Titratable Residues Research Articles

"Seeing Is Believing": How Neutron Crystallography Informs Enzyme Mechanisms by Visualizing Unique Water Species.

Hydrogen is the lightest atom and composes approximately half of the atomic content in macromolecules, yet their location can only be inferred or predicted in most macromolecular structures. This is because hydrogen can rarely be directly observed by the most common structure determination techniques (such as X-ray crystallography and electron cryomicroscopy). However, knowledge of hydrogen atom positions, especially for enzymes, can reveal protonation states of titratable active site residues, hydrogen bonding patterns, and the orientation of water molecules. Though we know they are present, this vital layer of information, which can inform a myriad of biological processes, is frustratingly invisible to us. The good news is that, even at modest resolution, neutron crystallography (NC) can reveal this layer and has emerged this century as a powerful tool to elucidate enzyme catalytic mechanisms. Due to its strong and coherent scattering of neutrons, incorporation of deuterium into the protein crystal amplifies the power of NC. This is especially true when solvation and the specific participation of key water molecules are crucial for catalysis. Neutron data allow the modeling of all three atoms in water molecules and have even revealed previously unobserved and unique species such as hydronium (D3O+) and deuteroxide (OD-) ions as well as lone deuterons (D+). Herein, we briefly review why neutrons are ideal probes for identifying catalytically important water molecules and these unique water-like species, limitations in interpretation, and four vignettes of enzyme success stories from disparate research groups. One of these groups was that of Dr. Chris G. Dealwis, who died unexpectedly in 2022. As a memorial appreciation of his scientific career, we will also highlight his interest and contributions to the neutron crystallography field. As both the authors were mentored by Chris, we feel we have a unique perspective on his love of molecular structure and admiration for neutrons as a tool to query those structures.

Assessment of amino acid charge states based on cryo-electron microscopy and molecular dynamics simulations of respiratory complex I

The charge states of titratable amino acid residues play a key role in the function of membrane-bound bioenergetic proteins. However, determination of these charge states both through experimental and computational approaches is extremely challenging. Cryo-EM density maps can provide insights on the charge states of titratable amino acid residues. By performing classical atomistic molecular dynamics simulations on the high resolution cryo-EM structures of respiratory complex I from Yarrowia lipolytica, we analyze the conformational and charge states of a key acidic residue in its ND1 subunit, aspartic acid D203, which is also a mitochondrial disease mutation locus. We suggest that in the native state of respiratory complex I, D203 is negatively charged and maintains a stable hydrogen bond to a conserved arginine residue. Alternatively, upon conformational change in the turnover state of the enzyme, its sidechain attains a charge-neutral status. We discuss the implications of this analysis on the molecular mechanism of respiratory complex I.

Absence of a link between stabilized charge-separated state and structural changes proposed from crystal structures of a photosynthetic reaction center

Structural differences between illuminated and unilluminated crystal structures led to the proposal that the charge-separated state was stabilized by structural changes in its membrane extrinsic protein subunit H in a bacterial photosynthetic reaction center [Katona, G. et al. Nat. Struct. Mol. Biol. 2005, 12, 630–631]. Here, we explored the proposal by titrating all titratable sites and calculating the redox potential (Em) values in these crystal structures. Contrary to the expected charge-separated states, Em for quinone, Em(QA/QA•–), is even lower in the proposed charge-separated structure than in the ground-state structure. The subunit-H residues, which were proposed to exhibit electron-density changes in the two crystal structures, contribute to an Em(QA/QA•–) difference of only <0.5 mV. Furthermore, the protonation states of the titratable residues in the entire reaction center are practically identical in the two structures. These findings indicate that the proposed structural differences are irrelevant to explaining the significant prolongation of the charge-separated-state lifetime.

