Titratable Residues Research Articles

Role of water in the determination of protonation states of titratable residues.

Water is the fundamental unit for living being, and its contribution in variety of crucial cellular functions is widely accepted. The presence of water molecules in protein's environment also accounts for structural optimization, in which highly conserved water molecules ensure structural stability of the biomolecule by providing protein-water (solute-solvent) hydrogen-bond interaction networks. Similarly, protonation states and pKa values of individual amino acid residues are also influenced by neighboring water molecules present in the protein's vicinity. In the present study, we have highlighted the role of water molecules in hydrogen-bond optimization, in determining pKa values and protonation states of titratable residues in JH2 domain of JAK2 apo protein. We found that inclusion or exclusion of water molecules while calculating pKa and assigning protonation states to amino acid residues during the molecular system build-up step resulted in slight differences in pKa values of few titratable residues and alternative protonation states of a certain residue. Accordingly, different protonation states of ionizable residues offer differing interaction patterns. Thus, we inferred that the presence of water optimizes the hydrogen-bond interactions by forming direct protein-water interactions and by linking via protein-protein bridging interactions. However, in the absence of water, the interaction pattern is somewhat disrupted. We assume that water molecules could modulate the plausibility of a particular protonation state of titratable residues on the basis of its fit with the local environment, by utilizing some particular hydrogen-bond contacts that would remain unexploited in the absence of water.

PK a Calculations in Membrane Proteins from Molecular Dynamics Simulations.

The conformational changes of membrane proteins are crucial to their function and usually lead to fluctuations in the electrostatic environment of the protein surface. A very effective way to quantify these changes is by calculating the pK a values of the protein's titratable residues, which can be regarded as electrostatic probes. To achieve this, we need to take advantage of the fast and reliable pK a calculators developed for globular proteins and adapt them to include the explicit effects of membranes. Here, we provide a detailed linear response approximation protocol that uses our own software (PypKa) to calculate reliable pK a values from short MD simulations of membrane proteins.

PH-Dependent Interactions of Apolipophorin-III with a Lipid Disk

Apolipophorin-III (ApoLp-III) is required for stabilization of molecular shuttles of lipid fuels in insects and is found to contribute to the insect immune reaction. Rearrangement of its five [Formula: see text]-helices enables ApoLp-III to reversibly associate with lipids. We investigate computationally the conformational changes of ApoLp-III and the pH-dependence of the binding free energy of ApoLp-III association with a lipid disk. A dominant binding mode along with several minor, low population, modes of the ApoLp-III binding to a lipid disk was identified. The pH-dependence of the binding energy for ApoLp-III with the lipid disk is predicted to be significant, with the pH-optimum at pH[Formula: see text]. The calculations suggest that there are no direct interactions between the lipid head groups and titratable residues of ApoLp-III. In the physiological pH range from 6.0 to 9.0, the binding free energy of ApoLp-III with the lipid disk decreases significantly with respect to its optimal value at pH 8.0 (at pH[Formula: see text], it is 1.02[Formula: see text]kcal/mol and at pH[Formula: see text] it is 0.23[Formula: see text]kcal/mol less favorable than at the optimal pH[Formula: see text]), indicating that the pH is an important regulator of ApoLp-III lipid disk association.

PH-dependent interactions of Apolipophorin-III with a lipid disk

Apolipophorin-III (ApoLp-III) is required for stabilization of molecular shuttles of lipid fuels in insects and is found to contribute to the insect immune reaction. Rearrangement of its five [Formula: see text]-helices enables ApoLp-III to reversibly associate with lipids. We investigate computationally the conformational changes of ApoLp-III and the pH-dependence of the binding free energy of ApoLp-III association with a lipid disk. A dominant binding mode along with several minor, low population, modes of the ApoLp-III binding to a lipid disk was identified. The pH-dependence of the binding energy for ApoLp-III with the lipid disk is predicted to be significant, with the pH-optimum at pH[Formula: see text]. The calculations suggest that there are no direct interactions between the lipid head groups and titratable residues of ApoLp-III. In the physiological pH range from 6.0 to 9.0, the binding free energy of ApoLp-III with the lipid disk decreases significantly with respect to its optimal value at pH 8.0 (at pH[Formula: see text], it is 1.02[Formula: see text]kcal/mol and at pH[Formula: see text] it is 0.23[Formula: see text]kcal/mol less favorable than at the optimal pH[Formula: see text]), indicating that the pH is an important regulator of ApoLp-III lipid disk association.

