Figure 1.
Schematic representation of the mycolic acid biosynthesis pathway.
The biochemical reactions necessary to achieve the biosynthesis of mature mycolic acids are symbolized with black arrows. The enzymes responsible for each reaction are named in colored squares. The cofactors necessary for certain reactions to occur (NADPH, NADH) were omitted for clarity. The proximal and distal position of the meromycolic chain modifications by the MA-Mtfs are indicated as P and D respectively. The interrupted arrows are used to symbolize the existence of intermediates biochemical reactions that are not detailed on the figure.
Figure 2.
The FAS-II specialized complexes.
The FAS-II initiation complex (I-FAS-II), elongation complex 1 and 2 (E1-FAS-II and E2-FAS-II), and the termination complex (T-FAS-II) are represented as filled grey boxes. The condensing enzymes KasA, KasB and MtFabH (labeled as FabH) are in orange. The reductases InhA and MabA are respectively represented in green and yellow. The malonylCoA ACP transacylase MtFabD is represented as a red box and labeled as FabD. The interactions between KasA and KasB with either the MA-Mtf (in violet) or the condensing enzyme Pks13 (in blue) are symbolized by curved arrows. The traffic of the substrates and products of each complex is also symbolized by curved arrows and the numbers represent the sequence of the events. MA is for mycolic acids, FAS-I for fatty acid synthase of type-I and FAS-II for FAS of type-II. The product of FAS-I and substrate of FAS-II is an Acyl-CoA. The product of FAS-II is an Acyl-ACP.
Table 1.
Y2H analysis of interactions between the Had monomers.
Table 2.
Y3H analysis of protein-protein interactions between the Had heterodimers and the FAS-II proteins.
Figure 3.
In vitro Co-IP between Mtb FAS-II proteins and MA-Mtfs.
The L-[35S]-methionine labeled h-proteins (HA tagged; h-protein) and c-proteins (c-myc tagged; c-protein) from in vitro transcription translation reactions and Co-IP reactions products were fractionated by SDS-PAGE (10%) followed by phosphor-imaging analysis. In column 1, the gels contained the h-protein alone. In even numbered column, the gels contained the c-protein alone. In the other columns (odd numbered except 1), the gel contained the Co-IP reaction products. Each row corresponds to a distinct h-protein analysis and the names of proteins are indicated in front of their position of migration. For CmaA1, the complete gel lines are presented. For the other Mtfs only the gel region of interest is presented. The black filled arrows represent a Co-IP band corresponding to a positive interaction. The open arrows mark the position of the absent Co-IP band and correspond to a negative interaction.
Table 3.
Protein-protein interactions between MA-Mtf and FAS-II: a compilation of Y2H and Co-IP.
Figure 4.
Summary of protein-protein interactions within the MA biosynthesis interactome.
The protein-protein interactions between the components of the specialized interconnected FAS-II complexes of Mtb are represented as defined in the present work and before [36], [37]. The condensing enzymes (in orange) interact with each other and with the core composed of MabA (in yellow),InhA (in dark green) and FabD (in red). For clarity, the core is depicted in each type of complex. KasA and KasB interact preferentially with HadAB (in light green) and HadBC respectively. The interactions between the MA-Mtfs (in violet) and KasA, HadAB, FabD, and InhA, or KasB, HadBC InhA, and FabD are represented with black curved arrows. Pks13 (in blue) interact with KasB and with the MA-Mtfs MmaA3 and MmaA4. Each type of complex is represented as a grey rectangle; I-FAS-II is the initiation complex, E1-FAS-II and E2-FAS-II the elongation complexes of type 1 and 2 respectively and T-FAS-II is the termination complex.
Table 4.
Y3H analysis of protein-protein interactions between the Had heterodimers and the MA-Mtfs.
Figure 5.
Modular organization of Mtb FAS-II and mFAS-I.
(A) Schematic representation of type-I (E1-FAS-II) and type-II (E2-FAS-II) FAS-II elongation complexes as defined by the analysis of protein interactions in the present work and before [36], [37]. The interactions between the different MA-Mtfs (in violet) and each complex are represented by curved arrows. (B) Schematic representation of a dimer of m-FAS-I adapted from Maier and colleagues [6] and drawn from the 3D structure. For both panels, the enoyl reductase domains (ER) and proteins (InhA) are in dark green, the keto-reductase domains (KR) or pseudo keto-reductase domains (Ψ-KR) and proteins (MabA) are in yellow, the keto-synthase domains (KS) or proteins (KasA, KasB) are in orange, the acyl transferase domains or the MtFabD protein (FabD) are in red, the pseudo-methyltransferase domains (Ψ-Mtf) or the MA-Mtf proteins (CmaA1, CmaA2, MmaA1 to MmaA4, UmaA, PcaA) are in violet, the dehydratase domains (DH) or proteins (HadA, HadB, HadC) are in light green and the mammalian FAS-I linker region (L) are in grey. The links between the domains of mFAS-I in its primary structure are symbolized by straight lines.