Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules.

Ignacio Boron,Juan Pablo Bustamante,Kanak Dikshit,Mariana Tinajero-Trejo,Dario A Estrin,Lesley Ah Bowman,Robert K Poole,Rafael Radi,Leonardo Boechi,Sandip Singh,Kelly S Davidge,Marcelo A Marti,Sebastián Carballal

doi:10.12688/f1000research.5921.1

Abstract

Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and (•)NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify (•)NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, (•)NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

Highlights

Mycobacterium tuberculosis, the causative agent of human tuberculosis, affects approximately two billion people world-wide, causing over three millions deaths each year[1]
By performing carbon monoxide (CO) association kinetic constant measurements, NO decomposition, and molecular dynamics (MD) simulations, we addressed molecular mechanisms that control ligand association in M. tuberculosis truncated hemoglobins
CO association kinetic constant measurements as well as MD simulations of Mt-trHbN wild type and site-specific mutants were performed to analyze the role of tunnels and water molecules in the ligand association process

Summary

Introduction

Mycobacterium tuberculosis, the causative agent of human tuberculosis, affects approximately two billion people world-wide, causing over three millions deaths each year[1]. The genome of this pathogenic organism includes two genes, glbN and glbO, which encode for two proteins, termed here truncated hemoglobin N (Mt-trHbN) and truncated hemoglobin O (Mt-trHbO), belonging to the truncated hemoglobin (trHb) family of heme proteins, widely distributed in eubacteria, cyanobacteria, microbial eukaryotes and plants[2,3]. The truncated hemoglobin family exhibits a three-dimensional structure similar to the common globin fold of myoglobin, but significantly smaller. Phylogenetic analysis has distinguished three different groups of truncated hemoglobins, classified as groups I, II and III, called N, O and P, respectively

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Conclusion