proteins STRUCTURE O FUNCTION O BIOINFORMATICS Structure of the Mycobacterium tuberculosis OmpATb protein: A model of an oligomeric channel in the mycobacterial cell wall Yinshan Yang,1,2 Daniel Auguin,1,2,3 Ste´phane Delbecq,4 Emilie Dumas,1,2 Ge´rard Molle,1,2 Virginie Molle,5 Christian Roumestand,1,2* and Nathalie Saint1,2 1 Centre de Biochimie Structurale, CNRS UMR 5048, Universite´ Montpellier 1 et 2, F34090 Montpellier, France 2 INSERM U554 F34090 Montpellier, France 3 INRA, USC2030 ‘Arbres et Re´ponses aux Contraintes Hydrique et Environnementales’ (ARCHE), F-45067 Orle´ans Cedex 02, France 4 Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS UMR 5235, Universite´ Montpellier 2, F34095 Montpellier Cedex 5, France 5 Institut de Biologie et de Chimie des Prote´ines, Universite´ de Lyon, CNRS UMR 5086, F69367 Lyon, France ABSTRACT INTRODUCTION The pore-forming outer membrane protein The etiological agent of tuberculosis (TB), Mycobacterium OmpATb from Mycobacterium tuberculosis is a viru- tuberculosis, causing nearly 2 million deaths per year, is presently lence factor required for acid resistance in host one of the most important infectious agents implicated in mortal- phagosomes. In this study, we determined the 3D ity worldwide. TB has emerged as a major public health threat structure of OmpATb by NMR in solution. We because of a significant increase in multiple-drug-resistant TB and found that OmpATb is composed of two independ- synergism between human immunodeficiency virus and M. tuber- ent domains separated by a proline-rich hinge culosis infection.1,2 One of the principle problems in TB therapy region. As expected, the high-resolution structure of is the slow uptake of drugs across the thick mycobacterial cell the C-terminal domain (OmpATb198–326) revealed a wall made of unique lipid and glycolipid moities.3,4 This cell wall module structurally related to other OmpA-like pro- is extremely hydrophobic and forms an exceptionally strong per- teins from Gram-negative bacteria. The N-terminal meability barrier, 100-fold less permeable than the outer mem- domain of OmpATb (73–204), which is sufficient to brane of Escherichia coli.5,6 Water-filled protein channels, called form channels in planar lipid bilayers, exhibits a porins, are considered to represent the main pathway for entry of fold, which belongs to the a1b sandwich class fold. hydrophilic drugs through the outer membrane of Gram-negative Its peculiarity is to be composed of two overlapping 7 subdomains linked via a BON (Bacterial OsmY and bacteria. Porin-like proteins have also been identified in the cell wall of several mycobacterial species such as M. chelonae,8 M. Nodulation) domain initially identified in bacterial 9 10 5,11 12 proteins predicted to interact with phospholipids. phlei, M. smegmatis, M. bovis BCG, and M. tuberculosis Although OmpATb73–204 is highly water soluble, by assaying organic solvent- or detergent-solubilized cell wall frac- current–voltage measurements demonstrate that it tions for channel-forming activity in artificial lipid bilayers. In is able to form conducting pores in model mem- most cases, the reported studies were in agreement concerning branes. A HADDOCK modeling of the NMR data the paucity of porins in mycobacterial membranes, which could gathered on the major monomeric form and on the contribute to the low permeability observed in vivo. The low level minor oligomeric populations of OmpATb73–204 sug- of mycobacterial porins in solubilized cell wall fractions has also gest that OmpATb73–204 can form oligomeric rings impeded their biochemical and structural characterization. How- able to insert into phospholipid membrane, similar ever, the most advanced characterization of a mycobacterial porin to related proteins from the Type III secretion sys- tems, which form multisubunits membrane-associ- ated rings at the basal body of the secretion ma- Additional Supporting Information may be found in the online version of this article. Yinshan Yang and Daniel Auguin contributed equally to this work. chinery. Daniel Auguin’s current address is Universite´ d’Orle´ans, UFR-Faculte´ des Sciences, Laboratoire de Biologie des Ligneux et des Grandes Cultures, UPRES EA 1207, rue de Chartres, BP 6759, 45067 Proteins 2011; 79:645–661. Orle´ans Cedex 02, France. VC 2010 Wiley-Liss, Inc. *Correspondence to: Christian Roumestand, Centre de Biochimmie Structurale, 29 rue de Navacelles, 34090 Montpellier Cedex 5, France. E-mail: [email protected]. Key words: membrane protein; porin; NMR struc- Received 27 July 2010; Revised 23 September 2010; Accepted 24 September 2010 Published online 12 October 2010 in Wiley Online Library (wileyonlinelibrary.com). ture; oligomeric assembly; HADDOCK calculation. DOI: 10.1002/prot.22912 VC 2010 WILEY-LISS, INC. PROTEINS 645 Y. Yang et al. was achieved on the MspA protein representing the channels with similar pore properties to those of entire major general diffusion pathway for hydrophilic com- OmpATb were observed, suggesting that the 72 N-termi- pounds in the fast growing Mycobacterium smegmatis.13 nal proximal residues are not necessary for the pore- In 2004, Faller et al.14 succeeded in resolving the 3D forming activity. Similarly, it was shown that the C-ter- structure of this mycobacterial porin by using a recombi- minal part of OmpATb was not essential for the channel nant MspA protein expressed in E. coli cells. The MspA activity because an OmpATb73–220 construct was able to crystal structure revealed a homooctameric goblet-like form pores in lipid bilayers.22 In a very recent report, architecture with a single central channel of 10 nm in the pore-forming activity of OmpATb was questioned length. This structure is completely different from that of because the protein is apparently not folded in a beta- the known trimeric porins of Gram-negative bacteria, barrel.25 which have one channel per monomer and are 4nm In this study, with the goal of characterizing the archi- long.15–17 Homologs of MspA have been identified in tecture of OmpATb responsible for its channel activity, other rapid-growing mycobacterial species such as M. we carried out NMR studies on the soluble form of the phlei but not in the slow-growing mycobacteria M. tuber- protein lacking the first 72 N-terminal residues, 18 culosis and M. bovis BCG. OmpATb73–326. Based on the complete NMR assignment In the slow-growing pathogen M. tuberculosis, a pro- of this 254-residue protein, extensive analysis of NOESY tein with significant sequence homology to the major spectrum revealed that OmpATb is composed of two in- outer membrane protein OmpA of E. coli was identified dependent domains separated by a proline-rich hinge about 10 years ago.19 This protein, OmpATb, has been region. The C-terminal module is structurally related to shown to form ion channels in planar lipid bilayers and other OmpA-like proteins, whereas the structure of the was considered as a porin-like protein from M. tuberculo- N-terminal domain displays an original fold unexpected sis.20 Furthermore, it was proposed that OmpATb corre- for a porin-like proteins family member. These results sponds to the major functioning porin at low pH are in agreement with the very recent NMR study of because deletion of the ompATb gene caused a decrease OmpATb proposed by Teriete et al.,25 which appeared in of the permeability to several small water-soluble sub- the literature during the drafting process of this article. stances under reduced pH conditions.21 Recent electro- The authors report the characterization of the secondary physiological studies with recombinant OmpATb pro- structure and the dynamics of the two domains duced in E. coli demonstrated that the channel activity of (OmpATb73–220 and OmpATb196–326) as well as the OmpATb is modulated by pH.22 It was also observed detailed 3D structure of the N-terminal domain that OmpATb channels exhibit more frequent and more (OmpATb73–220). In addition to this previously reported prolonged closure events at acidic pH. This particular structure of the N-terminal domain, we report here the behavior of OmpATb at low pH conditions was proposed 3D structure of the C-terminal domain, apparently to be beneficial to M. tuberculosis survival in the mildly related to the crystal structure of the OmpA-like domain acidic environment encountered in the phagocytotic of the RmpM protein from N. meningitidis.26 The struc- vacuole of host macrophages. Moreover, it was shown ture described in these two different studies cannot that OmpATb appears to be expressed only in pathogenic explain the porin activity of OmpATb: this highly soluble species (i.e., the members of the M. tuberculosis complex) protein is unlikely to insert into phospholipidic mem- underscoring its role in the virulence of these mycobacte- branes as is. From careful inspection of the gel chroma- rial strains.22 tography elution profile of the recombinant protein, we Since its identification in the genome of M. tuberculo- found that oligomeric minor species of OmpATb N-ter- sis, the structural characterization of OmpATb has been a minal domain coexist in solution with the monomeric crucial objective. Initial circular dichroism experiments major species. Based on NMR data combined with HAD- revealed that OmpATb presents a significant a-helical DOCK (High Ambiguity Driven DOCKing) bases model- and b-sheet content, in agreement with that was ing calculations, we propose here a multimeric model of observed for OmpA of E. coli.20,23 More recent studies OmpATb that can form a pore inside the cell wall of M. revealed that the N-terminal proximal residues of tuberculosis. OmpATb are essential to target the protein to the mem- brane. Indeed, the production of a recombinant MATERIALS AND METHODS OmpATb73–326 protein in mycobacterial strains such as M. smegmatis and M. bovis yielded to the expression of Bacterial strains the protein essentially in the cytosol of the mycobacte- 24 ria.