PypKa: A Flexible Python Module for Poisson-Boltzmann-Based pKa Calculations.

The protonation of titratable residues has a significant impact on the structure and function of biomolecules, influencing many physicochemical and ADME properties. Thus, the importance of the estimation of protonation free energies (pKa values) is paramount in different scientific communities, including bioinformatics, structural biology, or medicinal chemistry. Here, we introduce PypKa, a flexible tool to predict Poisson-Boltzmann/Monte Carlo-based pKa values of titratable sites in proteins. This application was benchmarked using a large data set of experimental values to show that our single structure-based method is fast and has a competitive performance. This is a free and open-source tool that provides a simple, reusable, and extensible Python API and CLI for pKa calculations with a valuable trade-off between fast and accurate predictions. PypKa allows pKa calculations in existing protocols with the addition of a few extra lines of code. PypKa supports CPU parallel computing on solvated proteins obtained from the PDB repository but also from MD simulations using three common naming schemes: GROMOS, AMBER, and CHARMM. The code and documentation to this open-source project is publicly available at https://github.com/mms-fcul/PypKa.

PKa Calculations with the Polarizable Drude Force Field and Poisson-Boltzmann Solvation Model.

Electronic polarization effects have been suggested to play an important role in proton binding to titratable residues in proteins. In this work, we describe a new computational method for pKa calculations, using Monte Carlo (MC) simulations to sample protein protonation states with the Drude polarizable force field and Poisson-Boltzmann (PB) continuum electrostatic solvent model. While the most populated protonation states at the selected pH, corresponding to residues that are half-protonated at that pH, are sampled using the exact relative free energies computed with Drude particles optimized in the field of the PB implicit solvation model, we introduce an approximation for the protein polarization of low-populated protonation states to reduce the computational cost. The highly populated protonation states used to compute the polarization and pKa's are then iteratively improved until convergence. It is shown that for lysozyme, when considering 9 of the 18 titratable residues, the new method converged within two iterations with computed pKa's differing only by 0.02 pH units from pKa's estimated with the exact approach. Application of the method to predict pKa's of 94 titratable side chains in 8 proteins shows the Drude-PB model to produce physically more correct results as compared to the additive CHARMM36 (C36) force field (FF). With a dielectric constant of two assigned to the protein interior the Root Mean Square (RMS) deviation between computed and experimental pKa's is 2.07 and 3.19 pH units with the Drude and C36 models, respectively, and the RMS deviation using the Drude-PB model is relatively insensitive to the choice of the internal dielectric constant in contrast to the additive C36 model. At the higher internal dielectric constant of 20, pKa's computed with the additive C36 model converge to the results obtained with the Drude polarizable force field, indicating the need to artificially overestimate electrostatic screening in a nonphysical way with the additive FF. In addition, inclusion of both syn and anti orientations of the proton in the neutral state of acidic groups is shown to yield improved agreement with experiment. The present work, which is the first example of the use of a polarizable model for the prediction of pKa's in proteins, shows that the use of a polarizable model represents a more physically correct model for the treatment of electrostatic contributions to pKa shifts in proteins.

The complex role of the N-terminus and acidic residues of HdeA as pH-dependent switches in its chaperone function

HdeA is a small acid-stress chaperone protein found in the periplasm of several pathogenic gram-negative bacteria. In neutral pH environments HdeA is an inactive folded homodimer but when exposed to strong acidic environments it partially unfolds and, once activated, binds to other periplasmic proteins, protecting them from irreversible aggregation. Here we use a combination of hydrogen/deuterium exchange NMR experiments and constant pH molecular dynamics simulations to elucidate the role of HdeA’s N-terminus in its activation mechanism. Previous work indicates that the N-terminus is flexible and unprotected at high pH while exhibiting interactions with some HdeA client binding site residues. It, however, becomes partially solvent-protected at pH 2.6 – 2.8 and then loses protection again at pH 2.0. This protection is not due to the appearance of new secondary structure, but rather increased contacts between N-terminal residues and the C-terminus of the other protomer in the dimer, as well as concurrent loosening of its hold on the client binding site residues, priming HdeA for interactions with periplasmic client proteins. This work also uncovers unusual protonation profiles of some titratable residues and suggests their complex role in chaperone function.