Proton transfer in cytochrome bd-I from E. coli involves Asp-105 in CydB

Cytochrome bds are bacterial terminal oxidases expressed under low oxygen conditions, and they are important for the survival of many pathogens and hence potential drug targets. The largest subunit CydA contains the three redox-active cofactors heme b558, heme b595 and the active site heme d. One suggested proton transfer pathway is found at the interface between the CydA and the other major subunit CydB. Here we have studied the O2 reduction mechanism in E. coli cyt. bd-I using the flow-flash technique and focused on the mechanism, kinetics and pathway for proton transfer.Our results show that the peroxy (P) to ferryl (F) transition, coupled to the oxidation of the low-spin heme b558 is pH dependent, with a maximum rate constant (~104 s−1) that is slowed down at higher pH. We assign this behavior to rate-limitation by internal proton transfer from a titratable residue with pKa ~ 9.7. Proton uptake from solution occurs with the same P➔F rate constant. Site-directed mutagenesis shows significant effects on catalytic turnover in the CydB variants Asp58B➔Asn and Asp105B➔Asn variants consistent with them playing a role in proton transfer. Furthermore, in the Asp105B➔Asn variant, the reactions up to P formation occur essentially as in the wildtype bd-I, but the P➔F transition is specifically inhibited, supporting a direct and specific role for Asp105B in the functional proton transfer pathway in bd-I. We further discuss the possible identity of the high pKa proton donor, and the conservation pattern of the Asp-105B in the cyt. bd superfamily.

Rational Design of Far-Red Archaerhodopsin-3-Based Fluorescent Genetically Encoded Voltage Indicators: from Elucidation of the Fluorescence Mechanism in Archers to Novel Red-Shifted Variants.

Genetically encoded voltage indicators (GEVIs) have found wide applications as molecular tools for visualization of changes in cell membrane potential. Among others, several classes of archaerhodopsin-3-based GEVIs have been developed and have proved themselves promising in various molecular imaging studies. To expand the application range for this type of GEVIs, new variants with absorption band maxima shifted toward the first biological window and enhanced fluorescence signal are required. Here, we integrate computational and experimental strategies to reveal structural factors that distinguish far-red bright archaerhodopsin-3-based GEVIs, Archers, obtained by directed evolution in a previous study (McIsaac et al., PNAS, 2014) and the wild-type archaerhodopsin-3 with an extremely dim fluorescence signal, aiming to use the obtained information in subsequent rational design. We found that the fluorescence can be enhanced by stabilization of a certain conformation of the protein, which, in turn, can be achieved by tuning the pK a value of two titratable residues. These findings were supported further by introducing mutations into wild-type archeorhodopsin-3 and detecting the enhancement of the fluorescence signal. Finally, we came up with a rational design and proposed previously unknown Archers variants with red-shifted absorption bands (λmax up to 640 nm) and potential-dependent bright fluorescence (quantum yield up to 0.97%).

Role of Protonation States in the Stability of Molecular Dynamics Simulations of High-Resolution Membrane Protein Structures.

Classical molecular dynamics (MD) simulations provide unmatched spatial and time resolution of protein structure and function. However, the accuracy of MD simulations often depends on the quality of force field parameters and the time scale of sampling. Another limitation of conventional MD simulations is that the protonation states of titratable amino acid residues remain fixed during simulations, even though protonation state changes coupled to conformational dynamics are central to protein function. Due to the uncertainty in selecting protonation states, classical MD simulations are sometimes performed with all amino acids modeled in their standard charged states at pH 7. Here, we performed and analyzed classical MD simulations on high-resolution cryo-EM structures of two large membrane proteins that transfer protons by catalyzing protonation/deprotonation reactions. In simulations performed with titratable amino acids modeled in their standard protonation (charged) states, the structure diverges far from its starting conformation. In comparison, MD simulations performed with predetermined protonation states of amino acid residues reproduce the structural conformation, protein hydration, and protein-water and protein-protein interactions of the structure much better. The results support the notion that it is crucial to perform basic protonation state calculations, especially on structures where protonation changes play an important functional role, prior to the launch of any conventional MD simulations. Furthermore, the combined approach of fast protonation state prediction and MD simulations can provide valuable information about the charge states of amino acids in the cryo-EM sample. Even though accurate prediction of protonation states in proteinaceous environments currently remains a challenge, we introduce an approach of combining pKa prediction with cryo-EM density map analysis that helps in improving not only the protonation state predictions but also the atomic modeling of density data.

Phage display assisted discovery of a pH-dependent anti-α-cobratoxin antibody from a natural variable domain library.

