Structure and inhibition mechanisms of Mycobacterium tuberculosis essential transporter efflux protein A.

A broad chemical genetics screen in Mycobacterium tuberculosis (Mtb) to identify inhibitors of established or previously untapped targets for therapeutic development yielded compounds (BRD-8000.3 and BRD-9327) that inhibit the essential efflux pump EfpA. To understand the mechanisms of inhibition by these compounds, we determined the structures of EfpA with inhibitors bound at 2.7 - 3.4 Å resolution. Our structures reveal different mechanisms of inhibition for the two inhibitors. BRD-8000.3 binds in a tunnel making contact with the lipid bilayer and extending toward the central cavity to displace the fatty acid chain of a lipid molecule bound in the apo structure, suggesting its blocking of an access route for a natural lipidic substrate, in contrast to its uncompetitive mechanism for the small molecule substrate ethidium bromide which likely enters through an alternative tunnel. Meanwhile, BRD-9327 binds in the outer vestibule without complete blockade of the substrate path to the outside, suggesting its possible inhibition of the dynamical motion necessary for "alternate access" to the two different sides of the membrane, as is characteristic of major facilitator superfamily (MFS) transporters. Both inhibitors may have a role in inhibiting the "alternate access" mechanism that could account for the uncompetitive nature of their efflux of some substrates. Our results explain the basis of the synergy of these inhibitors and their potential for combination in a multi drug strategy for anti-tuberculosis therapy. They also potentially point to a possible function for this essential efflux pump as a lipid transporter. The structures provide a foundation for rational modification of these inhibitors to increase potency.

Transition Path Sampling Study of Engineered Enzymes That Catalyze the Morita-Baylis-Hillman Reaction: Why Is Enzyme Design so Difficult?

It is hoped that artificial enzymes designed in laboratories can be efficient alternatives to chemical catalysts that have been used to synthesize organic molecules. However, the design of artificial enzymes is challenging and requires a detailed molecular-level analysis to understand the mechanism they promote in order to design efficient variants. In this study, we computationally investigate the mechanism of proficient Morita-Baylis-Hillman enzymes developed using a combination of computational design and directed evolution. The powerful transition path sampling method coupled with in-depth post-processing analysis has been successfully used to elucidate the different chemical pathways, transition states, protein dynamics, and free energy barriers of reactions catalyzed by such laboratory-optimized enzymes. This research provides an explanation for how different chemical modifications in an enzyme affect its catalytic activity in ways that are not predictable by static design algorithms.

Connecting Conformational Motions to Rapid Dynamics in Human Purine Nucleoside Phosphorylase.

The influence of protein motions on enzyme catalysis remains a topic of active discussion. Protein motions occur across a variety of time scales, from vibrational fluctuations in femtoseconds, to collective motions in milliseconds. There have been numerous studies that show conformational motions may assist in catalysis, protein folding, and substrate specificity. It is also known through transition path sampling studies that rapid promoting vibrations contribute to enzyme catalysis. Human purine nucleoside phosphorylase (PNP) is one enzyme that contains both an important conformational motion and a rapid promoting vibration. The slower motion in this enzyme is associated with a loop motion, that when open allows substrate entry and product release but closes over the active site during catalysis. We examine the differences between an unconstrained PNP structure and a PNP structure with constraints on the loop motion. To investigate possible coupling between the slow and fast protein dynamics, we employed transition path sampling, reaction coordinate identification, electric field calculations, and free energy calculations reported here.

Static polarizabilities within the generalized Kohn-Sham semicanonical projected random phase approximation (GKS-spRPA).

An analytical implementation of static dipole polarizabilities within the generalized Kohn-Sham semicanonical projected random phase approximation (GKS-spRPA) method for spin-restricted closed-shell and spin-unrestricted open-shell references is presented. General second-order analytical derivatives of the GKS-spRPA energy functional are derived using a Lagrangian approach. By resolution-of-the-identity and complex frequency integration methods, an asymptotic O(N4⁡log(N)) scaling of operation count and O(N3) scaling of storage is realized, i.e., the computational requirements are comparable to those for GKS-spRPA ground state energies. GKS-spRPA polarizabilities are assessed for small molecules, conjugated long-chain hydrocarbons, metallocenes, and metal clusters, by comparison against Hartree-Fock (HF), semilocal density functional approximations (DFAs), second-order Møller-Plesset perturbation theory, range-separated hybrids, and experimental data. For conjugated polydiacetylene and polybutatriene oligomers, GKS-spRPA effectively addresses the "overpolarization" problem of semilocal DFAs and the somewhat erratic behavior of post-PBE RPA polarizabilities without empirical adjustments. The ensemble averaged GKS-spRPA polarizabilities of sodium clusters (Nan for n = 2, 3, …, 10) exhibit a mean absolute deviation comparable to PBE with significantly fewer outliers than HF. In conclusion, analytical second-order derivatives of GKS-spRPA energies provide a computationally viable and consistent approach to molecular polarizabilities, including systems prohibitive for other methods due to their size and/or electronic structure.

Transition Path Sampling Based Calculations of Free Energies for Enzymatic Reactions: The Case of Human Methionine Adenosyl Transferase and Plasmodium vivax Adenosine Deaminase.

Transition path sampling (TPS) is widely used for the calculations of reaction rates, transition state structures, and reaction coordinates of condensed phase systems. Here we discuss a scheme for the calculation of free energies using the ensemble of TPS reactive trajectories in combination with a window-based sampling technique for enzyme-catalyzed reactions. We calculate the free energy profiles of the reactions catalyzed by the human methionine S-adenosyltransferase (MAT2A) enzyme and the Plasmodium vivax adenosine deaminase (pvADA) enzyme to assess the accuracy of this method. MAT2A catalyzes the formation of S-adenosine-l-methionine following a SN2 mechanism, and using our method, we estimate the free energy barrier for this reaction to be 16 kcal mol-1, which is in excellent agreement with the experimentally measured activation energy of 17.27 kcal mol-1. Furthermore, for the pvADA enzyme-catalyzed reaction we estimate a free energy barrier of 21 kcal mol-1, and the calculated free energy profile is similar to that predicted from experimental observations. Calculating free energies by employing our simple method within TPS provides significant advantages over methods such as umbrella sampling because it is free from any applied external bias, is accurate compared to experimental measurements, and has a reasonable computational cost.

Exploring the Solvation of Acetic Acid in Water Using Liquid Jet X-ray Photoelectron Spectroscopy and Core Level Electron Binding Energy Calculations.

Liquid jet X-ray photoelectron spectroscopy was used to investigate changes in the local electronic structure of acetic acid in the bulk of aqueous solutions induced by solvation effects. These effects manifest themselves as shifts in the difference in the carbon 1s binding energy (ΔBE) between the methyl and carboxyl carbons of acetic acid. Furthermore, molecular dynamics simulations, coupled with correlated electronic structure calculations of the first solvation sphere, provide insight into the number of water molecules directly interacting with the carboxyl group that are required to match the ΔBE from the photoelectron spectroscopy experiments. This comparison shows that a single water molecule in the first solvation shell describes the photoelectron ΔBE of acetic acid while at least 20 water molecules are required for the conjugate base, acetate, in aqueous solutions.

TURBOMOLE: Modular program suite for ab initio quantum-chemical and condensed-matter simulations.

TURBOMOLE is a collaborative, multi-national software development project aiming to provide highly efficient and stable computational tools for quantum chemical simulations of molecules, clusters, periodic systems, and solutions. The TURBOMOLE software suite is optimized for widely available, inexpensive, and resource-efficient hardware such as multi-core workstations and small computer clusters. TURBOMOLE specializes in electronic structure methods with outstanding accuracy-cost ratio, such as density functional theory including local hybrids and the random phase approximation (RPA), GW-Bethe-Salpeter methods, second-order Møller-Plesset theory, and explicitly correlated coupled-cluster methods. TURBOMOLE is based on Gaussian basis sets and has been pivotal for the development of many fast and low-scaling algorithms in the past three decades, such as integral-direct methods, fast multipole methods, the resolution-of-the-identity approximation, imaginary frequency integration, Laplace transform, and pair natural orbital methods. This review focuses on recent additions to TURBOMOLE's functionality, including excited-state methods, RPA and Green's function methods, relativistic approaches, high-order molecular properties, solvation effects, and periodic systems. A variety of illustrative applications along with accuracy and timing data are discussed. Moreover, available interfaces to users as well as other software are summarized. TURBOMOLE's current licensing, distribution, and support model are discussed, and an overview of TURBOMOLE's development workflow is provided. Challenges such as communication and outreach, software infrastructure, and funding are highlighted.

Formation of the End-on Bound Lanthanide Dinitrogen Complexes [(R2N)3Ln-N═N-Ln(NR2)3]2- from Divalent [(R2N)3Ln]1- Salts (R = SiMe3).