Catalytic Site pKa Values of Aspartic, Cysteine, and Serine Proteases: Constant pH MD Simulations.

Enzymatic function and activity of proteases is closely controlled by the pH value. The protonation states of titratable residues in the active site react to changes in the pH value, according to their pKa, and thereby determine the functionality of the enzyme. Knowledge of the titration behavior of these residues is crucial for the development of drugs targeting the active site residues. However, experimental pKa data are scarce, since the systems’ size and complexity make determination of these pKa values inherently difficult. In this study, we use single pH constant pH MD simulations as a fast and robust tool to estimate the active site pKa values of a set of aspartic, cysteine, and serine proteases. We capture characteristic pKa shifts of the active site residues, which dictate the experimentally determined activity profiles of the respective protease family. We find clear differences of active site pKa values within the respective families, which closely match the experimentally determined pH preferences of the respective proteases. These shifts are caused by a distinct network of electrostatic interactions characteristic for each protease family. While we find convincing agreement with experimental data for serine and aspartic proteases, we observe clear deficiencies in the description of the titration behavior of cysteines within the constant pH MD framework and highlight opportunities for improvement. Consequently, with this work, we provide a concise set of active site pKa values of aspartic and serine proteases, which could serve as reference for future theoretical as well as experimental studies.

Disease‐Causing Mutations Shift the pK of the pH‐Sensitive H+‐Conductor Slc4a11

SLC4A11 is a H+‐conducting membrane protein that is sensitive to changes in both intracellular and extracellular pH. It is expressed in corneal endothelial cells (CECs) where it functions alongside other acid/base transporters such as MCT1 and NBCe1. These proteins function to maintain the osmotic balance and clarity of the cornea. Mutations in SLC4A11 cause congenital hereditary endothelial dystrophy (CHED) and Fuchs’ endothelial corneal dystrophy (FECD). Signs of these corneal dystrophies include loss of normal CEC morphology and corneal edema. Previously—using the Xenopus oocyte expression system, two‐electrode voltage clamp circuitry (to monitor conductance), and H+‐selective microelectrodes (to monitor intracellular pH)—we determined that the intracellular pK of wild‐type (WT) mouse Slc4a11 activity is 7.15 ± 0.04 (n=13, SD, pHe=8.5). In this study we use the same experimental setup to explore whether disease‐causing mutations can affect the intracellular‐pH dependence (pK) of Slc4a11.We chose FECD mutation E399K and CHED mutations T401K and R804H for comparison to WT due to the loss or gain of a titratable residue. We find that Slc4a11‐‘E399K’ exhibits a pK value of 7.02 ± 0.03 (n = 6, SD) and that Slc4a11‐‘T401K’ exhibits a pK value of 7.09 ± 0.03 (n = 6, SD); both significantly acid‐shifted compared to WT. On the other hand, ‘R804H’‐Slc4a11 exhibits a significantly alkali‐shifted pK value of 7.29 ± 0.04 (n = 6, SD) [Statistics are the result of two‐tailed unpaired t‐tests with Bonferroni correction].Thus we describe three disease‐causing mutations that cause a shift in the intracellular pK value of Slc4a11. If the pH‐dependence of the H+ conductance mediated by Slc4a11 is critical to the physiological role of Slc4a11, these shifts in pK could contribute towards the etiology of corneal dystrophy.Support or Funding InformationThis work was supported by NIH R01‐EY028580 to MDP.

Beyond pKa: Experiments and Simulations of Nitrile Vibrational Probes in Staphylococcal Nuclease Show the Importance of Local Interactions.

Electric fields are fundamentally important to biological phenomena, but are difficult to measure experimentally or predict computationally. Changes in pKa of titratable residues have long been used to report on local electrostatic fields in proteins. Alternatively, nitrile vibrational probes are potentially less disruptive and more direct reporters of local electrostatic field, but quantitative interpretation is clouded by the ability of the nitrile to accept a hydrogen bond. To this end, we incorporated nitrile probes into 10 locations of staphylococcal nuclease (SNase) where pKa shifts had already been determined. We characterized the local environment of each nitrile probe experimentally, through temperature-dependent spectroscopy, and computationally, through molecular dynamics simulations, and show that hydrogen bonding interactions dominate the spectral line shapes. We demonstrate that the information provided by the line shape of the nitrile spectra, compared to scalar values of pKa shift or nitrile frequency shift, better describes local environments in proteins in a manner that will be useful for future computational efforts to predict electrostatics in complex biological systems.

