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Structures of the mycobacterial MmpL4 and MmpL5 transporters provide insights into their role in siderophore export and iron acquisition [1]

['Rakesh Maharjan', 'Department Of Pharmacology', 'Case Western Reserve University School Of Medicine', 'Cleveland', 'Ohio', 'United States Of America', 'Zhemin Zhang', 'Philip A. Klenotic', 'William D. Gregor', 'Marios L. Tringides']

Date: 2024-10

The Mycobacterium tuberculosis (Mtb) pathogen, the causative agent of the airborne infection tuberculosis (TB), harbors a number of mycobacterial membrane protein large (MmpL) transporters. These membrane proteins can be separated into 2 distinct subclasses, where they perform important functional roles, and thus, are considered potential drug targets to combat TB. Previously, we reported both X-ray and cryo-EM structures of the MmpL3 transporter, providing high-resolution structural information for this subclass of the MmpL proteins. Currently, there is no structural information available for the subclass associated with MmpL4 and MmpL5, transporters that play a critical role in iron homeostasis of the bacterium. Here, we report cryo-EM structures of the M. smegmatis MmpL4 and MmpL5 transporters to resolutions of 2.95 Å and 3.00 Å, respectively. These structures allow us to propose a plausible pathway for siderophore translocation via these 2 transporters, an essential step for iron acquisition that enables the survival and replication of the mycobacterium.

Thus far, there is no structural information of MmpL transporters that are affiliated with subclass I. To facilitate modeling protein structures of these important membrane proteins, we here present cryo-EM structures of the M. smegmatis MmpL4 and MmpL5 transporters to resolutions of 2.95 Å and 3.00 Å, respectively. We observed that both MmpL4 and MmpL5 form a channel that spans from the outer leaflet of the cytoplasmic transmembrane domain through the periplasmic domain, suggesting a plausible pathway for substrate translocation.

We and others have previously solved high-resolution structures of the MmpL3 transporter from M. smegmatis and M. tuberculosis using either X-ray crystallography or cryo-electron microscopy (cryo-EM) [ 25 – 31 ]. Importantly, we also determined the structures of MmpL3 bound with the lipid TMM [ 26 ]. These structures revealed that MmpL3 consists of at least 2 TMM lipid binding sites that promote the transport of lipids across the cytoplasmic membrane, allowing us to propose a mechanism of TMM translocation for cell wall biosynthesis [ 25 , 26 ].

The phylogenetic tree reveals that MmpL proteins can be separated into 2 distinct subclasses [ 24 ]. The majority of MmpLs, such as MmpL4, MmpL5, MmpL7, MmpL8, and MmpL10, belong to subclass I. It is anticipated that MmpL proteins in this subclass contain only 2 domains: a transmembrane domain and a periplasmic domain [ 24 ]. However, proteins within subclass II of the MmpL subfamily, including MmpL3 and MmpL11, are predicted to possess transmembrane and periplasmic domains with an additional C-terminal cytoplasmic domain [ 24 ] ( S1 Fig ).

In addition to exporting important lipid elements for cell wall biogenesis, select MmpL transporters have been reported to participate in the active efflux of anti-TB drugs. For example, up-regulation of MmpL5 confers increased resistance to bedaquiline [ 21 ], an FDA approved anti-TB drug, and MmpL7 contributes to isoniazid and ethionamide resistance when overexpressed [ 22 ]. Currently, MmpL3 is considered an important pharmacologic target for anti-TB drug discovery [ 23 ], making it a high priority to elucidate the molecular mechanisms of this transporter.

The genome of Mtb encodes 13 mycobacterial membrane protein large (MmpL) transporters [ 6 ]. These membrane proteins belong to a subfamily of the resistance-nodulation cell division (RND) superfamily of transporters [ 7 ] and are critical for mycobacterial physiology and pathogenesis, with several of these transporters involved in the export of fatty acids and lipid components to the cell envelope. MmpL3 is absolutely essential and transports TMM to the mycobacterial surface [ 8 – 10 ]. MmpL11 exports very long chain triacylglycerols (LC-TAG) and mycolate wax ester (MWE) [ 11 , 12 ]. MmpL4 and MmpL5 share similar functions in siderophore export and are required for iron acquisition [ 13 ]. MmpL7 and MmpL8 contribute to the transport of virulence-associated lipids PDIM and SL-1 [ 14 , 15 ], respectively, whereas MmpL10 is involved in the translocation of diacyltrehalose to the cell envelope [ 16 ]. Besides the essential gene mmpL3 in Mtb, it has been shown that disruption of mmpL4, mmpL5, mmpL7, mmpL8, mmpL10, and mmpL11 lead to significant virulence attenuation in a mouse model [ 8 , 17 – 20 ].

