Histidine is a versatile residue playing key roles in enzyme catalysis thanks to the chemistry of its imidazole group that can serve as nucleophile, general acid or base depending on its protonation ...state. In bacteria, signal transduction relies on two-component systems (TCS) which comprise a sensor histidine kinase (HK) containing a phosphorylatable catalytic His with phosphotransfer and phosphatase activities over an effector response regulator. Recently, a pH-gated model has been postulated to regulate the phosphatase activity of HisKA HKs based on the pH-dependent rotamer switch of the phosphorylatable His. Here, we have revisited this model from a structural and functional perspective on HK853-RR468 and EnvZ-OmpR TCS, the prototypical HisKA HKs. We have found that the rotamer of His is not influenced by the environmental pH, ruling out a pH-gated model and confirming that the chemistry of the His is responsible for the decrease in the phosphatase activity at acidic pH.
Protein phosphorylation is the most common type of post-translational modification in eukaryotes. The phosphoproteome is defined as the complete set of experimentally detectable phosphorylation sites ...present in a cell's proteome under various conditions. However, we are still far from identifying all the phosphorylation sites in a cell mainly due to the lack of information about phosphorylation events involving residues other than Ser, Thr and Tyr. Four types of phosphate–protein linkage exist and these generate nine different phosphoresidues—pSer, pThr, pTyr, pHis, pLys, pArg, pAsp, pGlu and pCys. Most of the effort in studying protein phosphorylation has been focused on Ser, Thr and Tyr phosphorylation. The recent development of 1- and 3-pHis monoclonal antibodies promises to increase our understanding of His phosphorylation and the kinases and phosphatases involved. Several His kinases are well defined in prokaryotes, especially those involved in two-component system (TCS) signaling. However, in higher eukaryotes, NM23, a protein originally characterized as a nucleoside diphosphate kinase, is the only characterized protein–histidine kinase. This ubiquitous and conserved His kinase autophosphorylates its active site His, and transfers this phosphate either onto a nucleoside diphosphate or onto a protein His residue. Studies of NM23 protein targets using newly developed anti-pHis antibodies will surely help illuminate the elusive His phosphorylation-based signaling pathways. This review discusses the role that the NM23/NME/NDPK phosphotransferase has, how the addition of the pHis phosphoproteome will expand the phosphoproteome and make His phosphorylation part of the global phosphorylation world. It also summarizes why our understanding of phosphorylation is still largely restricted to the acid stable phosphoproteome, and highlights the study of NM23 histidine kinase as an entrée into the world of histidine phosphorylation.
Novel peptide dendrimer with Lys-2His repeating units was recently synthesized, studied by NMR (Molecules, 2019, 24, 2481) and tested as a nanocontainer for siRNA delivery (Int. J. Mol. Sci., 2020, ...21, 3138). Histidine amino acid residues were inserted in the spacers of this dendrimer. Increase of their charge with a pH decrease turns a surface-charged dendrimer into a volume-charged one and should change all properties. In this paper, the molecular dynamics simulation method was applied to compare the properties of the dendrimer in water with explicit counterions at two different pHs (at normal pH with neutral histidines and at low pH with fully protonated histidines) in a wide interval of temperatures. We obtained that the dendrimer at low pH has essentially larger size and size fluctuations. The electrostatic properties of the dendrimers are different but they are in good agreement with the theoretical soft sphere model and practically do not depend on temperature. We have shown that the effect of pairing of side imidazole groups is much stronger in the dendrimer with neutral histidines than in the dendrimer with protonated histidines. We also demonstrated that the capacity of a nanocontainer based on this dendrimer with protonated histidines is significantly larger than that of a nanocontainer with neutral histidines.
Protein phosphorylation is a reversible post-translational modification. Nine of the 20 natural amino acids in proteins can be phosphorylated, but most of what we know about the roles of protein ...phosphorylation has come from studies of serine, threonine, and tyrosine phosphorylation. Much less is understood about the phosphorylation of histidine, lysine, arginine, cysteine, aspartate, and glutamate, so-called non-canonical phosphorylations. Phosphohistidine (pHis) was discovered 60 years ago as a mitochondrial enzyme intermediate; since then, evidence for the existence of histidine kinases and phosphohistidine phosphatases has emerged, together with examples where protein function is regulated by reversible histidine phosphorylation. pHis is chemically unstable and has thus been challenging to study. However, the recent development of tools for studying pHis has accelerated our understanding of the multifaceted functions of histidine phosphorylation, revealing a large number of proteins that are phosphorylated on histidine and implicating pHis in a wide range of cellular processes.
