Protonation State Of Histidine Research Articles

PH‐Dependent Assembly and Stability of Toll‐Like Receptor 3/dsRNA Signaling Complex: Insights from Constant pH Molecular Dynamics and Metadynamics Simulations

AbstractThe pH‐dependent assembly of Toll‐like receptors (TLRs), which triggers a threshold‐like response, is a key principle in immune signaling. While crystallography has revealed the intricate structure of these assembly complexes, the mechanisms underlying their pH dependency remain unclear. Herein, constant pH simulations and metadynamics are employed to investigate the pH‐dependent assembly and stability of the TLR3/dsRNA signaling complex. The findings demonstrate that system pH regulates complex assembly and stability by modulating the protonation and charge states of histidines. Histidines in TLR3 act as pH‐dependent, positively charged binding sites that capture negatively charged dsRNA. Additionally, these histidines form a [H682⁺]—[E626⁻] dipole, facilitating the assembly of two TLR3 molecules into an antisymmetric dimer through dipole–dipole interactions. Surprisingly, TLR3 can shift the pKa values of key histidines from their model pKa of 6.5, increasing protonation likelihood and enhancing ligand binding. Notably, the aromatic residue Phe84, located within the dsRNA binding site [His39⁺–His60⁺–Phe84–His108⁺], alters the pKa of His60 through cation‐π interactions with its protonated state. This study offers new insights into the molecular mechanisms underlying pH‐dependent immune signaling via higher‐order assemblies and suggests potential applications for histidine in self‐assembling biomaterials.

Histidine Protonation States Regulate the State Transition from R State Hemoglobin.

The objective of our work is to investigate the impact of pH on the structural changes of hemoglobin that affect its O2 affinity, known as the Bohr effect. We conducted molecular dynamics (MD) simulations to explore the transition between various hemoglobin states based on the protonation states (PSs) of two histidine residues (βHis143 and βHis146). We conducted the MD simulations from the R and R2 states with three sets of PSs assuming pH values of 7.0, 6.5, and 5.5, aiming to investigate the influence of pH on hemoglobin behavior. Our results demonstrated that the protonated His residues promote the state transition from the R state to the R2 state and encourage elongation of the distance between the β1-β2 subunits by weakening the inter-subunit interactions in the R state. These observations, aligning with the experimental evidence, indicate that the R2 state typically crystallizes under low pH conditions. Our findings suggest that the relationship between the PSs and the structural stability of the R state plays a role in the acid and alkaline Bohr effect.

Surface Charge Can Modulate Phase Separation of Multidomain Proteins.

Phase separation has emerged as an important mechanism explaining the formation of certain biomolecular condensates. Biological phase separation is often driven by the multivalent interactions of modular protein domains. Beyond valency, the physical features of folded domains that promote phase separation are poorly understood. We used a model system─the small ubiquitin modifier (SUMO) and its peptide ligand, the SUMO interaction motif (SIM)─to examine how domain surface charge influences multivalency-driven phase separation. Phase separation of polySUMO and polySIM was altered by pH via a change in the protonation state of SUMO surface histidines. These effects were recapitulated by histidine mutations, which modulated SUMO solubility and polySUMO-polySIM phase separation in parallel and were quantitatively explained by atomistic modeling of weak interactions among proteins in the system. Thus, surface charge can tune the phase separation of multivalent proteins, suggesting a means of controlling phase separation biologically, evolutionarily, and therapeutically.

Effect of histidine protonation state on ligand binding at the ATP-binding site of human protein kinase CK2

Histidine residues contribute to numerous molecular interactions, owing to their structure with the ionizable aromatic side chain with pKa close to the physiological pH. Herein, we studied how the two histidine residues, His115 and His160 of the catalytic subunit of human protein kinase CK2, affect the binding of the halogenated heterocyclic ligands at the ATP-binding site. Thermodynamic studies on the interaction between five variants of hCK2α (WT protein and four histidine mutants) and three ionizable bromo-benzotriazoles and their conditionally non-ionizable benzimidazole counterparts were performed with nanoDSF, MST, and ITC. The results allowed us to identify the contribution of interactions involving the particular histidine residues to ligand binding. We showed that despite the well-documented hydrogen bonding/salt bridge formation dragging the anionic ligands towards Lys68, the protonated His160 also contributes to the binding of such ligands by long-range electrostatic interactions. Simultaneously, His 115 indirectly affects ligand binding, placing the hinge region in open/closed conformations.

