Molecular Gas-Phase Conformational Ensembles.

Accurately determining the global minima of a molecular structure is important in diverse scientific fields, including drug design, materials science, and chemical synthesis. Conformational search engines serve as valuable tools for exploring the extensive conformational space of molecules and for identifying energetically favorable conformations. In this study, we present a comparison of Auto3D, CREST, Balloon, and ETKDG (from RDKit), which are freely available conformational search engines, to evaluate their effectiveness in locating global minima. These engines employ distinct methodologies, including machine learning (ML) potential-based, semiempirical, and force field-based approaches. To validate these methods, we propose the use of collisional cross-section (CCS) values obtained from ion mobility-mass spectrometry studies. We hypothesize that experimental gas-phase CCS values can provide experimental evidence that we likely have the global minimum for a given molecule. To facilitate this effort, we used our gas-phase conformation library (GPCL) which currently consists of the full ensembles of 20 small molecules and can be used by the community to validate any conformational search engine. Further members of the GPCL can be readily created for any molecule of interest using our standard workflow used to compute CCS values, expanding the ability of the GPCL in validation exercises. These innovative validation techniques enhance our understanding of the conformational landscape and provide valuable insights into the performance of conformational generation engines. Our findings shed light on the strengths and limitations of each search engine, enabling informed decisions for their utilization in various scientific fields, where accurate molecular structure determination is crucial for understanding biological activity and designing targeted interventions. By facilitating the identification of reliable conformations, this study significantly contributes to enhancing the efficiency and accuracy of molecular structure determination, with particular focus on metabolite structure elucidation. The findings of this research also provide valuable insights for developing effective workflows for predicting the structures of unknown compounds with high precision.

Getting zinc into and out of cells.

Ion channels are specialized proteins located on the plasma membrane and control the movement of ions across the membrane. Zn ion plays an indispensable role as a structural constituent of various proteins, moreover, it plays an important dynamic role in cell signaling. In this chapter, we discuss computational insights into zinc efflux and influx mechanism through YiiP (from Escherichia coli and Shewanella oneidensis) and BbZIP (Bordetella bronchiseptica) transporters, respectively. Gaining knowledge about the mechanism of zinc transport at the molecular level can aid in developing treatments for conditions such as diabetes and cancer by manipulating extracellular and intracellular levels of zinc ions.

Primer for Designing Main Protease (Mpro) Inhibitors of SARS-CoV-2.

The COVID-19 outbreak has been devastating, with hundreds of millions of infections and millions of deaths reported worldwide. In response, the application of structure-activity relationships (SAR) upon experimentally validated inhibitors of SARS-CoV-2 main protease (Mpro) may provide an avenue for the identification of new lead compounds active against COVID-19. Upon the basis of information gleaned from a combination of reported crystal structures and the docking of experimentally validated inhibitors, four "rules" for designing potent Mpro inhibitors have been proposed. The aim here is to guide medicinal chemists toward the most probable hits and to provide guidance on repurposing available structures as Mpro inhibitors. Experimental examination of our own previously reported inhibitors using the four "rules" identified a potential lead compound, the cathepsin inhibitor GB111-NH2, that was 2.3 times more potent than SARS-CoV-2 Mpro inhibitor N3.

Mechanism of Zinc Transport through the Zinc Transporter YiiP.

Zinc is an essential transition metal ion that plays as a structural, functional (catalytic), and a signaling molecule regulating cellular function. Unbalanced levels of zinc in cells can result in various pathological conditions. In the current work, all-atom molecular dynamics simulations were used to study the structure-function correlation between different YiiP states embedded in a lipid bilayer. This study enabled us to develop a hypothesis on the zinc efflux mechanism of YiiP. We have created six different models of YiiP representing the stages of the ion-transport process. We found that zinc ion plays a crucial role in restraining the transmembrane domains (TMDs) of the protein. In addition, H153, located in the TMD, has been proposed to guide the zinc ion toward the ZnA site of the YiiP transporter. Understanding the molecular-level Zn2+-transport process sheds light on the strategies affecting intracellular transition-metal ion concentrations in order to treat diseases like diabetes and cancer.

The BioFragment Database (BFDb): An open-data platform for computational chemistry analysis of noncovalent interactions.

Accurate potential energy models are necessary for reliable atomistic simulations of chemical phenomena. In the realm of biomolecular modeling, large systems like proteins comprise very many noncovalent interactions (NCIs) that can contribute to the protein's stability and structure. This work presents two high-quality chemical databases of common fragment interactions in biomolecular systems as extracted from high-resolution Protein DataBank crystal structures: 3380 sidechain-sidechain interactions and 100 backbone-backbone interactions that inaugurate the BioFragment Database (BFDb). Absolute interaction energies are generated with a computationally tractable explicitly correlated coupled cluster with perturbative triples [CCSD(T)-F12] "silver standard" (0.05 kcal/mol average error) for NCI that demands only a fraction of the cost of the conventional "gold standard," CCSD(T) at the complete basis set limit. By sampling extensively from biological environments, BFDb spans the natural diversity of protein NCI motifs and orientations. In addition to supplying a thorough assessment for lower scaling force-field (2), semi-empirical (3), density functional (244), and wavefunction (45) methods (comprising >1M interaction energies), BFDb provides interactive tools for running and manipulating the resulting large datasets and offers a valuable resource for potential energy model development and validation.

Reduction of urease activity by interaction with the flap covering the active site.

With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes, and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors.

Molecular Dynamics Study of Helicobacter pylori Urease.

Helicobacter pylori have been implicated in an array of gastrointestinal disorders including, but not limited to, gastric and duodenal ulcers and adenocarcinoma. This bacterium utilizes an enzyme, urease, to produce copious amounts of ammonia through urea hydrolysis in order to survive the harsh acidic conditions of the stomach. Molecular dynamics (MD) studies on the H. pylori urease enzyme have been employed in order to study structural features of this enzyme that may shed light on the hydrolysis mechanism. A total of 400 ns of MD simulation time were collected and analyzed in this study. A wide-open flap state previously observed in MD simulations on Klebsiella aerogenes [Roberts et al. J. Am. Chem. Soc.2012, 134, 9934] urease has been identified in the H. pylori enzyme that has yet to be experimentally observed. Critical distances between residues on the flap, contact points in the closed state, and the separation between the active site Ni2+ ions and the critical histidine α322 residue were used to characterize flap motion. An additional flap in the active site was elaborated upon that we postulate may serve as an exit conduit for hydrolysis products. Finally we discuss the internal hollow cavity and present analysis of the distribution of sodium ions over the course of the simulation.

Insights into the mechanistic dichotomy of the protein farnesyltransferase peptide substrates CVIM and CVLS.

Protein farnesyltransferase (FTase) catalyzes farnesylation of a variety of peptide substrates. (3)H α-secondary kinetic isotope effect (α-SKIE) measurements of two peptide substrates, CVIM and CVLS, are significantly different and have been proposed to reflect a rate-limiting S(N)2-like transition state with dissociative characteristics for CVIM, while, due to the absence of an isotope effect, CVLS was proposed to have a rate-limiting peptide conformational change. Potential of mean force quantum mechanical/molecular mechanical studies coupled with umbrella sampling techniques were performed to further probe this mechanistic dichotomy. We observe the experimentally proposed transition state (TS) for CVIM but find that CVLS has a symmetric S(N)2 TS, which is also consistent with the absence of a (3)H α-SKIE. These calculations demonstrate facile substrate-dependent alterations in the transition state structure catalyzed by FTase.

Finding a needle in the haystack: computational modeling of Mg2+ binding in the active site of protein farnesyltransferase.