The complex structure of the Mtb cell envelope plays a dominant role in the pathogenesis of the bacterium. The outer mycomembrane is very rigid and extremely impermeable to a wide range of compounds, including many antimicrobials [ 5 ]. It also provides a strong barrier against the host immune response. The outer mycomembrane is defined by an abundance of long chain mycolic acids (MAs). MAs are exported across the inner membrane of Mtb as trehalose monomycolates (TMMs) and then either covalently linked to the arabinogalactan-peptidoglycan layer as mycolyl arabinogalactan peptidoglycans (mAGPs) or incorporated into trehalose dimycolates (TDMs), which constitute the majority of the outer leaflet of this membrane. The outer leaflet also contains other non-covalently associated lipids, such as phthiocerol dimycocerosates (PDIMs) and sulfolipids (SLs) [ 5 ].

Mycobacterium tuberculosis (Mtb), the causative agent of the airborne infection tuberculosis (TB), is one of the deadliest human pathogens, exceeding both malaria and HIV [ 1 , 2 ]. Approximately 1/3 of the world’s population is infected by Mtb with most having the latent form of disease, yet approximately 10% progress to active TB [ 2 ]. The emergence and spread of multidrug-resistant (MDR)-TB compounds an increasingly difficult therapeutic challenge and severely threatens global public health. Unfortunately, TB is synergetic to both HIV [ 3 ] and COVID-19 [ 4 ] and can further escalate the severity of these infections.