Probing the Accuracy of Explicit Solvent Constant pH Molecular Dynamics Simulations for Peptides.

Protonation states of titratable amino acids play a key role in many biomolecular processes. Knowledge of protonatable residue charges at a given pH is essential for a correct understanding of protein catalysis, inter- and intramolecular interactions, substrate binding, and protein dynamics for instance. However, acquiring experimental values for individual amino acid protonation states of complex systems is not straightforward; therefore, several in silico approaches have been developed to tackle this issue. In this work, we assess the accuracy of our previously developed constant pH MD approach by comparing our theoretically obtained pKa values for titratable residues with experimental values from an equivalent NMR study. We selected a set of four pentapeptides, of adequately small size to ensure comprehensive sampling, but concurrently, due to their charge composition, posing a challenge for protonation state calculation. The comparison of the pKa values shows good agreement of the experimental and the theoretical approach with a largest difference of 0.25 pKa units. Further, the corresponding titration curves are in fair agreement, although the shift of the Hill coefficient from a value of 1 was not always reproduced in simulations. The phase space overlap in Cartesian space between trajectories generated in constant pH and standard MD simulations is fair and suggests that our constant pH MD approach reasonably well preserves the dynamics of the system, allowing dynamic protonation MD simulations without introducing structural artifacts.

A New Constant pH Method to Simultaneously Predict pH-Induced Conformational Changes and Individual PKA Values in Large Biomolecules

We describe a constant pH MD method that integrates a new FEP replica-exchange implementation for titratable residues into the Hamiltonian pH replica-exchange routine. Using the high performance OpenMM engine, during each FEP/REMD stage, a switch of protonation probabilities is attempted between 2 adjacent replicas (for a randomly chosen set of residues with a conventional Metropolis criterion). This FEP scheme enables a gradual evolution of the force field representation of protonation states for each residue independently, leading to a more precise calculation of protonation probabilities. Any net charge discrepancies arising from switches of protonation coupling parameters in systems with explicit solvent are addressed with an adaptive technique to preserve the overall zero charge for all replicas individually after each exchange step. Each replica runs on a single GPU, enabling linear scaling of N replicas on N GPU; this parallelization strategy, combined with advanced sampling, results in a significantly faster time-to-solution than other constant pH methods. Thus, the new method yields predictions of conformational states corresponding to given pH values with considerably reduced computational expense. Notably, the implementation's constant pH and MD modules are independent, rendering the method adaptable to a variety of propagation techniques and advanced sampling methods. The results obtained for small-, medium- and large-scale pH-dependent protein systems will be discussed to illustrate the performance of the current version of the method.

GPU-Accelerated Implementation of Continuous Constant pH Molecular Dynamics in Amber: pKa Predictions with Single-pH Simulations.

We present a GPU implementation of the continuous constant pH molecular dynamics (CpHMD) based on the most recent generalized Born implicit-solvent model in the pmemd engine of the Amber molecular dynamics package. To test the accuracy of the tool for rapid pKa predictions, a series of 2 ns single-pH simulations were performed for over 120 titratable residues in 10 benchmark proteins that were previously used to test the various continuous CpHMD methods. The calculated pKa's showed a root-mean-square deviation of 0.80 and correlation coefficient of 0.83 with respect to experiment. Also, 90% of the pKa's were converged with estimated errors below 0.1 pH units. Surprisingly, this level of accuracy is similar to our previous replica-exchange simulations with 2 ns per replica and an exchange attempt frequency of 2 ps-1 (Huang, Harris, and Shen J. Chem. Inf. Model. 2018 , 58 , 1372 - 1383 ). Interestingly, for the linked titration sites in two enzymes, although residue-specific protonation state sampling in the single-pH simulations was not converged within 2 ns, the protonation fraction of the linked residues appeared to be largely converged, and the experimental macroscopic pKa values were reproduced to within 1 pH unit. Comparison with replica-exchange simulations with different exchange attempt frequencies showed that the splitting between the two macroscopic pKa's is underestimated with frequent exchange attempts such as 2 ps-1, while single-pH simulations overestimate the splitting. The same trend is seen for the single-pH vs replica-exchange simulations of a hydrogen-bonded aspartyl dyad in a much larger protein. A 2 ns single-pH simulation of a 400-residue protein takes about 1 h on a single NVIDIA GeForce RTX 2080 graphics card, which is over 1000 times faster than a CpHMD run on a single CPU core of a high-performance computing cluster node. Thus, we envision that GPU-accelerated continuous CpHMD may be used in routine pKa predictions for a variety of applications, from assisting MD simulations with protonation state assignment to offering pH-dependent corrections of binding free energies and identifying reactive hot spots for covalent drug design.

