The full length MmpL3 from Mycobacterium smegmatis was expressed with a T4 lysozyme for protein purification and structural analysis methods. The protein was crystallized using the vapor diffusion hanging drop method under the conditions of 10%-20% (v/v) polyethylene glycol monomethyl ether 350 (PEGMME 350), 50 mM ADA (N-(2-Acetamido) iminodiacetic acid) (pH 6.0-7.0) and 7.5%-17.5% (v/v) polyethylene glycol 2000 (PEG2000) at 289K (4). The X-ray crystallization process resulted in an MmpL3 product with an unstable cytoplasmic C terminal domain, making it highly susceptible to degradation. By expressing, purifying, and crystallizing a recombinant MmpL3 structure using the first 748 residues with T4 lysozyme fused to its C terminus, a new crystal structure was produced at 2.7 angstroms resolution (1). The complete structure of MmpL3 was determined with X-ray diffraction (4).

Expasy determined that the MmpL3 protein has an isoelectric point of 6.40 and a molecular weight of 103894.87 Da (5). The asymmetric monomeric protein exists on mutant form (1). The MmpL3 protein has one main subunit  but can trimerically be divided into three domains: the periplasmic “porter” domain, the transmembrane proton translocation domain and the C-terminal cytoplasmic domain. The space between the transmembrane region in MmpL3 is  large enough for the protein to capture the TMM substrate from the lipid bilayer and cytoplasm in order to lead it into the vestibule. The periplasmic pore is made up of 2 subdomains: PN and PC that twist together to form a central cavity that connects to three openings: a top funnel (Gt), front opening (Gf) and back opening (Gb) of the headpiece (1). Residues Lys-34 through Lys-173 (PN) and Lys-417 through Ser-551 (PC) make up the porter domain (2). The periplasmic PN porter domain interacts on the surface with the adjacent subunit promoter in order to bind the TMM substrate.

Surface plasmon resonance and co-immunoprecipitation analysis displayed that MmpL3 is localized at the poles and septum of growing bacilli where synthesis of cell wall constituents occurs (6). From the poles, MmpL3 depends on the proton motive force dependent transporter that functions by way of the antiporter route through the cytoplasm (1). The mechanism by which MmpL3 moves TMM across the membrane into the cell wall remains unknown (3).

The MmpL3 molecule was produced by an internal gene duplication which combined two halves of a periplasmic headpiece of the molecule (N-/C- terminal pore subdomain) and a transmembrane domain (N-/C- terminal transmembrane subdomain) (1).  The secondary structure consists of 60% helical (43 helices; 570 residues), 6% beta sheet (21 strands; 61 residues). MmpL3 contains 943 residues (4). Within the tertiary structure of MmpL3, both the PN and PC domains are alpha helical, interacting with hydrophobic residues that cause conformational changes in the protein. MmpL3 only has a single subunit, therefore it does not form a quaternary structure (1).

The functionally important residues in MmpL3 are located in the central region of the transmembrane segments, the interface between the transmembrane, the porter domains vicinal to the pore structure, or the interface between the PN and PC loops of the porter domain that includes the channel feature. Hydrophilic residues Asp-58, Arg-63, Asp-144 hydrogen bond with and stabilize the trehalose head of the 6-DDTre near Gf (1). Hydrophilic residues, Asp-256 to Tyr-646 and Asp-645 to Tyr-257 reside in the middle of the TMH IV and TMH X helices that form the core bundle. The residues link the two alpha helices based on side chain interactions that form hydrogen bonds. Hydrophobic residues Asp-64, Tyr-44, Ser-54, and Leu-55 line the vestibule interior of the protein, enhancing the affinity for the TMM substrate (1). MmpL3 has two associated ligands. Carbamoylmethyl-carboxymethyl-amino-acetic Acid is a chelator and bacterial xenobiotic metabolite that helps MmpL3 move TMM esters across the membrane without altering their chemical structure (7).Alpha-D-glucopyranosyl 6-O-dodecyl-alpha-D-glucopyranoside is a non-ionic detergent solubilizes MmpL3, allowing for its movement through the cytoplasm and outer membrane (8).

There are currently four anti-tuberculosis drugs that specifically target the MmpL3 protein: Rimonabant, SQ109, AU1235, and ICA38. Rimonabant, a cannabinoid (CB1) receptor antagonist, and SQ109, a diamine, both target and inhibit MmpL3. Microscale thermophoresis assays revealed SQ109 binds MmpL3 in the TM domain and disrupts Asp and Try pairs. Rimonabant disrupts the residues: Gly-641,Leu642, and Ile-253. SQ109 forces conformational changes in the C terminal transmembrane helices of MmpL3. The conformational changes form a pocket with a volume of 282 Å in the proton flow passage of MmpL3. SQ109 inhibits MmpL3 by disrupting the proton relaying Asp-Tyr pairs and occupying the proton transportation channel in the protein. This inhibiting effect blocks the proton motive force for MmpL3 substrate translocation (1).

