Proteins from Mycobacterium tuberculosis

The structure of Mycobacterium tuberculosis HtrA reveals an auto-regulatory mechanism^[1]

There are three HtrA paralogues in M. tuberculosis viz., HtrA (Rv1223), PepD (Rv0983) and PepA (Rv0125). Among these, only HtrA is essential for bacterial survival. M. tuberculosis PepD participates in a two component signaling pathway involving MprAB and σ^E. It has been suggested that recognition of misfolded proteins by the PDZ domain in PepD activates this pathway. However, unlike DegS, this HtrA homologue is constitutively active and the PDZ domain positively modulates its enzymatic activity. An aspect that is less explored in this context is the role of the N-terminal cytoplasmic domain. Both HtrA and PepD are predicted to have a cytoplasmic polypeptide stretch of 100-150 residues.

A common structural feature of HtrA proteases is that of a trypsin-like serine protease domain attached to one or more PDZ domains. An in silico analysis of the topological arrangement of these proteases suggests that they are likely to adopt a similar N_in-C_out conformation with one transmembrane helix. Given substantial sequence and structural conservation, the precise roles as well as the rationale for multiple HtrA paralogues in a bacterium are difficult to predict. This aspect is of particular significance to M. tuberculosis as HtrA enzymes govern virulence but the molecular details remain unclear.

The crystal structure of M. tuberculosis HtrA (ΔTM HtrA) that was determined at 1.83 Å resolution. We note that this enzyme exhibits both monomeric as well as trimeric forms in solution. The structure reveals a conformation that would require minor structural alterations for proteolytic activity. Structural features thus suggest that M. tuberculosis HtrA is a regulated protease as opposed to the two other paralogues, PepD and PepA. This essential enzyme is thus likely to be involved in specific signal transduction role as opposed to housekeeping in the recognition and degradation of partially folded or misfolded proteins.

The was determined at a resolution of 1.83Å (PDB ID: 6ieo). In this crystal form, there is one molecule of HtrA in the asymmetric unit. The structure of the periplasmic domain reveals one (226-436; colored in royalblue) flexibly tethered to the (443-528; in gold) at the C-terminal end. The (Alpha Helices, Beta Strands ,  Loops , Turns) referred to as the N-terminal and C-terminal β-barrel. While the N-terminal β-barrel contains the active site residues His270 and Asp306, the C-terminal β-barrel has Ser387 from (colored in yellow). The substantial structural conservation across HtrA enzymes suggests a similar reaction mechanism as evident from the positive charge cavity (the oxyanion hole) which helps in stabilization of tetrahedral intermediate during the acylation step of catalysis. The side chain of the active site serine, Ser387, could be modelled in two alternate conformations with an occupancy of 0.53 and 0.47. Of note, that N_δ1 (His270) and O_δ1/O_δ2 (Asp306) are within (2.6Å /3.2Å). On the other hand, the orientation of active site histidine places N_ε2 of His270 from the O_ϒ of Ser387. The orientation of H270 (N_ε2) and Ser387 (O_γ) (separated by ca 8.0Å) suggests that this crystal structure represents an inactive conformation. The PDZ domain is linked to the protease domain by a . Based on extensive analysis of E. coli DegS, the L1 and L3 loops are essential for regulation of protease activity whereas the L2 loop governs substrate specificity.^[2] (loops are in green, active site residues are in yellow). These loops connecting helices or strands in protease domain. The movement of L3 loop away from PDZ domain has been shown to shift the equilibrium from the inactive to active state of DegS upon peptide binding to PDZ domain.^[3] In the M. tuberculosis HtrA structure, was noted that the L3 loop is displaced from the PDZ domain.

(M. tuberculosis HtrA active site residues are in yellow). The active site residues from bovine trypsin and proteases belonging to the HtrA family from different species were superposed with M. tuberculosis ΔTM HtrA. Two well characterized HtrA proteases (M. tuberculosis PepD (PDB ID: 2z9i; colored in salmon), E. coli DegS (PDB ID: 1soz; colored in cyan)) provided a basis for this comparison alongside bovine trypsin structures. Among these, one is a complex with phenylmethylsulfonyl fluoride (PMSF) (PDB ID: 1pqa; colored in dodgerblue) providing a reference for a covalently linked ligand to active site Ser-OH. The other representative model for a substrate bound form is the trypsin-peptide complex (AAPK) (PDB ID: 2agg; colored in violet). This structure provides a representation of the oxyanion hole wherein the peptide is bound to the active site Ser-OH providing a structural snapshot of the acyl enzyme intermediate. In both examples, the active site Histidine is flipped with χ¹ of 80.7° and -166.8° in the case of peptide bound (1pqa) or 89.4° and -174.8° in the case of the PMSF complex (2agg).^[4][5] For comparison, the Histidine rotamers with χ¹ of 80.7° and 89.4° represent the canonical catalytic triad alongside the active site Asp and Ser. Of note, that the χ¹ of His270 of M. tuberculosis HtrA is -80.9 leading a distorted catalytic triad. This conformation of the catalytic triad in M. tuberculosis HtrA thus represents either an inactive state or a distorted conformation mimicking Histidine flipping in the acylation step of catalysis.

The crystal structure of Acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb)^[6]

The crystal structure of AcpS from (Mtb) was solved at 1.95 Å (3hqj). It crystallized as one per asymmetric unit. Since Mtb AcpS has biologically active trimeric arrangement, (in green, blue, and (in orange) was constructed using the 3-fold crystallographic symmetry in the P23 space group. The 3′,5′-ADP moieties of the coenzyme A (CoA, colored magenta), are positioned in the cleft between each of two monomers forming three active sites within AcpS trimer. The is formed by the residues D9 (highly conserved), E58, L62, and S65 from monomer A and by R92, P93, R53, H116, and T115 from the neighboring monomer B. The residues labeled and shown as sticks (A and B in the brackets point on the name of the monomer). Hydrogen bonds are shown as dashed lines with interatomic distances in Å. The magnesium (Mg) atoms are shown in spacefill representation and colored in cyan. The CoA is shown in stick representation and colored magenta. Nitrogen and oxygen atoms of the CoA 3′,5′-ADP moiety and of the active site resudues are colored blue and red, respectively.

of the structures of the Mtb AcpS trimer (in green, blue, and orange) and the B. subtilis AcpS trimer (1f7t, in red, cyan, and yellow) reveals that the Mtb AcpS structure is similar to those of other members of group I phosphopantetheine transferase (PPT) family. The is that the extended α3 helix of Mtb AcpS has open conformation. Such open conformation permits to the extended loop of one monomer (green) to interact with adjacent monomer (blue). The considerably shorter α3 of one B. subtilis AcpS monomer (red) has closed conformation and this doesn't allow interaction with the neighboring monomer (cyan).

The B. subtilis AcpS trimer (1f80) three molecules of the acyl carrier protein (ASP). The interactions between B. subtilis AcpS and ACP are predominantly . The B. subtilis AcpS (white) is shown in spacefill representation, the agrinines, lysines, and histidines are colored blue, while aspartates and glutamates are colored red. The ACP molecule (green) is shown in ribbon representation with aspartates and glutamates as sticks and colored red. The B. subtilis AcpS has large with ASP. of Mtb AcpS surface using the similar orientation as B. subtilis AcpS, shows a moderate electronegative nature in the putative ACP binding site near the ASP 15. The Mtb ASPM structure (1klp, corresponding to ACP) demonstrates considerably lower negative charge. So, the electrostatic interactions between Mtb AcpS and ASPM are, probably, less important.