Results and discussion

Structure of the M. smegmatis MmpL4 transporter We were particularly interested in determining the structures of the MmpL4 and MmpL5 transporters, as they have been shown to be critical in the iron acquisition pathway of the mycobacterium [32]. Mycobacteria secrete mycobactin (Mbt) and carboxymycobactin (Cmb), siderophores with an extremely high affinity for Fe(III), via the MmpL4 and MmpL5 transporters to recruit this important metal that is essential for growth and survival [13]. We cloned the M. smegmatis full-length MmpL4 transporter (969 amino acids) into the Escherichia coli expression vector pET15b with a 6xHis tag at the C-terminus. This MmpL4 membrane protein was overproduced in E. coli BL21(DE3)ΔacrB cells and purified using Ni2+-affinity and Superdex 200 size exclusion columns. We then collected single-particle cryo-EM images of the MmpL4 transporter embedded in LMNG detergent micelles and solved its structure to a resolution of 2.95 Å (Figs 1 and S2 and S1 Table). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Structure of the M. smegmatis MmpL4 transporter. (A) Secondary structural topology of MmpL4. The topology was constructed based on the cryo-EM structure of MmpL4. The transmembrane helices (TMs) are colored pale green. The secondary structural elements of the periplasmic subdomains PD1 and PD2 are colored yellow and green, respectively. The missing subdomain PD3 is represented by red dotted lines. (B) Cryo-EM map of M. smegmatis MmpL4 at a resolution of 2.95 Å. (C) Ribbon diagram of the structure of MmpL4 viewed in the membrane plane. The secondary structural elements of the TM domain, PD1 subdomain, and PD2 subdomain are colored pale green, yellow, and green, respectively. The missing PD3 subdomain is represented by a red dotted curve. The PDB and EMDB accession codes of the structure of MmpL4 are 9B43 and EMD-44167, respectively. cryo-EM, cryo-electron microscopy; MmpL, mycobacterial membrane protein large. https://doi.org/10.1371/journal.pbio.3002874.g001 Our cryo-EM structure indicates that MmpL4 is monomeric in form with overall dimensions of 110 Å × 75 Å × 55 Å. This transporter consists of a large membrane-spanning domain formed by 12 transmembrane helices (TMs 1–12) and a large periplasmic domain formed by 2 hydrophilic loops (loops 1 and 2), consistent with the signature of the RND superfamily of proteins [7] (Fig 1A and 1B). Periplasmic loop 1 is located between TMs 1 and 2, whereas loop 2 is situated between TMs 7 and 8. These 2 loops coordinate and contribute to create the periplasmic subdomains PD1 and PD2. It should be noted that our cryo-EM structure contains most MmpL4 residues except for residues 495–689, which presumably form the PD3 subdomain located right above subdomain PD2. Interestingly, there appears to be a resemblance in the overall structure of MmpL4 to that of MmpL3, although MmpL3 and MmpL4 only share 22% protein sequence identity. These 2 MmpL transporters are superimposable; however, superimposition of the TM domain, and PD1 and PD2 subdomains of the structure of MmpL4 to those of MmpL3 (PDB ID: 7K8B) [26] gives rise to a very high root mean square deviation (RMSD) of 5.9 Å (for 673 Cα atoms), indicating the structures of MmpL3 and MmpL4 are very distinct from each other (S3 Fig). The N-terminal and C-terminal halves of MmpL4 are structurally related to each other by a pseudo 2-fold symmetry, but these 2 halves share a low protein sequence identity of only 16%. These 2 halves can be superimposed, but the RMSD is quite high for this superimposition (2.6 Å for 294 Cα atoms) (S3 Fig). It should be noted that a similar high RMSD of 2.6 Å has also been found when the 2 halves of the MmpL3 transporter are superimposed [25]. PD1 consists of 4 α-helices (α3, α4, α5, and α7), 4 β-strands (β1, β2, β3, and β4), and the first 10 N-terminal residues of TM2. The majority of the PD1 amino acids originate from loop 1. However, helix α7 (428–437) of loop 2 also participates in forming this subdomain. PD2 is composed of 5 α-helices (α2, α9, α10, α11, and α12), 4 β-strands (β5, β6, β7, and β8), and the first 10 N-terminal residues of TM8. The PD2 amino acids mainly originate from loop 2, but helix α2 (residues 72–83) of loop 1 also contributes to this periplasmic subdomain. The crossover of these 2 loops allows the 2 periplasmic subdomains to connect with each other in the periplasm. Notably, the periplasmic domains of MmpL4 are presumed to be flexible in nature as evidenced by the existence of long linkers. Residues 52–71 are predominantly unstructured and create a long linker connecting the C-terminus of TM1 of the transmembrane domain and the N-terminus of helix α2 of the PD2 periplasmic subdomain, although residues 55–61 form helix α1. Likewise, residues 413–427, which also include helix α6 (419–422), form a linker bridging the C-terminal end of TM7b and the N-terminal end of helix α7 of PD1 together. The TMs of MmpL4 are membrane embedded, but both TM2 and TM8 are significantly longer and protrude into the periplasmic region to participate in creating PD1 and PD2, respectively, similar to what is observed in the structures of MmpL3 [25–31]. These 2 TMs directly tether the 2 periplasmic subdomains and form part of the periplasmic structure of the protein. It should be noted that residues 495–689 are missing in our MmpL4 structure. Based on the structural information, these residues should form an independent periplasmic subdomain PD3, which may be critical for interacting with other periplasmic accessory proteins, such as mycobacterial membrane protein small 4 (MmpS4) and other periplasmic adaptor proteins including Rv0455c [33]. It should be noted that our MmpL4 protein is a full-length protein as indicated by size exclusion chromatography (SEC) and SDS-PAGE of the purified protein (S4 Fig). It is likely that subdomain PD3 is highly flexible, evidenced by the fact that we did not observe any cryo-EM densities arising from this subdomain even though the contour level of the map was set to a very low threshold (S4 Fig).