RecyclingIgG antibodies bind to their target antigen at physiological pH in the blood stream and release them upon endocytosis when pH levels drop, allowing the IgG antibodies to be recycled into circulation via FcRn-mediated cellular pathways, while the antigens undergo lysosomal degradation. This enables recycling antibodies to achieve comparable therapeutic effect at lower doses than their non-recycling counterparts. The development of such antibodies is typically achieved by histidine doping of their variable regions or by performing in vitro antibody selection campaigns utilizing histidine doped libraries. Both are strategies that may introduce sequence liabilities. Here, we present a methodology that employs a naïve antibody phage display library, consisting of natural variable domains, to discover antibodies that bind α-cobratoxin from the venom of Naja kaouthia in a pH-dependent manner. As a result, an antibody was discovered that exhibits a 7-fold higher off-rate at pH 5.5 than pH 7.4 in bio-layer interferometry experiments. Interestingly, no histidine residues were found in its variable domains, and in addition, the antibody showed pH-dependent binding to a histidine-devoid antigen mutant. As such, the results demonstrate that pH-dependent antigen-antibody binding may not always be driven by histidine residues. By employing molecular dynamics simulations, different protonation states of titratable residues were found, which potentially could be responsible for the observed pH-dependent antigen binding properties of the antibody. Finally, given the typically high diversity of naïve antibody libraries, the methodology presented here can likely be applied to discover recycling antibodies against different targets ab initio without the need for histidine doping.

A New Theory about Interfacial Proton Diffusion Revisited: The Commonly Accepted Laws of Electrostatics and Diffusion Prevail.

The high propensity of protons to stay at interfaces has attracted much attention over the decades. It enables long-range interfacial proton diffusion without relying on titratable residues or electrostatic attraction. As a result, various phenomena manifest themselves, ranging from spillover in material sciences to local proton circuits between proton pumps and ATP synthases in bioenergetics. In an attempt to replace all existing theoretical and experimental insight into the origin of protons' preference for interfaces, TELP, the "Transmembrane Electrostatically-Localized Protons" hypothesis, has been proposed. The TELP hypothesis envisions static H+ and OH- layers on opposite sides of interfaces that are up to 75 µm thick. Yet, the separation at which the electrostatic interaction between two elementary charges is comparable in magnitude to the thermal energy is more than two orders of magnitude smaller and, as a result, the H+ and OH- layers cannot mutually stabilize each other, rendering proton accumulation at the interface energetically unfavorable. We show that (i) the law of electroneutrality, (ii) Fick's law of diffusion, and (iii) Coulomb's law prevail. Using them does not hinder but helps to interpret previously published experimental results, and also helps us understand the high entropy release barrier enabling long-range proton diffusion along the membrane surface.

Absence of electron-transfer-associated changes in the time-dependent X-ray free-electron laser structures of the photosynthetic reaction center.

Using the X-ray free-electron laser (XFEL) structures of the photosynthetic reaction center from Blastochloris viridis that show light-induced time-dependent structural changes (Dods et al., (2021) Nature 589, 310-314), we investigated time-dependent changes in the energetics of the electron-transfer pathway, considering the entire protein environment of the protein structures and titrating the redox-active sites in the presence of all fully equilibrated titratable residues. In the dark and charge separation intermediate structures, the calculated redox potential (Em) values for the accessory bacteriochlorophyll and bacteriopheophytin in the electron-transfer-active branch (BL and HL) are higher than those in the electron-transfer-inactive branch (BM and HM). However, the stabilization of the charge-separated [PLPM]•+HL•- state owing to protein reorganization is not clearly observed in the Em(HL) values in the charge-separated 5 ps ([PLPM]•+HL•- state) structure. Furthermore, the expected chlorin ring deformation upon formation of HL•- (saddling mode) is absent in the HL geometry of the original 5 ps structure. These findings suggest that there is no clear link between the time-dependent structural changes and the electron-transfer events in the XFEL structures.

Absence of electron-transfer-associated changes in the time-dependent X-ray free-electron laser structures of the photosynthetic reaction center

Using the X-ray free-electron laser (XFEL) structures of the photosynthetic reaction center from Blastochloris viridis that show light-induced time-dependent structural changes (Dods et al., (2021) Nature 589, 310–314), we investigated time-dependent changes in the energetics of the electron-transfer pathway, considering the entire protein environment of the protein structures and titrating the redox-active sites in the presence of all fully equilibrated titratable residues. In the dark and charge separation intermediate structures, the calculated redox potential (Em) values for the accessory bacteriochlorophyll and bacteriopheophytin in the electron-transfer-active branch (BL and HL) are higher than those in the electron-transfer-inactive branch (BM and HM). However, the stabilization of the charge-separated [PLPM]•+HL•– state owing to protein reorganization is not clearly observed in the Em(HL) values in the charge-separated 5 ps ([PLPM]•+HL•– state) structure. Furthermore, the expected chlorin ring deformation upon formation of HL•– (saddling mode) is absent in the HL geometry of the original 5 ps structure. These findings suggest that there is no clear link between the time-dependent structural changes and the electron-transfer events in the XFEL structures.

Enhanced Uptake of Anticancer C6-Pep Dimer in a Model Membrane Caused by Differential pKa in Acidic Microenvironment.

Acidic tumor microenvironment (TME) presents a challenge for the action of antitumor drugs by acting as an additional barrier for the passive crossing of the cell membrane by chemotherapic agents playing a critical role in the proliferation of tumor cells. Anticancer lipopeptide C6-Pep dimer containing the leucine zipper motif shows an increased uptake into the model tumor membrane in TME, and application of external heat might lead to the uncoiling of the zipper, which could result in cell lysis. This work investigated the cause of this increased uptake of C6-Pep dimer into the bilayer model in TME. Accurate protonation states of all the titratable residues of the C6-Pep dimer in TME were determined using constant pH molecular dynamics. In TME, except for two terminal Glu5 residues, all other Glu residues in the C6-Pep dimer were permanently protonated. The remaining Glu5 residues had differential pKa values, leading to the construction of four possible dimers with different fixed protonation states, and molecular dynamics was used to study their interaction with the anionic bilayer. Except for the dimer at a physiological pH, the other dimers were positively charged and could readily adsorb on the membrane surface. The free energy of insertion of these dimers in the bilayer was lower for single and double protonated Glu5-containing dimers than for the others. After the insertion of the lipopeptides into the membrane, thinning of the bilayer in the vicinity of dimers and an increase in area per lipid of the bilayer were observed for all systems, indicating destabilization of the bilayer due to this intercalation. This study shows that the anticancer lipopeptide C6-Pep utilizes the TME around a tumor cell for insertion into the membrane.

GmXtal: Cooking Crystals with GROMACS

Molecular dynamics (MD) simulations are routinely performed of biomolecules in solution, because this is their native environment. However, the structures used in such simulations are often obtained with X-ray crystallography, which provides the atomic coordinates of the biomolecule in a crystal environment. With the advent of free electron lasers and time-resolved techniques, X-ray crystallography can now also access metastable states that are intermediates in a biochemical process. Such experiments provide additional data, which can be used, for example, to optimize MD force fields. Doing so requires that the simulation of the biomolecule is also performed in the crystal environment. However, in contrast to simulations of biomolecules in solution, setting up a crystal is challenging. In particular, because not all solvent molecules are resolved in X-ray crystallography, adding a suitable number of solvent molecules, such that the properties of the crystallographic unit cell are preserved in the simulation, can be difficult and typically is a trial-and-error based procedure requiring manual interventions. Such interventions preclude high throughput applications. To overcome this bottleneck, we introduce gmXtal, a tool for setting up crystal simulations for MD simulations with GROMACS. With the information from the protein data bank (rcsb.org) gmXtal automatically (i) builds the crystallographic unit cell; (ii) sets the protonation of titratable residues; (iii) builds missing residues that were not resolved experimentally; and (iv) adds an appropriate number of solvent molecules to the system. gmXtal is available as a standalone tool https://gitlab.com/pbuslaev/gmxtal.Graphical

AMOEBA Force Field Predicts Accurate Hydrogen Bond Counts of Nitriles in SNase by Revealing Water-Protein Interaction in Vibrational Absorption Frequencies.

Precisely quantifying the magnitude and direction of electric fields in proteins has long been an outstanding challenge in understanding biological functions. Nitrile vibrational Stark effect probes have been shown to be minimally disruptive to the protein structure and can be better direct reporters of local electrostatic field in the native state of a protein than other measures such as pKa shifts of titratable residues. However, interpretations of the connection between measured vibrational energy and electric field rely on the accurate molecular understanding of interactions of the nitrile group and its environment, particularly from hydrogen bonding. In this work, we compared the extent of hydrogen bonding calculated in two common force fields, the fixed charge force field Amber03 and polarizable force field AMOEBA, at 10 locations of cyanocysteine (CNC) in staphylococcal nuclease (SNase) against the experimental nitrile absorption frequency in terms of full width at half-maximum (FWHM) and frequency temperature line slope (FTLS). We observed that the number of hydrogen bonds correlated well in AMOEBA trajectories with respect to both the FWHM (r = 0.88) and the FTLS (r = -0.85), whereas the correlation of Amber03 trajectories was less reliable because the Amber03 force field predicted more hydrogen bonds in some mutants. Moreover, we demonstrated that contributions from the interactions between CNC and nearby water molecules were significant in AMOEBA trajectories but were not predicted by Amber03. We conclude that although the nitrile absorption peak shape could be qualitatively predicted by the fixed charge Amber03 force field, the detailed electrostatic environment measured by the nitrile probe in terms of the extent of hydrogen bonding could only be accurately observed in the AMOEBA trajectories, where the permanent dipole, quadrupole, and dipole-induced-dipole polarizable interactions were all taken into account. The significance of this finding to the goal of accurately predicting electric fields in complex biomolecular environments is discussed.

Benchmarking In Silico Tools for Cysteine pKa Prediction.

Accurate estimation of the pKa's of cysteine residues in proteins could inform targeted approaches in hit discovery. The pKa of a targetable cysteine residue in a disease-related protein is an important physiochemical parameter in covalent drug discovery, as it influences the fraction of nucleophilic thiolate amenable to chemical protein modification. Traditional structure-based in silico tools are limited in their predictive accuracy of cysteine pKa's relative to other titratable residues. Additionally, there are limited comprehensive benchmark assessments for cysteine pKa predictive tools. This raises the need for extensive assessment and evaluation of methods for cysteine pKa prediction. Here, we report the performance of several computational pKa methods, including single-structure and ensemble-based approaches, on a diverse test set of experimental cysteine pKa's retrieved from the PKAD database. The dataset consisted of 16 wildtype and 10 mutant proteins with experimentally measured cysteine pKa values. Our results highlight that these methods are varied in their overall predictive accuracies. Among the test set of wildtype proteins evaluated, the best method (MOE) yielded a mean absolute error of 2.3 pK units, highlighting the need for improvement of existing pKa methods for accurate cysteine pKa estimation. Given the limited accuracy of these methods, further development is needed before these approaches can be routinely employed to drive design decisions in early drug discovery efforts.

Residues involved in proton-coupled transport of antimalarial drugs by PfCRT.

A major setback in the fight against malaria has been the emergence of P. falciparum parasites resistant to chloroquine (CQ), the primary antimalarial drug. Neutral CQ enters the parasite's digestive vacuole (DV) and obtains two protons preventing its passive efflux through the membrane. The trapped CQ2+ kills the parasite by disrupting the detoxification process of heme released from proteolyzed host hemoglobin. CQ-resistant parasites show mutations in a DV membrane transporter, P. falciparum CQ resistance transporter (PfCRT), that enables active, proton-coupled efflux of CQ2+. While biochemical measurements established the coupling of PfCRT's transport function to the DV membrane proton gradient, the proton-coupled transport mechanism has remained elusive. Particularly it is still unclear what the proton binding site(s) is(are). Recently, the cryo-EM structure of PfCRT from a CQ-resistant variant was determined with a bound Fab fragment occupying its binding pocket. Using us-long all-atom molecular dynamics (MD) simulations we examined the stability of the transporter in the DV-open conformation in the absence of bound Fab fragments, discovering surprising protein stability upon removal of Fab. Next, utilizing constant pH (cpHMD) simulations at 7 different pH values, and considering Galvani potential offset corrections, we determined the pKa of the four titratable residues (H97, D137, D326, and D329) lining the binding pocket. These simulations allowed unambiguous determination of the primary site responding to DV's acidic environment. Finally, with protonation states compatible with the acidic pH of the DV for the aforementioned residues, we studied binding of CQ2+ to the transporter via a series of driven MD simulations. We then repeated the cpHMD simulations and determined the proton binding site in the presence of the bound CQ2+. Our results shed light on PfCRT's transport mechanism and open new possibilities to design new effective drugs against malaria.

