Role of Pre-A Motif in Nitric Oxide Scavenging by Truncated Hemoglobin, HbN, of Mycobacterium tuberculosis

Amrita Lama,Sudesh Pawaria,Axel Bidon-Chanal,Arvind Anand,José Luis Gelpí,Swati Arya,Marcelo Martí,Dario A Estrin,F Javier Luque,Kanak L Dikshit

doi:10.1074/jbc.m807436200

Abstract

Mycobacterium tuberculosis truncated hemoglobin, HbN, is endowed with a potent nitric-oxide dioxygenase activity and has been found to relieve nitrosative stress and enhance in vivo survival of a heterologous host, Salmonella enterica Typhimurium, within the macrophages. These findings implicate involvement of HbN in the defense of M. tuberculosis against nitrosative stress. The protein carries a tunnel system composed of a short and a long tunnel branch that has been proposed to facilitate diatomic ligand migration to the heme and an unusual Pre-A motif at the N terminus, which does not contribute significantly to the structural integrity of the protein, as it protrudes out of the compact globin fold. Strikingly, deletion of Pre-A region from the M. tuberculosis HbN drastically reduces its ability to scavenge nitric oxide (NO), whereas its insertion at the N terminus of Pre-A lacking HbN of Mycobacterium smegmatis improved its nitric-oxide dioxygenase activity. Titration of the oxygenated adduct of HbN and its mutants with NO indicated that the stoichiometric oxidation of protein is severalfold slower when the Pre-A region is deleted in HbN. Molecular dynamics simulations show that the excision of Pre-A motif results in distinct changes in the protein dynamics, which cause the gate of the tunnel long branch to be trapped into a closed conformation, thus impeding migration of diatomic ligands toward the heme active site. The present study, thus, unequivocally demonstrates vital function of Pre-A region in NO scavenging and unravels its unique role by which HbN might attain its efficient NO-detoxification ability.

Highlights

Lus to establish long term persistent infection within the human host lies in its ability to survive and resist hazardous environment of its intracellular niche
Extended molecular dynamics (MD) simulations showed that the essential motions of the protein backbone and the positioning of Phe(E15) gate changes significantly after O2 binding, which facilitates access of a diatomic ligand (NO) to the active site to carry out nitric oxide (NO) detoxification [16, 17]
JLS, M. flavescens, M. vanbaalenii, M. smegmatis, etc., lack the 12-residue N-terminal Pre-A motif observed in the x-ray structure of M. tuberculosis HbN (Ref. 13; Fig. 1A) and appears to be present in many slow growing species of mycobacterium such as M. bovis, Mycobacterium avium, Mycobacterium microti, Mycobacterium marinum, etc. that are associated with disease of various organisms

Summary

Introduction

Lus to establish long term persistent infection within the human host lies in its ability to survive and resist hazardous environment of its intracellular niche. It has been demonstrated that disruption of glbN gene, encoding HbN, in Mycobacterium bovis bacillus Calmette-Guerin causes a dramatic reduction in the NO-consuming activity of stationary phase cells, resulting in marked NO-induced inhibition of aerobic respiration relative to the wild type cells [10]. Overall, these studies strongly support the NO-scavenging and detoxification roles of HbN, which may be vital for in vivo survival and pathogenicity of M. tuberculosis. The critical question that emerges now is how M. tuberculosis HbN is able to achieve an efficient NO detoxification ability and what aspects of its structure and/or dynamics contribute to this property

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