapid proton exchange kinetics linked to a pK(a) value of 7.47 +/- 0.02.     

Nickel(II) complexes of the multihistidine peptide fragments of human prion protein

Nickel(II) complexes of the peptide fragments of human prion protein containing histidyl residues both inside and outside the octarepeat domain have been studied by the combined application of potentiometric, UV-visible and circular dichroism spectroscopic methods. The imidazole-N donor atoms of histidyl residues are the exclusive metal binding sites below pH 7.5, but the formation of stable macrochelates was characteristic only for the peptide HuPrP(76-114) containing four histidyl residues. Yellow colored square planar complexes were obtained above pH 7.5-8 with the cooperative deprotonation of three amide nitrogens in the [N_im,N⁻,N⁻,N⁻] coordination mode. It was found that the peptides can bind as many nickel(II) ions as the number of independent histidyl residues. All data supported that the complex formation processes of nickel(II) are very similar to those of copper(II), but with a significantly reduced stability for nickel(II), which shifts the complex formation reactions into the slightly alkaline pH range. The formation of coordination isomers was characteristic of the mononuclear complexes with a significant preference for the nickel(II) binding at the histidyl sites outside the octarepeat domain. The results obtained for the two-histidine fragments of the protein, HuPrP(91-115), HuPrP(76-114)H85A and HuPrP(84-114)H96A, made it possible to compare the binding ability of the His96 and His111 sites. These data reveal a significant difference in the nickel(II) and copper(II) binding sites of the peptides: His96 was found to predominate almost completely for nickel(II) ions, while the opposite order, but with comparable concentrations, was reported for copper(II).     

Structural characterization of the intra-membrane histidine kinase YbdK from Bacillus subtilis in DPC micelles

Bacterial histidine kinases (HKs) play a critical role in signal transduction for cellular adaptation to environmental conditions and stresses. YbdK from Bacillus subtilis is a 320-residue intra-membrane sensing HK characterized by a short input domain consisting of two transmembrane helices without an extracytoplasmic domain. While the cytoplasmic domains of HKs have been studied in detail, the intra-membrane sensing domain systems are still uncharacterized due to difficulties in handling the transmembrane domain. Here, we successfully obtained pure recombinant transmembrane domain of YbdK (YbdK-TM) from E. coli and analyzed the characteristics of YbdK-TM using nuclear magnetic resonance (NMR) and other biophysical methods. YbdK-TM was found to form homo-dimers in DPC micelles based on cross-linking assays and analytical ultracentrifugation analyses. We estimated the size of the YbdK-TM DPC complex to be 46 kDa using solution state NMR T₁/T₂ relaxation analyses in DPC micelles. These results provide information that will allow functional and structural studies of intra-membrane sensing HKs to begin.