Studies aimed at elucidating the unknown Mg2+ binding site in protein farnesyltransferase (FTase) are reported. FTase catalyzes the transfer of a farnesyl group to a conserved cysteine residue (Cys1p) on a target protein, an important step for proteins in the signal transduction pathways (e.g., Ras). Mg2+ ions accelerate the protein farnesylation reaction by up to 700-fold. The exact function of Mg2+ in catalysis and the structural characteristics of its binding remain unresolved to date. Molecular dynamics (MD) simulations addressing the role of magnesium ions in FTase are presented, and relevant octahedral binding motifs for Mg2+ in wild-type (WT) FTase and the Dß352A mutant are explored. Our simulations suggest that the addition of Mg2+ ions causes a conformational change to occur in the FTase active site, breaking interactions known to keep FPP in its inactive conformation. Two relevant Mg2+ ion binding motifs were determined in WT FTase. In the first binding motif, WT1, the Mg2+ ion is coordinated to D352ß, zinc-bound D297ß, two water molecules, and one oxygen atom from the α- and ß-phosphates of farnesyl diphosphate (FPP). The second binding motif, WT2, is identical with the exception of the zinc-bound D297ß being replaced by a water molecule in the Mg2+ coordination complex. In the Dß352A mutant Mg2+ binding motif, D297ß, three water molecules, and one oxygen atom from the α- and ß-phosphates of FPP complete the octahedral coordination sphere of Mg2+. Simulations of WT FTase, in which Mg2+ was replaced by water in the active site, recreated the salt bridges and hydrogen-bonding patterns around FPP, validating these simulations. In all Mg2+ binding motifs, a key hydrogen bond was identified between a magnesium-bound water and Cys1p, bridging the two metallic binding sites and, thereby, reducing the equilibrium distance between the reacting atoms of FPP Cys1p. The free energy profiles calculated for these systems provide a qualitative understanding of experimental results. They demonstrate that the two reactive atoms approach each other more readily in the presence of Mg2+ in WT FTase and mutant. The flexible WT2 model was found to possess the lowest barrier toward the conformational change, suggesting it is the preferred Mg2+ binding motif in WT FTase. In the mutant, the absence of D352ß makes the transition toward a conformational change harder. Our calculations find support for the proposal that D352ß performs a critical role in Mg2+ binding and Mg2+ plays an important role in the conformational transition step.

Importance of loop dynamics in the neocarzinostatin chromophore binding and release mechanisms.

Neocarzinostatin (NCS) is an antitumor chromophore carrier protein with many therapeutic applications. To characterize its binding and release mechanism, we have carried out molecular dynamics (MD) simulations for apo-NCS and holo-NCS. Although the beta-sheet regions of the protein exhibit restricted motion, large atomic fluctuations were observed in the loop regions, especially loop 99-104. This loop undergoes a "door-opening" motion that can facilitate chromophore binding and release. Calculated NMR order parameters confirm the simulated loop flexibility. We also provide a proposed explanation for the release rate difference for two mutants F78L and F78A through our simulation. The binding site structures of holo-NCS were also validated by chemical shift perturbations. Based on these results, a new binding and release mechanism for the NCS chromophore is proposed.

Apo and nickel-bound forms of the Pyrococcus horikoshii species of the metalloregulatory protein: NikR characterized by molecular dynamics simulations.

NikR is a homotetrameric nickel regulatory protein whose binding to free Ni(2+) increases its binding affinity for a gene that codes for a nickel transporter protein. It is comprised of a tetrameric nickel-binding domain, flanked by two dimeric DNA-binding domains. Though X-ray crystallography data for various species (Escherichia coli, Heliobacter pylori, and Pyrococcus horikoshii) of NikR reveal large conformational differences between nickel-bound, DNA-bound, and unbound forms, transitions between them have never been observed. We have run all-atom molecular dynamics simulations of three forms of the Pyrococcus horikoshii species of NikR including two apo-forms and one nickel-bound form. Though all 552 residues of this species occur naturally, quantum-mechanics-based force-field parametrization was required to accurately represent the four nickel-centers in the nickel-bound form. Global conformational analysis of the three 100-ns-long simulations indicates slow conformational kinetics and independent DNA binding domain motion. Correlation and flexibility analysis revealed regions of high structural and dynamical importance. A striking relationship was observed between regions with high levels of structural importance and regions with known biological importance. Mutation of key regions of P. horikoshii and analogous regions in both E. coli and H. pylori are suggested that might inhibit DNA-binding activity while not affecting nickel-binding.

Further analysis and comparative study of intermolecular interactions using dimers from the S22 database.

Accurate MP2 and CCSD(T) complete basis set (CBS) interaction energy curves (14 points for each curve) have been obtained for 20 of the dimers reported in the S22 set and analytical Morse curves have been fitted that can be used in developing updated density functional theory (DFT) and force field models. The magnitude and the effect of the basis set superposition error (BSSE) were carefully investigated. We found that going up to aug-cc-pVDZ and aug-cc-pVTZ basis sets is enough to obtain accurate CBS MP2 energies when BSSE corrected values are used but aug-cc-pVTZ and aug-cc-pVQZ basis sets are needed when the BSSE uncorrected total energies are used in CBS extrapolations. MP2 interaction energies with smaller basis sets such as 6-31G* are found to have very little dispersion energy and that the true source of dispersion attributed attractive interactions is almost entirely due to BSSE. MP2 and CCSD(T) CBS interaction energies are found to be very close to one another if aromatic systems are not involved. Comparative analyses have been performed with semiempirical and ab initio methods utilizing the moderate in size but affordable 6-31G* basis set both of which can be readily applied to macromolecular systems. The new M06-2X and M06-L DFT functionals were found to be more accurate than all methods tested herein. Interaction energy curves using the SG1 grid showed discontinuities for several of the dimer systems but this problem disappeared when finer DFT numerical grids were used.

Accurate benchmark calculations on the gas-phase basicities of small molecules.

Accurate benchmark calculations of gas-phase basicities of small molecules are presented and compared with available experimental results. The optimized geometries and thermochemical analyses were obtained from MP2/aug-cc-pVTZ calculations. Two different ab initio electron-correlated methods MP2 and CCSD(T) were employed for subsequent gas-phase basicity calculations, and the single-point energies were extrapolated to the complete basis set (CBS) limit. The overall accuracy for different ab initio methods is compared, and the accuracy in descending order is CCSD(T)_CBS > CCSD(T)/aug-cc-pVDZ > (MP2/aug-cc-pVQZ approximately MP2_CBS) > HF/aug-cc-pVQZ. The best root-mean-squared-error obtained was 1.0 kcal mol(-1) at the CCSD(T)_CBS//MP2/aug-cc-pVTZ level for a test set of 41 molecules. Clearly, accurate calculations for the electron correlation energy are important for the theoretical prediction of molecular gas-phase basicities. However, conformational effects were also found to be relevant in several instances when more complicated molecules were examined.

Protein NMR chemical shift calculations based on the automated fragmentation QM/MM approach.

An automated fragmentation quantum mechanics/molecular mechanics (AF-QM/MM) approach has been developed to routinely calculate ab initio protein NMR chemical shielding constants. The AF-QM/MM method is linear-scaling and trivially parallel. A general fragmentation scheme is employed to generate each residue-centric region which is treated by quantum mechanics, and the environmental electrostatic field is described with molecular mechanics. The AF-QM/MM method shows good agreement with standard self-consistent field (SCF) calculations of the NMR chemical shieldings for the mini-protein Trp cage. The root-mean-square errors (RMSEs) for 1H, 13C, and 15N NMR chemical shieldings are equal to or less than 0.09, 0.32, and 0.78 ppm, respectively, for all Hartree-Fock (HF) and density functional theory (DFT) calculations reported in this work. The environmental electrostatic potential is necessary to accurately reproduce the NMR chemical shieldings using the AF-QM/MM approach. The point-charge models provided by AMBER, AM1/CM2, PM3/CM1, and PM3/CM2 all effectively model the electrostatic field. The latter three point-charge models are generated via semiempirical linear-scaling SCF calculations of the entire protein system. The correlations between experimental 1H NMR chemical shifts and theoretical predictions are >0.95 for AF-QM/MM calculations using B3LYP with the 6-31G**, 6-311G**, and 6-311++G** basis sets. Our study, not unexpectedly, finds that conformational changes within a protein structure play an important role in the accurate prediction of experimental NMR chemical shifts from theory.

The intrinsic dynamics and function of nickel-binding regulatory protein: insights from elastic network analysis.

Nickel-responsive protein NikR regulates the nickel uptake in nickel-dependent bacteria by interacting with the operator of nikABCDE and subsequently repressing the transcription of NikABCDE, an ABC-type nickel transporter system. The function of NikR and its affinity for the operator DNA is highly conformation-dependent, which has been confirmed by three independent crystallographic studies on NikR proteins from different bacteria. Depending on the intracellular nickel concentration, NikR is able to adopt either the open form or one of the two closed forms (cis and trans) that differ in the domain-domain arrangement. Only the closed cis form is optimal for DNA binding. We examined the low-resolution vibrational spectrum of NikR in each conformational form using the elastic network model and observed large-scale domain-domain vibrations that are closely related to the conformational transitions required for function, particularly the symmetric bending mode and the asymmetric twisting mode. This analysis on the intrinsic dynamics coded in the three-dimensional molecular construct allows us to examine the proposed mechanisms of NikR regulation from the standpoint of protein collective motions. Our findings further support the three-state equilibrium hypothesis proposed by others, and imply that an isolated closed cis form may be dynamically unstable but can be stabilized by DNA binding. However, we also found that the simple C(alpha)-model used in the current analysis is insufficient to capture the impact of nickel binding on the protein dynamics, for which an all-atom model with detailed atom typing is more appropriate.

Insights into the strength and origin of halogen bonding: the halobenzene-formaldehyde dimer.

The observation of short halogen-carbonyl oxygen interactions in protein-ligand complexes has spurred us to use computational tools to better understand the strength of halogen bonding interactions. In this study we have produced potential energy curves for the halogen bonding interactions of several halobenzene-formaldehyde complexes. It was found that, for most halogen substituents, a halobenzene and formaldehyde form stable halogen bonded complexes with interaction energies that increase as the size of the halogen substituent increases.

Critical assessment of quantum mechanics based energy restraints in protein crystal structure refinement.

A critical evaluation of the performance of X-ray refinement protocols using various energy functions is presented using the bovine pancreatic trypsin inhibitor (BPTI) protein. The four potential energy functions we explored include: (1) fully quantum mechanical calculations; (2) one based on an incomplete molecular mechanics (MM) energy function employed in the Crystallography and NMR System (CNS) with empirical parameters developed by Engh and Huber (EH), which lacks electrostatic and attractive van der Waals terms; (3) one based on a complete MM energy function (AMBER ff99 parameter set); and (4) the same as 3, with the addition of a Generalized Born (GB) implicit solvation term. The R, R (free), real space R values of the refined structures and deviations from the original experimental structure were used to assess the relative performance. It was found that at 1 Angstrom resolution the physically based energy functions 1, 3, and 4 performed better than energy function 2, which we attribute to the better representation of key interactions, particularly electrostatics. The observed departures from the experimental structure were similar for the refinements with physically based energy functions and were smaller than the structure refined with EH. A test refinement was also performed with the reflections truncated at a high-resolution cutoff of 2.5 Angstrom and with random perturbations introduced into the initial coordinates, which showed that low-resolution refinements with physically based energy functions held the structure closer to the experimental structure solved at 1 Angstrom resolution than the EH-based refinements.

Exploring the role of the active site cysteine in human muscle creatine kinase.

