Assessing the accuracy and performance of implicit solvent models for drug molecules: conformational ensemble approaches.

The accuracy and performance of implicit solvent methods for solvation free energy calculations were assessed on a set of 20 neutral drug molecules. Molecular dynamics (MD) provided ensembles of conformations in water and water-saturated octanol. The solvation free energies were calculated by popular implicit solvent models based on quantum mechanical (QM) electronic densities (COSMO-RS, MST, SMD) as well as on molecular mechanical (MM) point-charge models (GB, PB). The performance of the implicit models was tested by a comparison with experimental water-octanol transfer free energies (ΔG(ow)) by using single- and multiconformation approaches. MD simulations revealed difficulties in a priori estimation of the flexibility features of the solutes from simple structural descriptors, such as the number of rotatable bonds. An increasing accuracy of the calculated ΔG(ow) was observed in the following order: GB1 ~ PB < GB7 ≪ MST < SMD ~ COSMO-RS with a clear distinction identified between MM- and QM-based models, although for the set excluding three largest molecules, the differences among COSMO-RS, MST, and SMD were negligible. It was shown that the single-conformation approach applied to crystal geometries provides a rather accurate estimate of ΔG(ow) for rigid molecules yet fails completely for the flexible ones. The multiconformation approaches improved the performance, but only when the deformation contribution was ignored. It was revealed that for large-scale calculations on small molecules a recent GB model, GB7, provided a reasonable accuracy/speed ratio. In conclusion, the study contributes to the understanding of solvation free energy calculations for physical and medicinal chemistry applications.

CO rebinding kinetics and molecular dynamics simulations highlight dynamic regulation of internal cavities in human cytoglobin.

Cytoglobin (Cygb) was recently discovered in the human genome and localized in different tissues. It was suggested to play tissue-specific protective roles, spanning from scavenging of reactive oxygen species in neurons to supplying oxygen to enzymes in fibroblasts. To shed light on the functioning of such versatile machinery, we have studied the processes supporting transport of gaseous heme ligands in Cygb. Carbon monoxide rebinding shows a complex kinetic pattern with several distinct reaction intermediates, reflecting rebinding from temporary docking sites, second order recombination, and formation (and dissociation) of a bis-histidyl heme hexacoordinated reaction intermediate. Ligand exit to the solvent occurs through distinct pathways, some of which exploit temporary docking sites. The remarkable change in energetic barriers, linked to heme bis-histidyl hexacoordination by HisE7, may be responsible for active regulation of the flux of reactants and products to and from the reaction site on the distal side of the heme. A substantial change in both protein dynamics and inner cavities is observed upon transition from the CO-liganded to the pentacoordinated and bis-histidyl hexacoordinated species, which could be exploited as a signalling state. These findings are consistent with the expected versatility of the molecular activity of this protein.

Biophysical characterisation of neuroglobin of the icefish, a natural knockout for hemoglobin and myoglobin. Comparison with human neuroglobin.

The Antarctic icefish Chaenocephalus aceratus lacks the globins common to most vertebrates, hemoglobin and myoglobin, but has retained neuroglobin in the brain. This conserved globin has been cloned, over-expressed and purified. To highlight similarities and differences, the structural features of the neuroglobin of this colourless-blooded fish were compared with those of the well characterised human neuroglobin as well as with the neuroglobin from the retina of the red blooded, hemoglobin and myoglobin-containing, closely related Antarctic notothenioid Dissostichus mawsoni. A detailed structural and functional analysis of the two Antarctic fish neuroglobins was carried out by UV-visible and Resonance Raman spectroscopies, molecular dynamics simulations and laser-flash photolysis. Similar to the human protein, Antarctic fish neuroglobins can reversibly bind oxygen and CO in the Fe(2+) form, and show six-coordination by distal His in the absence of exogenous ligands. A very large and structured internal cavity, with discrete docking sites, was identified in the modelled three-dimensional structures of the Antarctic neuroglobins. Estimate of the free-energy barriers from laser-flash photolysis and Implicit Ligand Sampling showed that the cavities are accessible from the solvent in both proteins.Comparison of structural and functional properties suggests that the two Antarctic fish neuroglobins most likely preserved and possibly improved the function recently proposed for human neuroglobin in ligand multichemistry. Despite subtle differences, the adaptation of Antarctic fish neuroglobins does not seem to parallel the dramatic adaptation of the oxygen carrying globins, hemoglobin and myoglobin, in the same organisms.

