Model of Type VII Secretion System Table 2. Functional assignments of the structural components involved in ESX-1 secretion pathway. Component name Multimeric assembly of secretory proteins Known function (from literature) Probable function (obtained from present study) Rv3868 Rv3870, Rv3871 Recognition of secretory proteins and motor activity Inner membrane translocation Rv3877 Rv3883c (MycP1) Mycomembrane translocation Rv3869 Rv3882c Proteolytic processing and secretion regulation at the periplasm-like space ATPase Delivery of ESAT-6/CFP-10 pair to the secretion machinery Pore forming protein located in the inner membrane Protease which cleaves EspB (Rv3881c) Membrane protein of unknown function Membrane protein predicted to face periplasm Initiation of the assembly of multiprotein complexes ATPase motor to recruit the secretory proteins to the inner membrane pore (Rv3877) and facilitate translocation Inner membrane pore. Regulates secretion of proteins through its interaction with MycP1 Cleaves the ubiquitin-like domain of Rv3877, thereby opening the gate and initiating translocation of secretory proteins Component required for the formation of the mycomembrane translocation machinery Component required for the formation of the mycomembrane translocation machinery Component required for the formation of the mycomembrane translocation machinery Function derived from Domain analysis, 3D fold analysis, literature Domain analysis, 3D fold analysis, literature Domain analysis, motif analysis, literature Motif analysis, literature Domain analysis, glycosylation site prediction, protein-protein association prediction, literature Protein-protein association prediction, literature Phylogenetic profile analysis, protein- protein association prediction, literature such four-helix-bundle structures is between 20–23 A˚. This diameter is similar to that of double stranded DNA molecules, which is observed to be around 20 A˚ [43,44]. These observations indicate that, the FtsK family proteins (Rv3870 and Rv3871) probably recruit and translocate the (four-helix-bundle) substrates in a manner similar to that used by FtsK for the translocation of double stranded DNA. Thus, the proteins Rv3870/Rv3871 of T7SS may provide a unique example wherein, a machinery initially believed to be used for translocation of double stranded DNA substrates, has been tuned to transport protein complexes. A similar component related to FtsK family, VirD4, has been shown to participate in the Type IV secretion system [28], which is responsible for translocation of both DNA as well as protein moieties. Inner membrane translocation: The subsequent step after recognition and recruitment of the secretory proteins, is movement of these proteins through the inner membrane pore. Rv3877 has previously been suggested to be a pore forming protein through which the secretory proteins cross the inner membrane [4]. As an additional analysis, we have determined the diameter of the pore probably formed by the TM helices of Rv3877. Considering a compact arrangement of the helices, the diameter of the pore was determined to be approximately 26 A˚ (Text S2). The above calculation assumes a tight packing among the constituent helices (ignoring the side chain lengths). Thus, the actual diameter is likely to increase upon incorporating the side-chain lengths and interactions. This suggests that, the pore formed by Rv3877 will probably be able to translocate the substrate pairs (in the form of four-helix-bundles) of the ESX-1 secretion pathway. Proteolytic processing and secretion regulation at the periplasm- like space: Our analysis identified MycP1 to be located in the periplasm-like space probably anchored to the inner membrane by a C-terminal transmembrane helix. The cleavage of EspB by MCE1 proteins (Mce1B, Mce1C, Unknown Mce1F, Rv0177) doi:10.1371/journal.pone.0027980.t002 10/ESAT-6 as well as the C-terminal of EspC [17,41]. Previous studies have also provided experimental evidences that CFP-10/ ESAT-6 and EspA/EspC pairs are dependent on each other for their secretion and stability [8,21], suggesting that, the dimeric pairs of CFP-10/ESAT-6 and EspA/EspC are probably secreted as a multimeric protein assembly. Thus, the results of our analysis along with the observations of the previous studies suggest that, Rv3868 probably helps in the multimerization of CFP-10/ESAT- 6/EspA/ESpC, which are subsequently secreted as a multimeric assembly. This multimeric protein assembly is probably recog- nized and translocated by a separate motor ATPase. Recognition of secretory proteins and motor activity: Rv3870 is annotated as a member of Ftsk/SpoIIIE family of DNA translo- case proteins [13]. It has been shown to contain two transmem- brane helices and a single FtsK ATPase domain [13]. It is known to interact with another cytoplasmic FtsK/SpoIIIE family protein Rv3871 [3,13], containing two FtsK ATPase domains. Earlier studies also provided evidence that, Rv3871 interacts with the C- terminal region of CFP-10, and recruits the CFP-10/ESAT-6 pair to the translocon unit [3,7,19,20]. The a and b units of the FtsK domain containing proteins are known to multimerize to produce a hexameric ring [42], with an inner diameter of ,30 A˚. This ring then acts as a DNA pump which translocates double stranded DNA during cell division [42]. Since Rv3870 contains one FtsK domain (one pair of a and b domain) and Rv3871 contains two such domains (two pairs of a and b domains), we propose that, the a and b domains contributed by two Rv3870/Rv3871 pairs could form a hexameric ring, similar to that formed by a hexamer of FtsK proteins. Moreover, we performed an estimation of the approximate diameters (summarized in Text S1) of four-helix-bundle structures (corresponding to the CFP-10/ESAT-6 structural homologues). Our structural analysis indicates that, the approximate diameter of PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27980