All known guanidino kinases contain a conserved cysteine residue that interacts with the non-nucleophilic eta1-nitrogen of the guanidino substrate. Site-directed mutagenesis studies have shown that this cysteine is important, but not essential for activity. In human muscle creatine kinase (HMCK) this residue, Cys283, forms part of a conserved cysteine-proline-serine (CPS) motif and has a pKa about 3 pH units below that of a regular cysteine residue. Here we employ a computational approach to predict the contribution of residues in this motif to the unusually low cysteine pKa. We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute approximately 1 pH unit, while the presence of Pro284 in the motif lowers the pKa of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pKa of the active site cysteine in WT HMCK and in the P284A, S285A, and C283S/S285C mutants was determined experimentally. The pKa values, although consistently about 0.5 pH unit lower, were in broad agreement with those predicted. The effect of each of these mutations on the pH-rate profile was also examined. The results show conclusively that, contrary to a previous report (Wang et al. (2001) Biochemistry 40, 11698-11705), Cys283 is not responsible for the pKa of 5.4 observed in the WT V/K(creatine) pH profile. Finally we use molecular dynamics simulations to demonstrate that, in order to maintain the linear alignment necessary for associative inline transfer of a phosphoryl group, Cys283 needs to be ionized.

Computational studies of the farnesyltransferase ternary complex part I: substrate binding.

Farnesyltransferase (FTase) catalyzes the transfer of farnesyl from farnesyl diphosphate (FPP) to cysteine residues at or near the C-terminus of protein acceptors with a CaaX motif (a, aliphatic; X, Met). Farnesylation is a critical modification to many switch proteins involved in the extracellular signal transduction pathway, which facilitates their fixation on the cell membrane where the extracellular signal is processed. The malfunction caused by mutations in these proteins often causes uncontrolled cell reproduction and leads to tumor formation. FTase inhibitors have been extensively studied as potential anticancer agents in recent years, several of which have advanced to different phases of clinical trials. However, the mechanism of this biologically important enzyme has not been firmly established. Understanding how FTase recruits the FPP substrate is the first and foremost step toward further mechanistic investigations and the design of effective FTase inhibitors. Molecular dynamic simulations were carried out on the ternary structure of FTase complexed with the FPP substrate and an acetyl-capped tetrapeptide (acetyl-CVIM), which revealed that the FPP substrate maintains an inactive conformation and the binding of the diphosphate group can be largely attributed to residues R291beta, K164alpha, K294beta, and H248beta. The FPP substrate assumes an extended conformation in the binding site with restricted rotation of the backbone dihedral angles; however, it does not have a well-defined conformation when unbound in solution. This is evident from multinanosecond MD simulations of the FPP substrate in a vacuum and solution. Our conclusion is further supported by theoretical J coupling calculations. Our results on the FPP binding are in good agreement with previous experimental kinetic studies on FTase mutants. The hypothetical conformational activation of the FPP substrate is currently under investigation.

Effects of fluorine substitution on the edge-to-face interaction of the benzene dimer.

To gain some insight into the effects of fluorination on the aromatic-aromatic interactions found in protein-ligand complexes, like those observed in the set of N-(4-sulfamylbenzoyl)benzylamine (SBB) inhibitors bound to Human Carbonic Anhydrase II (HCAII), we have produced potential energy curves for the edge-to-face interactions of a set of fluorinated benzene dimer compounds. All calculations were carried out at the MP2/aug-cc-pVDZ level of theory using the counterpoise method of Boys and Bernardi (Boys, S. F.; Bernardi, F. Mol. Phys. 1970, 19, 553) to account for the basis set superposition error. Fluorine substitutions are made onto the face molecule of the edge-to-face benzene dimer. As one might expect, the substitution of additional fluorines into this system generally resulted in a decrease of the binding energy. It was also found that the positioning of the fluorine substituents on isosubstituted compounds has a large effect on the total binding energy of these types of systems. More specifically, complexes with fluorines that are substituted closer to the hydrogen atoms of the edge benzene will tend to be stabilized by an electrostatic interaction between the partially negative fluorine atoms and the partially positive hydrogen atoms. However, our findings do not explain the recent crystallographic findings for the SBB-HCAII protein-ligand complex, where increased fluorination resulted in closer edge-to-face contacts, which suggests that there are factors, other than edge-to-face aromatic interactions, influencing this system's behavior.