Role of PheE15 gate in ligand entry and nitric oxide detoxification function of mycobacterium tuberculosis truncated hemoglobin N.

The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O(2) and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O(2)/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.

A Multilevel Strategy for the Exploration of the Conformational Flexibility of Small Molecules.

Predicting the conformational preferences of flexible compounds is still a challenging problem with important implications in areas such as molecular recognition and drug design. In this work, we describe a multilevel strategy to explore the conformational preferences of molecules. The method relies on the predominant-state approximation, which partitions the conformational space into distinct conformational wells. Moreover, it combines low-level (LL) methods for sampling the conformational minima and high-level (HL) techniques for calibrating their relative stability. In the implementation used in this study, the LL sampling is performed with the semiempirical RM1 Hamiltonian, and solvent effects are included using the RM1 version of the MST continuum solvation model. The HL refinement of the conformational wells is performed by combining geometry optimizations of the minima at the B3LYP (gas phase) or MST-B3LYP (solution) level, followed by single point MP2 computations using Dunning's augmented basis sets. Then, the effective free energy of a conformational well is estimated by combining the MP2 energy, supplemented with the MST-B3LYP solvation free energy for a conformational search in solution, with the local curvature of the well sampled at the semiempirical level. Applications of this strategy involve the exploration of the conformational preferences of 1,2-dichloroethane and neutral histamine in both the gas phase and water solution. Finally, the multilevel strategy is used to estimate the reorganization cost required for selecting the bioactive conformation of HIV reverse transcriptase inhibitors, which is estimated to be at most 1.3 kcal/mol.

Ligand migration in Methanosarcina acetivorans protoglobin: effects of ligand binding and dimeric assembly.

Protoglobin is the first globin found in Archaea. Its biological role is still unknown, although this protein can bind O(2), CO, and NO reversibly in vitro. The X-ray structure of Methanosarcina acetivorans protoglobin (MaPgb) has shown that access of ligands to the heme, which is completely buried within the protein matrix, can be granted by two apolar tunnels, which are mainly defined by helices G and B (tunnel 1), and helices B and E (tunnel 2). Here we analyze the structural and dynamical behavior of MaPgb through molecular dynamics and computational techniques aimed at shedding light on distinctive features of ligand migration through the tunnels that may be linked to functionality. While tunnel 2 is found to be accessible to diatomic ligands in both deoxygenated and oxygenated forms of the protein, the accessibility of tunnel 1 is controlled through the synergistic effect of both the protein dimeric state and the presence of the heme-bound ligand. Thus, dimerization mainly affects the spatial arrangement of helix G, which influences the shape of tunnel 1. Ligand accessibility through this tunnel is regulated by Phe(145)G8, which can adopt open and closed conformations. Noteworthy, the ratio between open and closed states is modulated by protein dimerization and more strikingly by ligand binding. In particular, sensing of the ligand is mediated by Phe(93)E11, and the steric hindrance between Phe(93)E11 and the heme-bound ligand alters the structural and dynamical behavior of helices B and E, which facilitates opening of tunnel 1. This functional mechanism provides a basis to understand the finding that ligation favors fast rebinding from ligand binding kinetic to MaPgb. Finally, it also suggests that MaPgb might be physiologically involved in a ligand-controlled bimolecular chemical process.

Comparing and combining implicit ligand sampling with multiple steered molecular dynamics to study ligand migration processes in heme proteins.

