The cationic dummy atom approach provides a powerful nonbonded description for a range of alkaline-earth and transition-metal centers, capturing both structural and electrostatic effects. In this work we refine existing literature parameters for octahedrally coordinated Mn2+, Zn2+, Mg2+, and Ca2+, as well as providing new parameters for Ni2+, Co2+, and Fe2+. In all the cases, we are able to reproduce both M2+–O distances and experimental solvation free energies, which has not been achieved to date for transition metals using any other model. The parameters have also been tested using two different water models and show consistent performance. Therefore, our parameters are easily transferable to any force field that describes nonbonded interactions using Coulomb and Lennard-Jones potentials. Finally, we demonstrate the stability of our parameters in both the human and Escherichia coli variants of the enzyme glyoxalase I as showcase systems, as both enzymes are active with a range of transition metals. The parameters presented in this work provide a valuable resource for the molecular simulation community, as they extend the range of metal ions that can be studied using classical approaches, while also providing a starting point for subsequent parametrization of new metal centers.

Odorants activate sensory transduction in olfactory receptor neurons (ORNs) via a cAMP-signaling cascade, which results in the opening of nonselective, cyclic nucleotide–gated (CNG) channels. The consequent Ca2+ influx through CNG channels activates Cl channels, which serve to amplify the transduction signal. We investigate here some general properties of this Ca-activated Cl channel in rat, as well as its functional interplay with the CNG channel, by using inside-out membrane patches excised from ORN dendritic knobs/cilia. At physiological concentrations of external divalent cations, the maximally activated Cl current was ∼30 times as large as the CNG current. The Cl channels on an excised patch could be activated by Ca2+ flux through the CNG channels opened by cAMP. The magnitude of the Cl current depended on the strength of Ca buffering in the bath solution, suggesting that the CNG and Cl channels were probably not organized as constituents of a local transducisome complex. Likewise, Cl channels and the Na/Ca exchanger, which extrudes Ca2+, appear to be spatially segregated. Based on the theory of buffered Ca2+ diffusion, we determined the Ca2+ diffusion coefficient and calculated that the CNG and Cl channel densities on the membrane were ∼8 and 62 μm−2, respectively. These densities, together with the Ca2+ diffusion coefficient, demonstrate that a given Cl channel is activated by Ca2+ originating from multiple CNG channels, thus allowing low-noise amplification of the olfactory receptor current.

The introduction of accelerator devices such as graphics processing units (GPUs) has had profound impact on molecular dynamics simulations and has enabled order-of-magnitude performance advances using commodity hardware. To fully reap these benefits, it has been necessary to reformulate some of the most fundamental algorithms, including the Verlet list, pair searching, and cutoffs. Here, we present the heterogeneous parallelization and acceleration design of molecular dynamics implemented in the GROMACS codebase over the last decade. The setup involves a general cluster-based approach to pair lists and non-bonded pair interactions that utilizes both GPU and central processing unit (CPU) single instruction, multiple data acceleration efficiently, including the ability to load-balance tasks between CPUs and GPUs. The algorithm work efficiency is tuned for each type of hardware, and to use accelerators more efficiently, we introduce dual pair lists with rolling pruning updates. Combined with new direct GPU–GPU communication and GPU integration, this enables excellent performance from single GPU simulations through strong scaling across multiple GPUs and efficient multi-node parallelization.