Probing the role of the protonation state of a minor groove-linker histidine in Exd-Hox–DNA binding

DNA recognition and targeting by transcription factors (TFs) through specific binding are fundamental in biological processes. Furthermore, the histidine protonation state at the TF-DNA binding interface can significantly influence the binding mechanism of TF-DNA complexes. Nevertheless, the role of histidine in TF-DNA complexes remains underexplored. Here, we employed all-atom molecular dynamics simulations using AlphaFold2-modeled complexes based on previously solved co-crystal structures to probe the role of the His-12 residue in the Extradenticle (Exd)-Sex combs reduced (Scr)-DNA complex when binding to Scr and Ultrabithorax (Ubx) target sites. Our results demonstrate that the protonation state of histidine notably affected the DNA minor-groove width profile and binding free energy. Examining flanking sequences of various binding affinities derived from SELEX-seq experiments, we analyzed the relationship between binding affinity and specificity. We uncovered how histidine protonation leads to increased binding affinity but can lower specificity. Our findings provide new mechanistic insights into the role of histidine in modulating TF-DNA binding.

Structural and dynamic properties of urinary human serum albumin fragments: a molecular dynamics study

A microalbuminuria level acts as a good index to screen and monitor diabetes and renal failure. However, the urinary albumin loss after sample preservation and storage is the major bottleneck to obtain the accurate microalbuminuria test. Such loss is due to the rapid albumin fragmentation by urinary proteases. Some fragments were suggested to be bioactive biomarkers of diabetes and renal disease, but no structural and dynamical properties of albumin fragments are available. Thus, in this work, the structural and dynamical properties of reported albumin fragments are revealed using molecular dynamics simulations. The properties of nine fragments (F1–F9) discovered recently were studied at the real pH conditions of urine samples (pH 4.5, 7 and 8). The complete loss of secondary structure is found in short fragments (F1–F6), while large-sized polypeptides (F7–F9) can somehow maintain their folds. Especially, F8 (subdomain IIIB) is the most stable fragment. The difference in histidine protonation states has no impact on the structural stability of albumin fragments. The ability of F8 (subdomain IIIB) to maintain its stability and folds suggests it as an alternative albumin biomarker in urine. An insight obtained here will become the fundamental importance for understanding clinical assays for albumin detection, sample stability and peptidomics analysis of urine. Communicated by Ramaswamy H. Sarma

Multiple Timescale Dynamic Analysis of Functionally-Impairing Mutations in Human Ileal Bile Acid-Binding Protein.

Human ileal bile acid-binding protein (hI-BABP) has a key role in the enterohepatic circulation of bile salts. Its two internal binding sites exhibit positive cooperativity accompanied by a site-selectivity of glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two most abundant bile salts in humans. To improve our understanding of the role of dynamics in ligand binding, we introduced functionally impairing single-residue mutations at two key regions of the protein and subjected the mutants to NMR relaxation analysis and MD simulations. According to our results, mutation in both the vicinity of the C/D (Q51A) and the G/H (Q99A) turns results in a redistribution of motional freedom in apo hI-BABP. Mutation Q51A, deteriorating the site-selectivity of GCA and GCDA, results in the channeling of ms fluctuations into faster motions in the binding pocket hampering the realization of key side chain interactions. Mutation Q99A, abolishing positive binding cooperativity for GCDA, leaves ms motions in the C-terminal half unchanged but by decoupling βD from a dynamic cluster of the N-terminal half displays an increased flexibility in the vicinity of site 1. MD simulations of the variants indicate structural differences in the portal region and mutation-induced changes in dynamics, which depend on the protonation state of histidines. A dynamic coupling between the EFGH portal, the C/D-region, and the helical cap is evidenced highlighting the interplay of structural and dynamic effects in bile salt recognition in hI-BABP.

Understanding the conformational changes in the influenza B M2 ion channel at various protonation states

The characterization of influenza (A/B M2) ion channels is very important as they are potential binding sites for the drugs. We report the all-atom molecular dynamics study of the influenza B M2 ion channel in the presence of explicit solvent and lipid bilayers using the high resolution solid-state NMR structures. The importance of the various protonation states of histidine in the activation of the ion channel is discussed. The conformational changes at the closed and the open structures clearly show that the increase in tilt angle is necessary for the activation of the ion channel. Additionally, the free energy surfaces of the eight systems show the importance of the protonation state of the histidine residues in the activation of the influenza B M2 ion channel. The protonation of the histidine residues increases the tilt angle and the intra-helix distance which is evident from the superimposition of the structures corresponding to the maxima and the minima in the free energy landscape. The findings imply differences in the singly protonated and double protonated conformational states of BM2 ion channel and provide insights to help further studies of these ion channels as the drug targets for the influenza virus.

Protonation State of an Important Histidine from High Resolution Structures of Lytic Polysaccharide Monooxygenases.

Lytic Polysaccharide Monooxygenases (LPMOs) oxidatively cleave recalcitrant polysaccharides. The mechanism involves (i) reduction of the Cu, (ii) polysaccharide binding, (iii) binding of different oxygen species, and (iv) glycosidic bond cleavage. However, the complete mechanism is poorly understood and may vary across different families and even within the same family. Here, we have investigated the protonation state of a secondary co-ordination sphere histidine, conserved across AA9 family LPMOs that has previously been proposed to be a potential proton donor. Partial unrestrained refinement of newly obtained higher resolution data for two AA9 LPMOs and re-refinement of four additional data sets deposited in the PDB were carried out, where the His was refined without restraints, followed by measurements of the His ring geometrical parameters. This allowed reliable assignment of the protonation state, as also validated by following the same procedure for the His brace, for which the protonation state is predictable. The study shows that this histidine is generally singly protonated at the Nε2 atom, which is close to the oxygen species binding site. Our results indicate robustness of the method. In view of this and other emerging evidence, a role as proton donor during catalysis is unlikely for this His.

Determination of Histidine Protonation States in Proteins by Fast Magic Angle Spinning NMR.

Histidine residues play important structural and functional roles in proteins, such as serving as metal-binding ligands, mediating enzyme catalysis, and modulating proton channel activity. Many of these activities are modulated by the ionization state of the imidazole ring. Here we present a fast MAS NMR approach for the determination of protonation and tautomeric states of His at frequencies of 40–62 kHz. The experiments combine 1H detection with selective magnetization inversion techniques and transferred echo double resonance (TEDOR)–based filters, in 2D heteronuclear correlation experiments. We illustrate this approach using microcrystalline assemblies of HIV-1 CACTD-SP1 protein.

Kinetics and Mechanism of Fentanyl Dissociation from the μ-Opioid Receptor.

Driven by illicit fentanyl, opioid related deaths have reached the highest level in 2020. Currently, an opioid overdose is resuscitated by the use of naloxone, which competitively binds and antagonizes the μ-opioid receptor (mOR). Thus, knowledge of the residence times of opioids at mOR and the unbinding mechanisms is valuable for assessing the effectiveness of naloxone. In the present study, we calculate the fentanyl-mOR dissociation time and elucidate the mechanism by applying an enhanced sampling molecular dynamics (MD) technique. Two sets of metadynamics simulations with different initial structures were performed while accounting for the protonation state of the conserved H2976.52, which has been suggested to modulate the ligand-mOR affinity and binding mode. Surprisingly, with the Nδ-protonated H2976.52, fentanyl can descend as much as 10 Å below the level of the conserved D1473.32 before escaping the receptor and has a calculated residence time τ of 38 s. In contrast, with the Nϵ- and doubly protonated H2976.52, the calculated τ are 2.6 and 0.9 s, respectively. Analysis suggests that formation of the piperidine–Hid297 hydrogen bond strengthens the hydrophobic contacts with the transmembrane helix (TM) 6, allowing fentanyl to explore a deep pocket. Considering the experimental τ of ∼4 min for fentanyl and the role of TM6 in mOR activation, the deep insertion mechanism may be biologically relevant. The work paves the way for large-scale computational predictions of opioid dissociation rates to inform evaluation of strategies for opioid overdose reversal. The profound role of the histidine protonation state found here may shift the paradigm in computational studies of ligand–receptor kinetics.