Effect of Ethephon on ripening and postharvest quality of mango

The experiment was conducted at the laboratory of Postharvest Technology Division, BARI to evaluate the effect of postharvest application of 6 concentrations (0, 250, 500, 750, 1000 & 10000 ppm) of ethephon on ripening and postharvest qualityof mango (cv. Langra) fruits harvested at mature green stage on 3rdweek of June in 2011 and 2012. The treated fruits were assessed for physiological changes such as ripening %, weight loss (%), biochemical aspects such as TSS (0Brix), titratable acidity (%), reducing sugar (%), total sugar (%), ascorbic acid content (mg/100g), total carotenoids (μg/100g), carbon di oxide production (ml/g fruit) and residual level of the applied ethephon during storage period. The observations were recorded at 2 days interval during 6 days storage at ambient condition (23±2°C with 80±5% RH). Complete yellow color (full ripe) was developed on the fruits treated with 500-1000 ppm ethephon at 4 days of storage while yellowish green and greenish yellow color was developed on 250 ppm treated and control fruits, respectively, and 10000 ppm ethephon treated fruits overripened at this period. At 6 days of storage, 250 ppm ethephon treated fruits got ripen and 500-1000 ppm ethephon treated fruits overripened whereas 10000 ppm treated fruits got rotten and control one was still unripe. Irrespective of ethephon treatments, weight loss of fruits, TSS, reducing sugar, total sugar, carbon di oxide production and total carotenoid showed increasing trends upto 6 days whereas titratable acidity, ascorbic acid and residue level of ethephon showed decreasing trends in both years. At 4 days of storage, 750-1000 ppm ethephon dipped fruits induced uniform attractive yellow color while untreated control fruits remained yellowish greenish (unripe) even after 6 days of storage. At 6 days of storage TSS, reducing sugar, toatal sugar, ascorbic acid and total carotenoid content were found maximum in 750-1000 ppm treated fruits compared to 250-500 ppm treated fruits. The residue level of ethephon in mango fruits treated with ethephon concentrations (250-1000 ppm) at 6 days of storage was found below 2 ppm (0.1 1ppm-0.54 ppm), which is safe for human consumption. Therefore, mangoes ripened by using ethephon @ 750-1000 ppm can be consumed safely without any health risk.
 Bangladesh J. Agril. Res. 44(3): 453-467, September 2019

How Electrostatic Coupling Enables Conformational Plasticity in a Tyrosine Kinase.

Protein kinases are important cellular signaling molecules involved in cancer and a multitude of other diseases. It is well-known that inactive kinases display a remarkable conformational plasticity; however, the molecular mechanisms remain poorly understood. Conformational heterogeneity presents an opportunity but also a challenge in kinase drug discovery. The ability to predictively model various conformational states could accelerate selective inhibitor design. Here we performed a proton-coupled molecular dynamics study to explore the conformational landscape of a c-Src kinase. Starting from a completely inactive structure, the simulations captured all major types of conformational states without the use of a target structure, mutation, or bias. The simulations allowed us to test the experimental hypotheses regarding the mechanism of DFG flip, its coupling to the αC-helix movement, and the formation of regulatory spine. Perhaps the most significant finding is how key titratable residues, such as DFG-Asp, αC-Glu, and HRD-Asp, change protonation states dependent on the DFG, αC, and activation loop conformations. Our data offer direct evidence to support a long-standing hypothesis that protonation of Asp favors the DFG-out state and explain why DFG flip is also possible in simulations with deprotonated Asp. The simulations also revealed intermediate states, among which a unique DFG-out/α-C state formed as DFG-Asp is moved into a back pocket forming a salt bridge with catalytic Lys, which can be tested in selective inhibitor design. Our finding of how proton coupling enables the remarkable conformational plasticity may shift the paradigm of computational studies of kinases which assume fixed protonation states. Understanding proton-coupled conformational dynamics may hold a key to further innovation in kinase drug discovery.