AU1235, an adamantly urea compound, also targets MmpL3. The compound binds in the TM domain of MmpL3  and disrupts the Asp-Tyr pairs. There is a bulky trifluorophenyl group in the AU1235 compound that produces more hydrophobic interactions upon binding to the MmpL3 protein than does the geranyl tail of SQ109 (1).

Among the anti-TB drugs, ICA, an indolcarboxamide provides the most in vitro anti-Mtb activity, but also has the poorest bioavailability. ICA has derivatives, NITD-304 and NITD-349 (novartis), which are better candidates for resisting TB. ICA38 has a bulky indole group that fits into the hydrophobic subsite of MmpL3 on top of the channel. This forms a stronger hydrophobic interaction than the geranyl tail of SQ109 and fluorophenyl group of AU1235. ICA38 has an amide group which hydrogen bonds to the side chain of Asp-645 in MmpL3. The carbonyl oxygen of ICA38 also hydrogen bonds with the side chain of Tyr646 in MmpL3. The carbocyclic spiro group in ICA38 locks itself into the bulky hydrophobic subsite at the bottom of the tunnel. ICA38 produces a greater set of hydrophobic interactions that oppose the TMM substrate in MmpL3 than does the adamantine group in SQ109 and AU1235 (1).

         The Blast Local Alignment Search Tool (BLAST) compares proteins with similar protein structures to a protein query. BLAST calculates the gaps between sequence homologies and produces an E value for each sequence comparison. The lower the E value, the more homologous protein sequences are to one another. An E value lower than 0.05 deems the analysis of the proteins as significant (9). The Dali server also compares the tertiary structures of proteins and uses the sums-of-pairs method to determine intramolecular distances in protein. The server assigns a Z score to proteins where a score above 2 indicates structural similarities (10). The crystal structure of the dynein motor domain in the AMPPNP-bound state from: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (PBD ID:4W8F) was compared with MmpL3 and assigned a Z Score of 24.1 and an E value of 2e-99, indicating a high similarity in structures and sequence homology (9,10,11).

The crystal structure of the dynein motor domain in the AMPPNP-bound state from: Saccharomyces cerevisiae (Dynein) has a sequence length longer than MmpL3 at 5322 residues. Dynein contains more alpha and beta sheet content than MmpL3, with an A chain consisting of  55% helical (125 helices; 1474 residues) and 10% beta sheet (63 strands; 268 residues) and a B chain consisting of 56% helical (126 helices; 1492 residues) and 9% beta sheet (64 strands; 262 residues) (11). AAA1 and AAA3 are ATPase domains that power microtubule motility in the dynein protein. ATP binds to AAA1, triggering a number of conformational changes that affects six AAA domains. This cascade of conformational changes causes movement of the “linker,” dynein’s motor domain (12).

Both MmpL3 and the dynein motor domain function to translocate necessary protons and polypeptides for growth in a biological structure. The AAA1 and AAA3 sites in dynein are similar to the PC and PN domains in MmpL3.  Both domains are asymmetric sites that trigger conformational changes in order to abolish the main function of the protein. Dynein uses a cascade of changes to transfer energy into molecules whereas MmpL3 uses antiporters to move protons through a pathway. Dynein has six distinct AAA domains centralized on a single polypeptide just as MmpL3 has the PC and PN domains localized on one chain. Dynein has an associated magnesium ligand which plays a role in its stability of the protein whereas MmpL3 has a cofactor protein, TtfA, that attaches and provides it with stability (1,13). The dynein protein contains a pseudo-two fold symmetry axis where the linker-faces of the AAA rings oppose one another (12). The transmembrane region of MmpL3 also contains a pseudo-two-fold symmetry axis that associates 6 N terminal helices to 6 C terminal helices (1). 

Overall, the biological significance of the transmembrane protein MmpL3 is found in its transport of mycolic acids in the form of trehalose monomycolate from the cytoplasm to the outer membrane or periplasm in order to promote TB resistance through hydrophobic interactions on the membrane. MmpL3 is the only mutant protein that cannot be knocked out by Mtb, making it a point of contention regarding resistance to tuberculosis (1). The ability of many potential therapeutics to block the linkages and transport of TMM to the cell wall through MmpL3 makes the membrane protein a critical TB drug target (3). MmpL3 translocates mycolic acids to the outer membrane at the pole and septum of viable growing cells, indicating that there may also be an association of MmpL3 with protein scaffolding of new mycolic acids and possibly various other cell envelope constituents attributed to cell elongation and division (6).