Determining structure and action mechanism of LBF14 by molecular simulation

The recently discovered, membrane–active peptide LBF14 contains several non–proteinogenic amino acids and is able to transform vesicles into tubule networks. The exact membrane interaction mechanism and detailed secondary structure are yet to be determined. We performed molecular dynamics simulations of LBF14 and let it fold de novo into its ensemble of native secondary structures. Histidine protonation state effects on secondary structure were investigated. An MD simulation of the peptide with a lipid bilayer was performed. Simulation results were compared to circular dichroism and electron paramagnetic resonance data of previous studies. LBF14 contains a conserved helical section in an otherwise random structure. Helical stability is influenced by histidine protonation. The peptide localized to the polar layer of the membrane, consistent with experimental results. While the overall secondary structure is unaffected by membrane interaction, Ramachandran plot analysis yielded two distinct peptide conformations during membrane interaction. This conformational change was accompanied by residue repositioning within the membrane. LBF14 only affected the local order in the membrane, and had no measurable effect on pressure. The simulation results are consistent with the previously proposed membrane interaction mechanism of LBF14 and can additionally explain the local interaction mechanism. Communicated by Ramaswamy H. Sarma

Histidine protonation states are key in the LigI catalytic reaction mechanism.

Lignin is one of the world's most abundant organic polymers, and 2-pyrone-4,6-dicarboxylate lactonase (LigI) catalyzes the hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) in the degradation of lignin. The pH has profound effects on enzyme catalysis and therefore we studied this in the context of LigI. We found that changes of the pH mostly affects surface residues, while the residues at the active site are more subject to changes of the surrounding microenvironment. In accordance with this, a high pH facilitates the deprotonation of the substrate. Detailed free energy calculations by the empirical valence bond (EVB) approach revealed that the overall hydrolysis reaction is more likely when the three active site histidines (His31, His33 and His180) are protonated at the ɛ site, however, protonation at the δ site may be favored during specific steps of the reaction. Our studies have uncovered the determinant role of the protonation state of the active site residues His31, His33 and His180 in the hydrolysis of PDC.

Inhibitor binding influences the protonation states of histidines in SARS-CoV-2 main protease.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of Mpro, a cysteine protease, have been determined, facilitating structure-based drug design. Mpro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41–Cys145, Mpro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nucleophile Cys145 have been debated in previous studies of SARS-CoV Mpro, but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 Mpro as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of Mpro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an α-ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored Nδ (HD) and Nϵ (HE) protonation of His41 and His164, respectively, the α-ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 Mpro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts.

Probing Microenvironmental Acidity in Lyophilized Protein and Vaccine Formulations Using Solid-state NMR Spectroscopy.

Biophysical and biochemical instability of therapeutic proteins in the solution state may necessitate the development of products in the solid form, due to their enhanced stability. Lyophilization is a widely used method to ensure dry state stabilization of biological products. A commonly encountered issue is the pH shifts that can occur due to undesired crystallization of a buffer component, resulting in loss of protein activities. However, it is technically challenging to noninvasively investigate the physicochemical environment in the lyophile matrix. In this work, we demonstrate an approach based on solid-state NMR to investigate the microenvironmental acidity in lyophilized protein formulations, using histidine, a commonly used buffer agent, as a molecular probe. The solid-state acidity in the lyophilized matrix can be assessed by monitoring the chemical shift changes of histidine. The protonation and tautomeric states of histidine lyophilized at a range of pH values from 4.5 to 11.0 were identified from full 13C and 15N resonance assignments in one-dimensional and two-dimensional NMR experiments. The results demonstrated a pH-dependence of histidine chemical shift in the amorphous state. Moreover, we successfully applied this protocol to investigate the microenvironmental pH in lyophilized formulations of the HPV vaccine and lactate dehydrogenase protein.

Deuterium-Enhanced Raman Spectroscopy for Histidine pKa Determination in a pH-Responsive Hydrogel

We report here a method for the determination of the pKa of histidine in complex or heterogeneous systems amenable to neither solid-state nor solution NMR spectroscopy. Careful synthesis of a fluorenylmethyloxycarbonyl- and trityl-protected, C2-deuterated histidine produces a vibrational-probe-equipped amino acid that can readily be incorporated into any peptide accessible by standard solid-phase methods. The frequency of the unique, Raman-active stretching vibration of this C2-D probe is a clear reporter of the protonation state of histidine. We investigate here a pH-sensitive peptide that self-assembles to form a hydrogel at neutral pH. The pKa of the lone histidine residue in the peptide, which is likely responsible for this pH-dependent behavior, cannot be investigated by NMR spectroscopy because of the supramolecular, soft nature of the gel. However, after synthesizing a C2-deuterated-histidine-containing peptide, we were able to follow the protonation state of histidine throughout a pH titration using Raman difference spectroscopy, thereby precisely determining the pKa of interest.