PH-Dependent Protonation of the Phl p 6 Pollen Allergen Studied by NMR and cpH-aMD.

We use state-of-the-art NMR experiments to measure apparent pKa values in the native protein environment and employ a cutting-edge combination of enhanced sampling and constant pH molecular dynamics (MD) simulations to rationalize strong pKa shifts. The major timothy grass pollen allergen Phl p 6 serves as an ideal model system for both methods due to its high number of titratable residues despite its comparably small size. We present a proton transition analysis as intuitive tool to depict the captured protonation state ensemble in atomistic detail. Combining microscopic structural details from MD simulations and macroscopic ensemble averages from NMR shifts leads to a comprehensive view on pH dependencies of protonation states and tautomers. Overall, we find striking agreement between simulation-based pKa predictions and experiment. However, our analyses suggest subtle differences in the underlying molecular origin of the observed pKa shifts. From accelerated constant pH MD simulations, we identify immediate proximity of opposite charges, followed by vicinity of equal charges as major driving forces for pKa shifts. NMR experiments on the other hand, suggest only a weak relation of pKa shifts and close contacts to charged residues, while the strongest influence derives from the dipolar character of α helices. The presented study hence pinpoints opportunities for improvements concerning the theoretical description of protonation state and tautomer probabilities. However, the coherence in the resulting apparent pKa values from simulations and experiment affirms cpH-aMD as a reliable tool to study allergen dynamics at varying pH levels.

Exploring the Mechanism of Action of Membrane Fusion Protein IncA Using Molecular Dynamics Simulations

Infecting millions of people each year, Chlamydia trachomatis is currently the leading cause of non‐congenital blindness and pelvic inflammatory disease. Incapable of replicating on its own, this bacterium survives by entering a host cell through endocytosis and subsequently modifying the vacuole in which it is enclosed to allow for replication. The newly formed replicative compartment, referred to as an inclusion, has numerous inclusion membrane proteins (Inc proteins) that promote growth and maintenance of the developing C. trachomatis bodies. One Inc in particular, known as IncA, exponentially enhances growth of C. trachomatis by initiating the homotypic fusion of multiple inclusions within a host cell. Although the structure and significance of IncA are known, its mechanism of action remains a mystery. The present research aims to elucidate the structure‐function relationship of IncA; offering insight into fusion protein functionality and potential targets for drug development. Analysis of IncA was accomplished by running Constant pH Molecular Dynamics (CpH MD) simulations with a refined focus on aspartic acids, glutamic acids, and histidines. Similar to conventional MD, CpH MD creates conditions that mimic the natural environment of IncA but with the additional capability to explore protonation states of residues. By running the CpH MD system across a range of pHs, the necessary data are generated to determine dissociation constants for specific residues. Combining proton dissociation with tests examining the solvent accessibility of IncA residues, results suggested that there is a correlation between accessibility of a residue and its proton dissociation. Further analysis of residue fluctuation at varying pHs shows that there appears to be a relationship between a residue's protonation state and the degree to which that residue fluctuated throughout a simulation. These findings demonstrate that the location of titratable residues influence the activity of IncA and furthermore, that IncA's titratable residues affect inclusion membrane fusion.Support or Funding InformationNational Science Foundation, Grant No. 1560325.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigation of a Juxtamembrane Lysine‐rich Region as a Determinant of Intracellular pH Sensitivity in the H+‐conducting Transmembrane Protein Slc4a11