Constant pH molecular dynamics simulations of membrane proteins.

Environment-driven dynamic protonation of titratable residues in a protein plays a key role in membrane transport and pH response in various biological processes. The dominant protonation state of a titratable amino-acid residue depends on environmental conditions like pH, dielectric environment (ionic strength, proximity to water-membrane interfaces), other amino-acid residues in its vicinity, etc. Constant pH Molecular Dynamics (CpHMD) techniques enable the study of dynamic protonation events in a molecular simulation and can be regarded as an in-silico titration process. In CpHMD techniques, the protonation state of a titratable residue is allowed to propagate between the different ionization states. As the protonation states of different residues change, the charge of the total system fluctuates. In hybrid solvent-based schemes, where the changes in the protonation states are handled using an implicit solvent representation, these charge fluctuations do not pose a problem. However, in case of an explicit-solvent representation, where the electrostatics are handled using a particle mesh ewald (PME) scheme, the varying charge poses a problem in reliable evaluation of energetics and thereby the pKa of the titratable residues. A large aqueous fraction in the simulation box is known to mitigate the charge artifacts. However, compared to soluble proteins, membrane protein simulation systems have a large non-aqueous volume fraction, owing to the lipid membrane. In this study, we compare different CpHMD methodologies for estimation of pKa in membrane proteins. These methods include both an explicit solvent-based CpHMD as well as hybrid solvent-based CpHMD. We present the pKa values of the acidic residues (aspartic acid, glutamic acid) from these simulations. In general, the hybrid solvent-based scheme showed a faster convergence compared to the explicit solvent-based scheme. However, in case of buried residues, the hybrid solvent-based scheme took a longer time to converge.

Membrane composition may regulate proton wave propagation.

Lateral proton diffusion along membrane surfaces, specifically between a proton source and a sink, is a fundamental process in many biological systems. It may play a significant part in the uptake of cations, sugars, amino acids, and ATP synthesis. Fast interfacial diffusion cannot rely on titratable membrane residues, as a comparison with the relatively slow proton release reaction shows [1]. Yet, an entropy-dominated free energy barrier prevents surface protons from equilibrating with bulk protons [2]. The dependence of barrier height on membrane compositions thus far remained enigmatic. To establish the barrier-modulating role of glycolipids, we released protons to a small spot on the surface of planar lipid bilayers by Ca2+ microinjection. The membranes contained a calcium ionophore (A23187) which facilitated the transmembrane transport of Ca2+ ions in exchange for protons [3]. A lipid-anchored pH-sensor allowed detection of the arrival of the locally triggered proton wave on a distant membrane patch as a change in fluorescence intensity. Our results indicated that interfacial sugar moieties might be potent regulators of proton migration between membrane-embedded proton sources and sinks. This project was supported by the Marie Skłodowska-Curie grant 860592 (Horizon 2020). [1] Springer, et al. Proc. Natl. Acad. Sci. U.S.A, 2011, 108, 14461-14466. [2] Weichselbaum, et al. Scientific Reports, 2017, 7, 4553. [3] Peter Pohl, Yuri N. Antonenko, Lev S. Yaguzhinsky, Biochimica et Biophysica Acta, 1990, 1027, 295-300.

Charactering ionizable residue networks at SH2 interfaces in pH-sensitive proteins.