Lanthanide-based dinitrogen reduction chemistry has been expanded by the discovery of the first end-on Ln2(µ-η1:η1-N2) complexes, whose synthesis and reactivity help explain the reduction of N2 by the combination of trivalent Ln(NR2)3 complexes (R = SiMe3) and potassium. The formation of end-on versus the more common side-on Ln2(µ-η2:η2-N2) complexes is possible by using recently discovered Ln(II) complexes ligated by three NR2 amide ligands (R = SiMe3). The isolated Ln(II) tris(amide) complex [K(crypt)][Tb(NR2)3] (crypt = 2.2.2-cryptand), 1-Tb, reacts with dinitrogen in Et2O at -35 °C to form the end-on bridging dinitrogen complex [K(crypt)]2{[(R2N)3Tb]2[µ-η1:η1-N2]}, 2-Tb. The 18-crown-6 (18-c-6) Tb(II) analogue, [K(18-c-6)2][Tb(NR2)3], 3-Tb, also reacts with N2 to form an end-on product, [K2(18-c-6)3]{[(R2N)3Tb]2[µ-η1:η1-N2]}, 4-Tb. The reaction of 1-Gd with dinitrogen forms a complex with the same composition as 2-Tb but with both side-on and end-on bonding of the N2 unit in the same crystal, [K(crypt)]2{[(R2N)3Gd]2[µ-ηx:ηx-N2]} (x = 1 and 2), 5-Gd. Similarly, the 18-c-6 Gd(II) complex, 3-Gd, generates a product with both binding modes: [K2(18-c-6)3]{[(R2N)3Gd]2[µ-ηx:ηx-N2]} (x = 1, 2), 6-Gd. All of these new reduced dinitrogen complexes, 2-Tb, 4-Tb, 5-Gd, and 6-Gd, have three ancillary amide ligands per metal. In contrast, the side-on bound complexes, [(THF)(R2N)2Ln]2[µ-η2:η2-N2], 7-Ln, observed previously in Ln(NR2)3/K/N2 reactions, have only two amides per metal. A connection between these systems related to their formation was observed in the structure of the bimetallic penta-amide complex, [K(THF)6]{[(THF)(R2N)2Gd][µ-η2:η2-N2][Gd(NR2)3]}, 8-Gd, synthesized at -196 °C. Reaction conditions are crucial in this dinitrogen reaction system. When 5-Gd and 6-Gd are warmed above -15 °C, they reform Gd(II) complexes. If 1-Gd is dissolved in THF instead of Et2O under N2, the irreversible formation of an (N2)3- complex [K(crypt)][(THF)(R2N)2Gd]2[µ-η2:η2-N2], 9-Gd, is observed.

Synthesis, Structure, and Magnetism of Tris(amide) [Ln{N(SiMe3 )2 }3 ]1- Complexes of the Non-traditional +2 Lanthanide Ions.

A new series of Ln2+ complexes has been synthesized that overturns two previous generalizations in rare-earth metal reduction chemistry: that amide ligands do not form isolable complexes of the highly reducing non-traditional Ln2+ ions, and that yttrium is a good model for the late lanthanides in these reductive reactions. Reduction of Ln(NR2 )3 (R=SiMe3 ) complexes in THF under Ar with M=K or Rb in the presence of 2.2.2-cryptand (crypt) forms crystallographically characterizable [M(crypt)][Ln(NR2 )3 ] complexes not only for the traditional Tm2+ ion and the configurational crossover ions, Nd2+ and Dy2+ , but also for the non-traditional Gd2+ , Tb2+ , Ho2+ , and Er2+ ions. Crystallographic data as well as UV/Vis, magnetic susceptibility, and density functional theory studies are consistent with the accessibility of 4fn 5d1 configurations for Ln2+ ions in this tris(silylamide) ligand environment. The Dy2+ complex, [K(crypt)][Dy(NR2 )3 ], has a higher magnetic moment than previously observed for any monometallic complex: 11.67 µB .

Random-Phase Approximation Methods.

Random-phase approximation (RPA) methods are rapidly emerging as cost-effective validation tools for semilocal density functional computations. We present the theoretical background of RPA in an intuitive rather than formal fashion, focusing on the physical picture of screening and simple diagrammatic analysis. A new decomposition of the RPA correlation energy into plasmonic modes leads to an appealing visualization of electron correlation in terms of charge density fluctuations. Recent developments in the areas of beyond-RPA methods, RPA correlation potentials, and efficient algorithms for RPA energy and property calculations are reviewed. The ability of RPA to approximately capture static correlation in molecules is quantified by an analysis of RPA natural occupation numbers. We illustrate the use of RPA methods in applications to small-gap systems such as open-shell d- and f-element compounds, radicals, and weakly bound complexes, where semilocal density functional results exhibit strong functional dependence.

Noble gas encapsulation into carbon nanotubes: predictions from analytical model and DFT studies.

The energetics for the interaction of the noble gas atoms with the carbon nanotubes (CNTs) are investigated using an analytical model and density functional theory calculations. Encapsulation of the noble gas atoms, He, Ne, Ar, Kr, and Xe into CNTs of various chiralities is studied in detail using an analytical model, developed earlier by Hill and co-workers. The constrained motion of the noble gas atoms along the axes of the CNTs as well as the off-axis motion are discussed. Analyses of the forces, interaction energies, acceptance and suction energies for the encapsulation enable us to predict the optimal CNTs that can encapsulate each of the noble gas atoms. We find that CNTs of radii 2.98 - 4.20 Å (chiral indices, (5,4), (6,4), (9,1), (6,6), and (9,3)) can efficiently encapsulate the He, Ne, Ar, Kr, and Xe atoms, respectively. Endohedral adsorption of all the noble gas atoms is preferred over exohedral adsorption on various CNTs. The results obtained using the analytical model are subsequently compared with the calculations performed with the dispersion-including density functional theory at the M06 - 2X level using a triple-zeta basis set and good qualitative agreement is found. The analytical model is however found to be computationally cheap as the equations can be numerically programmed and the results obtained in comparatively very less time.