SLC4A11 is a unique H+‐transporting member of the SLC4 family of bicarbonate transporters that is expressed in a variety of cells, including corneal endothelia. The H+ conductance of SLC4A11 increases in response to rises in both intracellular pH (pHi) and extracellular pH (pHe). By aligning the amino‐acid sequences of mammalian Slc4a11 and their bicarbonate‐transporting paralogs, we have identified a unique and highly‐conserved insertion ‘KxKxVGK’ in Slc4a11 at the juxtamembrane boundary between its cytosolic amino‐terminal‐domain and its first transmembrane‐span. The third lysine in this motif (‘K343’ in mouse Slc4a11) brings to mind the lysine at a comparable location in the renal K+‐channel ROMK that underlies the pHi sensitivity of ROMK currents (Fakler et al. EMBO J, 1996). Thus, we hypothesized that K343 might contribute to the pHi sensitivity of Slc4a11.To probe the role of K343, we generated a mutant ‘K343Q’, Q being the non‐titratable residue glutamine that is found at this location in other SLC4s. We expressed wild‐type (WT) or K343Q Slc4a11 from cRNA injected into Xenopus oocytes. We then compared the pH‐sensitivities of the expressed H+ conductances (Gm) at pHe=7.5 by using two‐electrode voltage‐clamp circuitry while raising oocyte pHi (monitored by a H+‐selective microelectrode) using a micro‐injector loaded with 1.2M NaHCO3 solution. As controls we used oocytes that had been injected with water rather than cRNA (‘H2O‐injected’) and oocytes that had been injected with cRNA encoding the mouse equivalent of the human corneal‐dystrophy‐causing SLC4A11 mutant ‘R125H’.ANOVA shows that the Gm of R125H‐expressing cells is indistinguishable from that of H2O‐injected cells over the entire tested pHI range (7.2–8.1), whereas the Gm of WT‐expressing cells is significantly greater than that of H2O‐injected cells over the entire range (n=4–12 data points). On the other hand, the Gm of K343Q‐expressing cells is indistinguishable from H2O‐injected cells over the pHi range 7.2–7.5, greater than H2O‐injected cells but not as great as WT‐expressing cells over the pHi range 7.5–7.9, and ultimately indistinguishable from WT‐expressing cells over the pHi range 7.9–8.1 (n = 4–35 data points). Thus the pHi‐responsiveness of K343Q is altered compared to WT.The H+ conductance of Slc4a11 is much enhanced at pHe=8.5 such that the pHi vs. Gm relationship becomes sigmoidal and can be fit to the Hill equation to determine an apparent pK and Hill constant. We assessed K343Q at pHe=8.5 and calculated a pK of 7.15 ± 0.02 and a Hill constant of 5 ± 1 (n = 4). Both of these values are significantly different from the WT values that we had previously determined (pK= 7.25 ± 0.03, Hill = 8 ± 1, n = 10). The lowered Hill and pK for K343Q are consistent with the loss of a titratable residue that contributes to the pHi sensitivity of Slc4a11. Further work is underway to identify other determinants of pHi and pHe sensitivity.Support or Funding InformationThis work was supported by NIH R01‐EY028580 to MDPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigating the Intracellular and Extracellular pH Sensitivities of the H+‐conducting Transmembrane Protein Slc4a11

SLC4A11 is the only member of the SLC4 family that transports H+ rather than bicarbonate (HCO3−) or carbonate (CO3=). SLC4A11 is expressed in a variety of cells including the corneal endothelial cells (CECs) in which it is presumed to mediate H+ influx. CECs help to maintain the optimal hydration status and optical clarity of the cornea by pumping fluid from the corneal stroma. This pump function requires the joint action of NBCe1 (Na+/CO3= influx), MCT1 (H+/lactate− influx), and SLC4A11. Notably, SLC4A11 mutations are sufficient to cause pump failure and the subsequent corneal swelling and opacification that are characteristic of corneal endothelial dystrophy. The role of SLC4A11 in corneal health and disease remains unclear, although we have hypothesized that SLC4A11 action could promote CEC health by correcting pH disturbances caused by imbalanced NBCe1 (base) and MCT1 (acid) import. Consistent with this hypothesis, our laboratory has previously demonstrated that the magnitude of the H+ conductance mediated by SLC4A11 is increased by rises in intracellular pH (pHi) as well as rises in extracellular pH (pHe).To better understand the features of SLC4A11 activation, we studied the pH‐sensitivity of mouse Slc4a11 expressed from cRNA in Xenopus oocytes. Using two electrode voltage clamp circuitry in conjunction with a H+‐selective microelectrode and a micro‐injector loaded with 1.2M NaHCO3 solution, we caused incremental increases in oocyte pHi and followed the accompanying increase in membrane conductance (Gm) while pHe was maintained at 7.5 or 8.5. We find, when pHe = 7.5, that Gm rises steadily over the pHi range 7.1–8.1, reaching a maximum observed conductance (Gm(max)) of 82 ± 5 μS (n = 6, SE) with an apparent intracellular pK ~7.6. On the other hand, when pHe= 8.5, Gm exhibits a steeper dependence on pHi and rises in a sigmoidal fashion over the pHi range 7.0–7.4 achieving a Gm(max) of 78 ± 8 μS (n = 14, SE) at pHi ≥ 7.4. [In comparable experiments performed on oocytes that were not expressing Slc4a11, Gm for was not greater than 4 μS (n=6) at pHi > 7.4.] The data gathered from Slc4a11‐expressing oocytes at pHe=8.5 can be fit to the Hill equation to reveal an average intracellular pK of 7.15 ± 0.02 (n = 10, SE) and an average Hill constant of 8 ± 1 (n = 10, SE).These data reveal a complex connection between pHe and pHi− sensitivity which highlights the challenge of working with such a dynamic substrate. Specifically, our data indicate that the kinetics of pHi sensing by Slc4a11 are modulated by pHe and are governed by numerous titratable residues. Knowledge of the kinetics governing the pH‐sensitivity of SLC4A11 H+ conductance will be valuable for assessing the physiological relevance of pH sensing by SLC4A11, for implementing SLC4A11 in mathematical models of CEC action, and for assessing the consequences of SLC4A11 mutations.Support or Funding InformationThis work was supported by NIH R01‐EY028580 to MDPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Use of NMR to probe changes in the flexibility of acid‐stress chaperone HdeB from its inactive to active state

HdeB is an intrinsically disordered periplasmic chaperone essential to the acid resistance of numerous pathogenic bacteria. It, along with its homolog, HdeA, binds to vulnerable periplasmic proteins at low pH and prevents them from irreversibly aggregating in the acidic stomach. The activity of these two proteins is responsible for the survivability of pathogenic bacteria and resulting proliferation of dysentery. At physiological pH, HdeB is an inactive folded homodimer and remains a structurally unchanged, fully folded dimer in its active state (at pH 4–5), suggesting that the mechanism of chaperone activation is a result of changes in its dynamics (internal motions). The long‐term goal of this study is to gain insight into HdeB's unique chaperoning mechanism by characterizing its pH dependent changes in structure and dynamics. In order to achieve atomic level specificity and detect subtle conformational changes, we are utilizing Nuclear Magnetic Resonance (NMR) spectroscopy to compare HdeB's flexibility and structure in its inactive and active states at pH 6.0 and 4.5, respectively. Isotopically labeled HdeB was recombinantly expressed and purified. Subsequently, protein backbone and side chain dynamics were examined at pH 6 and pH 4.5 utilizing R2 and R1 relaxation experiments, which identify fast (ps–ns) time scale motions within the structure; this indicates local motions, such as methyl rotations. Additionally, intermediate (μs‐ms) time scale motions, which reflect global conformational changes and flexibility, were identified using CPMG relaxation dispersion experiments. Notable changes in dynamics were observed with activation. At pH 6.0, most residues with μs‐ms motions are clustered at the dimer binding interface (BC loop and helix B). With the transition to pH 4.5, μs‐ms motions expand within the dimer binding interface (W27, T33, G37, T40, K53) and extend beyond it (L16δ, I55δ). Furthermore, at pH 4.5, Helix B decreases in solvent protection, suggesting decreased burial and a loosening of the dimer binding interface. Since this helix is extremely hydrophobic, loosening exposes residues that are well suited to interacting with the exposed hydrophobic regions of an unfolding client protein, likely enabling interactions crucial to substrate binding. Interestingly, the largest changes with activation were observed near multiple titratable residues, making us suspect a protonation change. Continuing research will focus on experimentally determining acid dissociation constants to confirm this.Support or Funding InformationWe gratefully acknowledge the support of the NIH for research funds (SC3‐GM116745) and BUILD PODER for research support to L.A., as well as the NSF for funding the purchase of our NMR spectrometer (CHE‐1040134).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.