A Hierarchical Approach to Predict Conformation-Dependent Histidine Protonation States in Stable and Flexible Proteins.

Solution acidity measured by pH is an important environmental factor that affects protein structure. It influences the protonation state of protein residues, which in turn may be coupled to protein conformational changes, unfolding, and ligand binding. It remains difficult to compute and measure the p Ka of individual residues, as well as to relate them to pH-dependent protein transitions. This paper presents a hierarchical approach to compute the p Ka of individual protonatable residues, specifically histidines, coupled with underlying structural changes of a protein. A fast and efficient free energy perturbation (FEP) algorithm has also been developed utilizing a fast implementation of standard molecular dynamics (MD) algorithms. Specifically, a CUDA version of the AMBER MD engine is used in this paper. Eight histidine p Ka's are computed in a diverse set of pH stable proteins to demonstrate the proposed approach's utility and assess the predictive quality of the AMBER FF99SB force field. A reference molecule is carefully selected and tested for convergence. A hierarchical approach is used to model p Ka's of the six histidine residues of the diphtheria toxin translocation domain (DTT), which exhibits a diverse ensemble of individual conformations and pH-dependent unfolding. The hierarchical approach consists of first sampling equilibrium conformational ensembles of a protein with protonated and neutral histidine residues via long equilibrium MD simulations (Flores-Canales, J. C.; et al. bioRxiv, 2019, 572040). A clustering method is then used to identify sampled protein conformations, and p Ka's of histidines in each protein conformation are computed. Finally, an ensemble averaging formalism is developed to compute weighted average histidine p Ka's. These can be compared with an apparent experimentally measured p Ka of the DTT protein and thus allows us to propose a mechanism of pH-dependent unfolding of the DTT protein.

Assessment of a Large Enzyme-Drug Complex by Proton-Detected Solid-State NMR Spectroscopy without Deuteration.

Solid-state NMR spectroscopy has recently enabled structural biology with small amounts of non-deuterated proteins, largely alleviating the classical sample production demands. Still, despite the benefits for sample preparation, successful and comprehensive characterization of complex spin systems in the few cases of higher-molecular-weight proteins has thus far relied on traditional 13 C-detected methodology or sample deuteration. Herein we show for a 29 kDa carbonic anhydrase:acetazolamide complex that different aspects of solid-state NMR assessment of a complex spin system can be successfully accessed using a non-deuterated, 500 μg sample in combination with adequate spectroscopic tools. The shown access to protein structure, protein dynamics, as well as biochemical parameters in amino acid sidechains, such as histidine protonation states, will be transferable to proteins that are not expressible in E. coli.

Conformational Effects of a Disease-Associated Hydrophobic-to-Hydrophobic Substitution and Histidine Protonation State Located at the Midpoint of the Intrinsically Disordered Region of proBDNF

Conformational Effects of a Disease-Associated Hydrophobic-to-Hydrophobic Substitution and Histidine Protonation State Located at the Midpoint of the Intrinsically Disordered Region of proBDNF

PKa Determination of a Histidine Residue in a Short Peptide Using Raman Spectroscopy.

Determining the pKa of key functional groups is critical to understanding the pH-dependent behavior of biological proteins and peptide-based biomaterials. Traditionally, 1H NMR spectroscopy has been used to determine the pKa of amino acids; however, for larger molecules and aggregating systems, this method can be practically impossible. Previous studies concluded that the C-D stretches in Raman are a useful alternative for determining the pKa of histidine residues. In this study, we report on the Raman application of the C2-D probe on histidine’s imidazole side chain to determining the pKa of histidine in a short peptide sequence. The pKa of the tripeptide was found via difference Raman spectroscopy to be 6.82, and this value was independently confirmed via 1H NMR spectroscopy on the same peptide. The C2-D probe was also compared to other Raman reporters of the protonation state of histidine and was determined to be more sensitive and reliable than other protonation-dependent signals. The C2-D Raman probe expands the tool box available to chemists interested in directly interrogating the pKa’s of histidine-containing peptide and protein systems.