In this perspective, Hunter reviews the history of protein-histidine phosphorylation, the current status of the field, and the prospects for further advances in understanding the functions of histidine phosphorylation in regulating cellular processes.
An interesting phenomenon is described that the fluorescence signal of poly(adenine) (A) DNA-templated gold nanoclusters (AuNCs) is greatly improved in the presence of
l
-histidine by means of
l
...-histidine-DNA interaction. The modified nanoclusters display strong fluorescence emission with excitation/emission maxima at 290/475 nm. The fluorescence quantum yield (QY) is improved from 1.9 to 6.5%. Fluorescence enhancement is mainly ascribed to the
l
-histidine-DNA interaction leading to conformational changes of the poly(A) DNA template, which offer a better microenvironment to protect AuNCs. The assay enables
l
-histidine to be determined with good sensitivity and a linear response that covers the 1 to 50 nM
l
-histidine concentration range with a 0.3 nM limit of detection. The proposed method has been applied to the determination of imidazole-containing drugs in pharmaceutical samples.
Graphical abstract
A turn-on fluorescent method has been designed for the sensitive detection of
l
-histidine as well as imidazole-containing drugs on the basis of the
l
-histidine-DNA interaction.
Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes ...switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (Agp1). Whereas Agp1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While Agp1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes suggest the use of light-controllable histidine kinases and phosphatases for optogenetics.
The availability of an expanded genetic code opens exciting new opportunities in enzyme design and engineering. In this regard histidine analogues have proven particularly versatile, serving as ...ligands to augment metalloenzyme function and as catalytic nucleophiles in designed enzymes. The ability to genetically encode multiple functional residues could greatly expand the range of chemistry accessible within enzyme active sites. Here, we develop mutually orthogonal translation components to selectively encode two structurally similar histidine analogues. Transplanting known mutations from a promiscuous Methanosarcina mazei pyrrolysyl‐tRNA synthetase (MmPylRSIFGFF) into a single domain PylRS from Methanomethylophilus alvus (MaPylRSIFGFF) provided a variant with improved efficiency and specificity for 3‐methyl‐L‐histidine (MeHis) incorporation. The MaPylRSIFGFF clone was further characterized using in vitro biochemical assays and x‐ray crystallography. We subsequently engineered the orthogonal MmPylRS for activity and selectivity for 3‐(3‐pyridyl)‐L‐alanine (3‐Pyr), which was used in combination with MaPylRSIFGFF to produce proteins containing both 3‐Pyr and MeHis. Given the versatile roles played by histidine in enzyme mechanisms, we anticipate that the tools developed within this study will underpin the development of enzymes with new and enhanced functions.
Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. Three domains, termed PAS, GAF, and PHY domains, ...constitute the N‐terminal photosensory chromophore module (PCM); the C‐terminus is often a histidine kinase module. In the Agrobacterium fabrum phytochrome Agp1, the autophosphorylation activity of the histidine kinase is high in the Pr and low in the Pfr form. Crystal structure analyses of PCMs suggest flexibility around position 308 in the Pr but not in the Pfr form. Here, we performed time‐resolved fluorescence anisotropy measurements with different Agp1 mutants, each with a single cysteine residue at various positions. The fluorophore label Atto‐488 was attached to each mutant, and time‐resolved fluorescence anisotropy was measured in the Pr and Pfr forms. Fluorescence anisotropy curves were fitted with biexponential functions. Differences in the amplitude A2 of the second component between the PCM and the full‐length variant indicate a mechanical coupling between position 362 and the histidine kinase. Pr‐to‐Pfr photoconversion induced no significant changes in the time constant t2 at any position. An intermediate t2 value at position 295, which is located in a compact environment, suggests flexibility around the nearby position 308 in Pr and in Pfr.
