Rv3868 (EccA1), an essential component of the Mycobacterium tuberculosis ESX-1 secretion system, is thermostable Amit Luthra 1, 2 , Amit Gaur 2 , Ravishankar Ramachandran ⁎ Molecular & Structural Biology Division, CSIR-Central Drug Research Institute, P.O. Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow-226001, India abstract article info Article history: Received 20 July 2012 Received in revised form 1 February 2013 Accepted 4 February 2013 Available online 13 February 2013 Keywords: ESX-1 secretion system AAA + ATPase Thermostability Charged and aliphatic amino acids Rv3868 (EccA1) is an essential CbxX/CfqX-family ATPase of the Mycobacterium tuberculosis ESX-1 secretion system. Previously, we demonstrated that Rv3868 is composed of two domains; a regulatory N-terminal domain (NT-Rv3868) and an ATP binding C-terminal domain (CT-Rv3868). In the present report, chemical denaturation studies show that electrostatic interactions stabilize the Rv3868. Interestingly, Rv3868 has notable heat stability and retains about 50% of ATPase activity even at 60 °C. The C-terminal domain was found to be important for the heat stability as demonstrated by both enzymatic activity assays and thermal denaturation experiments. Furthermore a structure-sequence analysis based on the content of charged and aliphatic amino acids rationalizes the higher propensity of Rv3868 for thermophilic characteristics. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Mycobacterium tuberculosis is the causative agent of the tuber- culosis, which is one of the leading causes of death by bacterial infection [1]. Evolution of drug resistant strains has renewed the pathogen's threat and in extreme cases makes the disease untreatable by cur- rent antibiotic regimens [2]. The pathogenesis of Mycobacterium involves a variety of virulence factors and little is known about how mycobacterium secretes virulence factors across the cell enve- lope. M. tuberculosis possesses a secretion system dedicated to the export of major secreted antigens: ESX (ESAT-6 secretion) system [3]. The proteins corresponding to this secretion system are encoded by genes of the region of difference (RD1) [4] which is absent in Mycobacterium bovis BCG [5]. Five major virulent factors, namely, CFP-10, ESAT-6, EspA, EspC, and EspR, have been associated with ESX-1 secretion system [6]. Of note, all of the substrates in the ESX-1 system are mutually dependent upon each other [7]. A few genes such as Rv3868, Rv3869 and Rv3872 are vital for the secretion of these virulent proteins [8]. Rv3868, now annotated as EccA1 [9] is also essential for survival within macrophages highlighting the importance of this gene [10]. The earlier detailed investigation of Rv3868 by our group revealed that the full-length protein is a weak ATPase and assembles into a hexamer or higher order multimers, with each subunit consisting of two structural domains [11]. The CT-Rv3868 domain at the C-terminus is the catalytic and oligomeri- zation domain that exhibits the catalytic function more efficiently on its own. Based on the results obtained by dynamic fluorescence quenching and tryptophan fluorescence, we also hypothesized that both structural domains move closer to each other upon the ad- dition of the nucleotide [11]. The earlier findings lead us to assess the structural and stability properties of recombinant full length Rv3868 and its domains. Very recently the Mycobacterium marinum homolog of the protein was found to be involved in the synthesis of mycolic acids [12]. This highlights the importance of Rv3868 as a new therapeutic target with the possibility of being exploited in the identification of new inhibitors with the potential ability to overcome some of the present drug resistance issues. We report here the investigation of the structural and function- al alterations associated with chemical and temperature induced unfolding of Rv3868 and its domains. The data reported here demonstrates that Rv3868 behaves as a thermostable molecule where CT-Rv3868 is important for this thermal stability. In an attempt to rationalize the basis of the observed thermal stability, we performed comparative bioinformatics analysis of the amino acid composition of Rv3868 to that of other thermostable proteins of M. tuberculosis and the essential genes needed for intracellular sur- vival within macrophage. Overall, the results highlight for the first time the presence of a thermostable protein in any secretion system of M. tuberculosis. Biochimica et Biophysica Acta 1834 (2013) 1181–1186 Abbreviations: NT-Rv3868, N-terminal domain of Rv3868; CT-Rv3868, C-terminal ATP-binding domain of Rv3868; CD, circular dichroism; GdmCl, guanidine hydrochloride ⁎ Corresponding author at: Molecular & Structural Biology Division, Central Drug Research Institute (CSIR), Lucknow-226001, U.P., India. Tel.: +91 522 2612411x4442; fax: +91 522 2623405. E-mail address: r_ravishankar@cdri.res.in (R. Ramachandran). 1 Present address: Department of Medicine, The University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3715. 2 These authors contributed equally to the work. 1570-9639/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbapap.2013.02.004 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap