Ligand Binding Rate Constants in Heme Proteins Using Markov State Models and Molecular Dynamics Simulations

Computer simulation studies of the molecular basis for ligand migration in proteins allow the description and quantification of the key events implicated in this process as, such as the transition between docking sites, displacements of existing ligands and solvent molecules, and open/closure of specific 'gates', among other factors. In heme proteins, especially in globins, these phenomena are related to the regulation of protein function, since ligand migration from the solvent to the active site preludes ligand binding to the iron in the distal cavity, which in turn triggers the different globin functions. In this work, a combination of molecular dynamics simulations with a Markov-state model of ligand migration is used to the study the migration of O2 and ·NO in two truncated hemoglobins of Mycobacterium tuberculosis (truncated hemoglobin N -Mt-TrHbN- and O -Mt-TrHbO). The results indicate that the proposed model provides trends in kinetic association constants in agreement with experimental data. In particular, for Mt-TrHbN, we show that the difference in the association constant in the oxy and deoxy states relies mainly in the displacement of water molecules anchored in the distal cavity by O2 in the deoxy form, whereas the conformational transition of PheE15 between open and closed states plays a minor role. On the other hand, the results also show the relevant effect played by easily diffusive tunnels, as the ones present in Mt-TrHbN, compared to the more impeded passage in Mt-TrHbO, which contributes to justify the different .NO dioxygenation rates in these proteins. Altogether, the results in this work provide a valuable approach to study ligand migration in globins using molecular dynamics simulations and Markov-state model analysis

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. © 2011 Wiley Periodicals, Inc.Fil: Forti, Flavio. Universidad de Barcelona; EspañaFil: Boechi, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentin

Impact of the Pre-a Motif on Truncated Hemoglobin N Activity

Tuberculosis (TB) remains the leading cause of death by an infectious agent and therefore a global health crisis, according to the most recent report by the World Health Organization. This is due, in part, to Mycobacterium tuberculosis’ impressive defensive mechanisms against immune response, as well as the rise of Multi-Drug Resistant strains that have recently developed. Towards the turn of the century, a small heme protein called truncated hemoglobin N (trHbN) was discovered to protect the bacteria against reactive nitrogen species by converting nitric oxide (NO) to nitrate at rates far exceeding those of myoglobin and closer to those of the well-known NO dioxygenase flavohemoglobin. Ferrous oxygenated trHbN (oxy-trHbN) first converts NO to nitrate, which leaves the protein in a ferric state (met-trHbN). Met-trHbN is re-reduced to give a 5-coordinate ferrous species (red-trHbN), which is then re-oxygenated to oxy-trHbN. Recently, a unique 12-amino acid motif at the trHbN N-terminus was identified, the so-called pre-A tail, that appears to enhance the organism’s ability to convert NO to nitrate. The results presented herein show that the pre-A tail of trHbN affects every step of the putative NO dioxygenation catalytic cycle, but it affects the rate of met-trHbN re-reduction most profoundly. In a variant that lacks the pre-A tail (trHbNdelN), met-trHbNdelN was reduced about 40 times more slowly than met-trHbNWT by the non-specific reductant RuII. By comparison, the reactions of oxy-trHbN or red-trHbN with NO were only 2x – 4x slower in the trHbNdelN variant than in the wild type (the reaction of red-trHbN with NO is a good surrogate for the reaction of red-trHbN with O2). Importantly, the effect of the pre-A tail is completely lost in variants that lack distal site residues Tyr33 and Gln 58. These residues help to hold O2 firmly on the heme in oxy-trHbN, and a water molecule on the heme of met-trHbN. They also anchor a non-coordinated water molecule in the distal site of red-trHbN that blocks access by incoming diatomic gases. In a variant that lacks Tyr33 and Gln 58 (trHbNDM), met-trHbNDM is reduced 5x more rapidly by RuII than is met- trHbNWT because the distal site is now either vacant or occupied by weakly bound water, so rate-limiting water loss upon heme reduction is accelerated. A variant that lacks Tyr33, Gln 58, and the pre-A tail (met-trHbNTM), is reduced by RuII at the same rate as is met-trHbNDM, showing that tail loss does not affect the reduction rate if the distal site amino acids are absent. This is strong evidence that the pre-A tail’s primary function is to facilitate release of the distal water molecule from met-trHbN, a function that is less important in met-trHbNDM and met-trHbNTM than it is in trHbNWT