Human liver glycerol-3-phosphate dehydrogenase (hlGPDH) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to form glycerol 3-phosphate, using the binding energy associated with the nonreacting phosphodianion of the substrate to properly orient the enzymesubstrate complex within the active site. Herein, we report the crystal structures for unliganded, binary E•NAD, and ternary E•NAD•DHAP complexes of wild type hlGPDH, illustrating a new position of DHAP, and probe the kinetics of multiple mutant enzymes with natural and truncated substrates. Mutation of Lys120, which is positioned to donate a proton to the carbonyl of DHAP, results in similar increases in the activation barrier to hlGPDH-catlyzed reduction of DHAP and to phosphite dianion activated reduction of glycolaldehyde, illustrating that these transition states show similar interactions with the cationic K120 side chain. The K120A mutation results in a 5.3 kcal/mol transition state destabilization, and 3.0 kcal/mol of the lost transition state stabilization is rescued by 1.0 M ethylammonium cation. The 6.5 kcal/mol increase in the activation barrier observed for the D260G mutant hlGPDH-catalyzed reaction represents a 3.5 kcal/mol weakening of transition state stabilization by the K120A side chain, and a 3.0 kcal/mol weakening of the interactions with other residues. The interactions, at the enzyme active site, between the K120 side chain and the Q295 and R269 side chains was likewise examined by double mutant analyses. These results provide strong evidence that the enzyme rate acceleration is due mainly or exclusively to transition state stabilization by electrostatic interactions with polar amino acid side chains.

Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis.

Cyclic nucleotide-gated (CNG) channels play a central role in the conversion of sensory stimuli into electrical signals. CNG channels form heterooligomeric complexes built of A and B subunits. Here, we study the subunit stoichiometry of the native rod CNG channel by chemical crosslinking. The apparent molecular weight (M(w)) of each crosslink product was determined by SDS-PAGE, and its composition was analyzed by Western blotting using antibodies specific for the A1 or B1 subunit. The number of crosslink products and their M(w) as well as the immunological identification of A1 and B1 subunits in the crosslink products led us to conclude that the native rod CNG channel is a tetramer composed of three A1 and one B1 subunit. This is an example of violation of symmetry in tetrameric channels.

Monoamine oxidases (MAOs) A and B are flavoenzymes responsible for the metabolism of biogenic amines such as dopamine, serotonin and noradrenaline. In this work, we present a comprehensive study of the rate-limiting step of dopamine degradation by MAO B, which consists in the hydride transfer from the methylene group of the substrate to the flavin moiety of the FAD prosthetic group. This article builds on our previous quantum chemical study of the same reaction using a cluster model (Vianello et al., Eur J Org Chem 2012; 7057), but now considering the full dimensionality of the hydrated enzyme with extensive configurational sampling. We show that MAO B is specifically tuned to catalyze the hydride transfer step from the substrate to the flavin moiety of the FAD prosthetic group and that it lowers the activation barrier by 12.3 kcal mol⁻¹ compared to the same reaction in aqueous solution, a rate enhancement of more than nine orders of magnitude. Taking into account the deprotonation of the substrate prior to the hydride transfer reaction, the activation barrier in the enzyme is calculated to be 16.1 kcal mol⁻¹, in excellent agreement with the experimental value of 16.5 kcal mol⁻¹. Additionally, we demonstrate that the protonation state of the active site residue Lys296 does not have an influence on the hydride transfer reaction.

It is becoming widely accepted that catalytic promiscuity, i.e., the ability of a single enzyme to catalyze the turnover of multiple, chemically distinct substrates, plays a key role in the evolution of new enzyme functions. In this context, the members of the alkaline phosphatase superfamily have been extensively studied as model systems in order to understand the phenomenon of enzyme multifunctionality. In the present work, we model the selectivity of two multiply promiscuous members of this superfamily, namely the phosphonate monoester hydrolases from Burkholderia caryophylli and Rhizobium leguminosarum. We have performed extensive simulations of the enzymatic reaction of both wild-type enzymes and several experimentally characterized mutants. Our computational models are in agreement with key experimental observables, such as the observed activities of the wild-type enzymes, qualitative interpretations of experimental pH-rate profiles, and activity trends among several active site mutants. In all cases the substrates of interest bind to the enzyme in similar conformations, with largely unperturbed transition states from their corresponding analogues in aqueous solution. Examination of transition-state geometries and the contribution of individual residues to the calculated activation barriers suggest that the broad promiscuity of these enzymes arises from cooperative electrostatic interactions in the active site, allowing each enzyme to adapt to the electrostatic needs of different substrates. By comparing the structural and electrostatic features of several alkaline phosphatases, we suggest that this phenomenon is a generalized feature driving selectivity and promiscuity within this superfamily and can be in turn used for artificial enzyme design.