The ubiquitous heme proteins perform a wide variety of tasks that rely on the subtle regulation of their affinity for small ligands like O2, CO, and NO. Ligand affinity is characterized by kinetic association and dissociation rate constants, that partially depend on ligand migration between the solvent and active site, mediated by the presence of internal cavities or tunnels. Different computational methods have been developed to study these processes which can be roughly divided in two strategies: those costly methods in which the ligand is treated explicitly during the simulations, and the free energy landscape of the process is computed; and those faster methods that use prior computed Molecular Dynamics simulation without the ligand, and incorporate it afterwards, called implicit ligand sampling (ILS) methods. To compare both approaches performance and to provide a combined protocol to study ligand migration in heme proteins, we performed ILS and multiple steered molecular dynamics (MSMD) free energy calculations of the ligand migration process in three representative and well theoretically and experimentally studied cases that cover a wide range of complex situations presenting a challenging benchmark for the aim of the present work. Our results show that ILS provides a good description of the tunnel topology and a reasonable approximation to the free energy landscape, while MSMD provides more accurate and detailed free energy profile description of each tunnel. Based on these results, a combined strategy is presented for the study of internal ligand migration in heme proteins.

Role of heme distortion on oxygen affinity in heme proteins: the protoglobin case.

The chemical properties of heme proteins largely reflect the electronic properties of their heme group. Often, the porphyrin ring of the heme exhibits significant distortions from its isolated structure, but the impact of these distortions on the chemical properties of the heme is yet uncertain. A systematic study focused on the effects of the distortion of the macrocycle on the binding affinity for oxygen is presented. The results show that out-of-plane distortions decrease the binding affinity, while in-plane distortions can increase or decrease it. Among in-plane distortions, only the breathing mode, which involves the symmetric compression-expansion of the porphyrin ring, strongly modulates the binding affinity. These findings shed light into the peculiar binding affinity of Methanosarcina acetivorans protoglobin, a protein that contains a highly distorted heme. Overall, the results highlight that in-plane distortions might be exploited by certain classes of heme proteins to modulate the ligand affinity.

Performance of the IEF-MST solvation continuum model in a blind test prediction of hydration free energies.

The IEF-MST continuum solvation model is used to predict the hydration free energies of a set of 63 multifunctional compounds very recently compiled as a blind test (denoted SAMPL1) for computational solvation methods (Guthrie, J. P. J. Phys. Chem. B 2009, 113, 4501). Computations were performed using the IEF-MST versions parametrized at both HF/6-31G(d) and B3LYP/6-31G(d) levels. For direct comparison with other methods, computations were performed using the frozen geometries provided with the SAMPL1 data set, as well as the gas phase optimized geometries following the implementation of the IEF-MST model. Comparison with experimental data yields a root-mean square deviation for the whole set of compounds of 2.7 and 2.4 kcal/mol at both HF and B3LYP levels. The agreement between IEF-MST and experimental data is then quite remarkable, especially considering the reduced set of training compounds (72 data in water) used in the parametrization of the IEF-MST method.

Extension of the MST continuum solvation model to the RM1 semiempirical Hamiltonian.

The need to simulate large-sized molecules or to deal with large series of compounds is a challenging topic in computational chemistry, which has stimulated the development of accurate semiempirical methods, such as the recently reported Recife Model 1 (RM1; J Comput Chem 2006, 27, 1101). Even though RM1 may prove to be of value simply due to the enhanced quantitative accuracy in gas phase, it is unclear how the new parameters optimized for RM1 affect the suitability of this semiempirical Hamiltonian to study chemical processes in condensed phases. To address this question, we report the parametrization of the MST/RM1 continuum model for neutral solutes in water, octanol, chloroform and carbon tetrachloride, and for ions in water. The results are used to discuss the transferability of the solvation parameters implemented in previous MST/AM1 and MST/PM3 models.