Active Site

Figure 1. Topology and architecture of B. subtilis MraY protein. The N- and C-termini of the structure appear on the periplasmic side. Each protomer consists of 10 transmembrane segments, 4 periplasmic loops, and the 5 cytoplasmic loops that are labelled I –V. The conserved residues are represented by circles and squares for residues that share similar characteristics. (Al-Dabbagh et al.)

The conserved residues identified through mutagenesis

The importance of the residues in the catalytic process were assessed by site-directed mutagenesis, performed on Bacillus subtilis MraY to gain functional insights. The results show that 19 charged and/or polar residues exposed toward the cytoplasm, located in the five cytoplasmic segments of MraY are invariants in the sequences of MraY orthologous. Some of these residues are even conserved throughout the superfamily. The highest degrees of conservation are identified around the cleft of TM3, TM4, TM8 and TM9b. 14 of those residues were thought to be essential for the MraY activity by in vivo functional complementation assay and in vitro enzymatic assay.

Figure 2. 90 degrees rotation of MraY translocase representing the view from the cytoplasmic side of the membrane. Each helix of one of the molecules in the dimer is colour coded and the dimensions of the structure is present. At the interface between the two dimers there is an oval-shaped tunnel surrounded by hydrophobic residues, which is large enough to accommodate lipids, suggesting this has a role in the overall mechanism.

TM9 is composed of two helical fragments, TM9a and TM9b. TM9b is tilted in a 50° angle relative to the other transmembrane segments, protruding 20 Å into the lipid bilayer. This orientation is stabilised by interaction with TM5. A cleft can also be seen in the region of TM8 due to the surrounding of TM5 by TM3, TM4, TM8 and TM9b. This cleft is significant in the function of the protein, suggesting this region serves as the active site.

The significant residues that contribute to the active site 

There are three aspartate residues in the protein sequence that are conserved in the entire PNPT family. They are located at position 117, 118 and 265. In addition, His³²⁴ and His³²⁵ are also observed to be invariant in the MraY family based on the results of mutational mapping. However, theses residues are not involved in keeping the putative active site in shape, this suggests they must be catalytically active residues. Further investigation on these 5 residues by individual mutation found that a complete loss of activity of MraY upon mutating Asp¹¹⁷, Asp¹¹⁸, Asp²⁶⁵ and His³²⁴. Thus, we can conclude the importance of theses 4 residues in contributing to the catalysis of MraY

The role of Mg²⁺  ion hypothesis

Mg²⁺ ion is absolutely essential for the activity of the protein. However, the involvement of the aspartate residues binding with the metal ion has always been an assumption. This hypothesis is based on the sequence similarity to the Mg²⁺ binding motif DDXD/N of the farnesyl diphosphate synthetases where the chelation of Mg²⁺ is syntheisised by a pair of Asp residues. Another study has replaced Mg²⁺ with Mn²⁺ to study the scattering pattern of the substituted MraY_AA crystals. The Fourier density maps display peaks at Asp²⁶⁵ when Mn²⁺ is soaked or co-crystallised with the protein. The unassigned density peaks are probably signals from the coordination of Mg²⁺ with Asp residues.

Figure 3. Electron density map of loop E and TM9b determined by the phase-extended crystallographic method. The two different angles of the protruding TM9b are shown and the position of the Nickel ion. (Chung et al., 2013)

Figure 4. TM9b and loop E are shown in sausage representation with the thicker magenta region more conserved and the thinner cyan region more varieties.(Chung et al., 2013)

TM9b unique structure and its relationship with the active site

TM9b has another role in shaping the active site. Its unique protruding structure together with the loop connecting TM9b to TM10 makes the active site cleft deeper for substrates. TM9b and the loop form the HHH motif that is highly conserved with polar amino acid residues localizing and pointing toward the Mg²⁺ active site. This is physiologically relevant as the substrate UDP-MurNAx-pentapeptide is hydrophilic.

Figure 5. Image on the left is a zoomed-in view of TM9b and loop E (HHH motif). The conserved amino acids are showed as sticks, nickel ion and magnesium ion coloured in yellow and green respectively. Graph on the right is taken from (Chung et al., 2013) which shows the results of TLC-based translocase assay. The level of activity varies with different divalent ions and their concentrations.