Intracellular pH (pHi) dynamics (7.0 to 7.8) regulate myriad cell processes but the molecular mechanisms driving these behaviors are largely unknown. While some pH-sensitive proteins use titratable histidine residues to sense physiological changes in pHi, others have unknown or histidine-independent mechanisms. Charged amino acids (Asp, Glu, and Lys) can have pKas that are up- or down-shifted into the physiological range depending on protein environment, but the role for these residues in regulating pH-dependent protein function is understudied. Here, we use computational and biochemical approaches to investigate pH sensors that use networks of cooperative ionizable residues with a cumulative physiological pKa. We performed computational analyses of two pH sensitive signaling proteins with unknown mechanisms: (phosphatidylinositol-3-kinase (PI3K) and SHP-2 tyrosine-protein phosphatase). In both proteins, we identified networks of ionizable residues with upshifted pKas at the inhibitory SH2 domain interface. We next used biochemical mutational analyses to probe the accuracy of our computational approaches by measuring pH sensitive activity of wild-type and mutant proteins in vitro. Importantly, because SH2 domains are structurally conserved, this pH-sensing mechanism could be conserved in a subset of kinases and phosphatases with inhibitory SH2 domains. We tested this hypothesis by identifying a network of ionizable residues in other signaling proteins that contain inhibitory SH2 domains. We analyzed JAK2 kinase, which has not been shown to have pH sensitive function, and identified an ionizable network at the SH2 domain interface. Future work will validate this JACK2 result biochemically and explore whether our computational approaches can be applied to identify pH sensitive proteins across the human proteome. Improved understanding of how ionizable networks respond to pH could reveal paths to better therapeutic targeting of these proteins in diseases with dysregulated pHi.

Computational fragment screening of dimeric SARS-CoV-2 main protease.

During the coronavirus pandemic the lack of antiviral therapeutics drove intense interest in rapid drug discovery by the scientific, and medical communities. The Voelz lab took part in the COVID Moonshot initiative, which focused on virtually screening small drug-like fragments as leads for inhibitors of the main protease (Mpro) of SARS-CoV-2, identified using high throughput crystallographic techniques. The Voelz lab performed absolute binding free energy calculations on the [email protected] distributed computing platform to massively parallelize the molecular dynamic (MD) simulations. Crystallographic fragment-based screening techniques can be time consuming, costly, and dependent on the availability of protein crystals. Could fragment based drug discovery be performed completely in silico? To address this question, we aim to perform simulations to virtually screen the original fragment library used in the COVID Moonshot, and assess the accuracy of the predictions. The GROMACS simulation package was used to construct a simulation topology of the apo form of dimeric Mpro from its crystal structure. The correct protonation states of titratable residues were taken from published experimental results. As a first step, we prepared and performed a set of simulations of Mpro in solution, each with one of 11 drug fragments selected from the crystallographic fragment screen, all of which are known to co-crystallize with Mpro experimentally. The simulations are analyzed to determine where the drug fragments like to bind to the Mpro receptor. These results are compared results to the experimental co-crystal structures to determine how accurately MD simulations can predict the known binding pose of small drug like fragments.

The Structural Basis for Metal Ion Transport in the SLC11/NRAMP Family

The SLC11/NRAMP proteins constitute a conserved family of metal ion transporters that are expressed in all kingdoms of life. In humans, the two paralogs DMT1 and NRMP1 play an important role in iron homeostasis and the defense against pathogens. SLC11 transporters have evolved an exquisite selectivity for transition metal ions, which facilitates their efficient transport from a large background of Ca2+ and Mg2+. This is accomplished by the evolution of a conserved binding site, which contains besides promiscuous hard ligands, a methionine acting as soft ligand that exclusively coordinates transition metals and thus contributes to the exclusion of alkaline earth metal ions. This site is altered in a branch of prokaryotic family members, which are capable of transporting Mg2+, where the removal of the coordinating methionine and the accompanying expansion of the binding pocket captures this small ion in a hydrated state. The disposition of titratable residues in H+-coupled transition metal ion transporters, that are absent in uncoupled Mg2+ transporters, sheds light on potential coupling mechanisms. In combination, the discussed work has revealed detailed insight into transition metal ion transport and provides a basis for the development of inhibitors of DMT1 as strategy against iron overload disorders.