The cartoon shows a structural model of the phytochrome Agp1 from Agrobacterium fabrum. The colored spheres indicate positions where Cys residues were introduced for labeling with the fluorescent Atto‐488. Flexibility of protein regions was determined at the different positions in the Pr and Pfr forms of the phytochrome by time‐resolved fluorescence anisotropy. Green, PAS domain; blue, GAF domain; yellow, PHY domain; orange, Dhp domain of histidine kinase; magenta, ATPase domain of histidine kinase. Flexibility parameters differed significantly between the different regions and between full‐length and protein without histidine kinase, but not between Pr and Pfr.
In this study, Cu
modulated silver nanoclusters were constructed for the turn-on, label-free detection of L-histidine. Six Ag NCs protected by oligonucleotides (DNA-Ag NCs) were tested in a series of ...experiments. Finally, A-DAN-Ag NCs were chosen as the best candidate due to their excellent fluorescent properties. The fluorescence of A-DAN-Ag NCs was quenched using Cu
through energy or electron transfer. However, quenched fluorescence could be restored dramatically in the presence of L-histidine due to Cu
liberation from A-DAN-Ag NCs and because of the chelation between the imidazole group of L-histidine and Cu
. The proposed sensor exhibited high selectivity towards L-histidine over other amino acids, with a limit of detection (LOD) of 0.096 μM ranging from 0 to 8 μM. The proposed sensor succeeded in detecting L-histidine in diluted human urine. Therefore, the sensor has promising practical applications in biological systems.
The objective of this experiment was to evaluate the effect of supplementing a metabolizable protein (MP)-deficient diet with rumen-protected (RP) Lys, Met, and specifically His on dairy cow ...performance. The experiment was conducted for 12wk with 48Holstein cows. Following a 2-wk covariate period, cows were blocked by DIM and milk yield and randomly assigned to 1 of 4 diets, based on corn silage and alfalfa haylage: control, MP-adequate diet (ADMP; MP balance: +9 g/d); MP-deficient diet (DMP; MP balance: −317g/d); DMP supplemented with RPLys (AminoShure-L, Balchem Corp., New Hampton, NY) and RPMet (Mepron; Evonik Industries AG, Hanau, Germany; DMPLM); and DMPLM supplemented with an experimental RPHis preparation (DMPLMH). The analyzed crude protein content of the ADMP and DMP diets was 15.7 and 13.5 to 13.6%, respectively. The apparent total-tract digestibility of all measured nutrients, plasma urea-N, and urinary N excretion were decreased by the DMP diets compared with ADMP. Milk N secretion as a proportion of N intake was greater for the DMP diets compared with ADMP. Compared with ADMP, dry matter intake (DMI) tended to be lower for DMP, but was similar for DMPLM and DMPLMH (24.5, 23.0, 23.7, and 24.3kg/d, respectively). Milk yield was decreased by DMP (35.2kg/d), but was similar to ADMP (38.8kg/d) for DMPLM and DMPLMH (36.9 and 38.5kg/d, respectively), paralleling the trend in DMI. The National Research Council 2001model underpredicted milk yield of the DMP cows by an average (±SE) of 10.3±0.75kg/d. Milk fat and true protein content did not differ among treatments, but milk protein yield was increased by DMPLM and DMPLMH compared with DMP and was not different from ADMP. Plasma essential amino acids (AA), Lys, and His were lower for DMP compared with ADMP. Supplementation of the DMP diets with RP AA increased plasma Lys, Met, and His. In conclusion, MP deficiency, approximately 15% below the National Research Council requirements from 2001, decreased DMI and milk yield in dairy cows. Supplementation of the MP-deficient diet with RPLys and RPMet diminished the difference in DMI and milk yield compared with ADMP and additional supplementation with RPHis eliminated it. As total-tract fiber digestibility was decreased with the DMP diets, but DMI tended to increase with RP AA supplementation, we propose that, similar to monogastric species, AA play a role in DMI regulation in dairy cows. Our data implicate His as a limiting AA in high-producing dairy cows fed corn silage- and alfalfa haylage-based diets, deficient in MP. The MP-deficient diets clearly increased milk N efficiency and decreased dramatically urinary N losses.
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