Structure of the M. smegmatis MmpL5 transporter MmpL5 is homologous to MmpL4. These 2 membrane proteins share 65% protein sequence identity, perform similar functions in siderophore export and are required for iron acquisition [13,34]. However, only MmpL5 appears to confer increased resistance to bedaquiline [21], an FDA approved anti-TB drug. As the structure of MmpL4 is missing the periplasmic subdomain PD3 of approximately 200 residues, we thought that the structure of MmpL5 could provide the structural information of this missing subdomain. We therefore used a similar approach to clone the full-length M. smegmatis MmpL5 transporter into the vector pET15b with a 6xHis tag at the C-terminus to generate pET15bΩmmpL5. This MmpL5 membrane protein was overproduced in E. coli BL21(DE3)ΔacrB cells and purified using Ni2+-affinity and Superdex 200 size exclusion columns. The cryo-EM structure of MmpL5 embedded in LMNG detergent micelles was solved to a nominal resolution of 3.00 Å (Figs 2 and S5 and S1 Table). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Structure of the1 M. smegmatis MmpL5 transporter. (A) Secondary structural topology of MmpL5. The topology was constructed based on the cryo-EM structure of MmpL5. The transmembrane helices (TMs) are colored pink. The secondary structural elements of the periplasmic subdomains PD1 and PD2 are colored brown and yellow, respectively. The missing subdomain PD3 is represented by blue dotted lines. (B) Cryo-EM map of M. smegmatis MmpL5 at a resolution of 3.00 Å. (C) Ribbon diagram of the structure of MmpL5 viewed in the membrane plane. The secondary structural elements of the TM domain, PD1 subdomain, and PD2 subdomain are colored pink, brown, and yellow, respectively. The missing PD3 subdomain is represented by a blue dotted curve. The PDB and EMDB accession codes of the structure of MmpL5 are 9B46 and EMD-44171, respectively. cryo-EM, cryo-electron microscopy; MmpL, mycobacterial membrane protein large. https://doi.org/10.1371/journal.pbio.3002874.g002 The cryo-EM structure of MmpL5 is nearly identical to that of MmpL4. Superimposition of these 2 structures gives rise to an RMSD of 0.9 Å (for 681 Cα atoms). Like MmpL4, TM1-TM12 of the transmembrane domain and PD1-PD2 of the periplasmic domain are clearly defined in the cryo-EM map. However, cryo-EM densities for the majority of residues making up the periplasmic subdomain PD3 are missing, similar to what was seen with MmpL4. This result adds additional evidence that PD3 is highly flexible, which may allow this subdomain to have a higher degree of freedom for finding binding partners in the periplasm. PD1 is composed of 4 α-helices and 4 β-strands, including α3, α4, α5, α7, β1, β2, β3, and β4. The majority of these structural elements belong to periplasmic loop 1 of MmpL5, but helix α7 of loop 2 also contributes to form this subdomain. PD2 contains 5 α-helices and 4 β-strands, which include α2, α9, α10, α11, α12, β5, β6, β7, and β8. Most of these structural elements, except helix α2, arise from loop 2 of the protein sequence. Like MmpL4, the periplasmic domain of MmpL5 possesses several linkers to tether these 2 periplasmic subdomains together as well as attach them to the transmembrane domain. These findings indeed highlight the flexible nature of the MmpL5 transporter.

The MmpL4 and MmpL5 channels Within the structure of MmpL3, a channel-like cavity spanning the outer leaflet of the inner membrane up through the periplasmic domain was observed. Interestingly, it was previously found that MmpL3 possesses 2 TMM lipid-binding sites [35]. The first bound TMM molecule was located within the pocket at the outer leaflet of the cytoplasmic membrane. This binding pocket, which forms the beginning of the elongated channel, is created by TMs 7–10 of the transmembrane region of MmpL3. The second bound TMM lipid was found to sandwich in a cavity generated between subdomains PD1 and PD2 of the periplasmic domain of MmpL3. This cavity is situated within the passageway of the MmpL3 channel. Coupled together, the structural data and computational simulations have led to a proposed mechanism for substrate translocation, where the MmpL3 transporter exports TMM lipids from the outer leaflet of the transmembrane up through the periplasm [35]. We used the program CAVER (https//www.caver.cz/) to identify channel formation in MmpL4. Similar to MmpL3, the structure of the MmpL4 transporter also forms an elongated channel spanning the pocket surrounded by TMs 7–10 at the outer leaflet of the cytoplasmic membrane and the cavity generated between subdomains PD1 and PD2 in the periplasm (Fig 3A). The pocket located at the transmembrane domain and the cavity sandwiched between the 2 periplasmic subdomains potentially harbor substrate binding sites for anchoring siderophore substrates such as Mbts. This elongated channel is continuously open and directly connects these 2 potential siderophore-binding sites. Therefore, this conformation of MmpL4 may represent the open-channel state of this transporter, and this channel is likely used to export siderophore substrates. Additionally, both the pocket surrounded by TMs 7–10 and the cavity between PD1 and PD2 can bind Mbt in the micromolar range based on docking calculations (see below). It should be noted that we also observed a channel with an opening orchestrated by TMs 1–4 of MmpL4 (S6 Fig). However, this pocket is not capable of binding Mbt based on docking calculations (see below). Therefore, it is unlikely that the pocket surrounded by TMs 1–4 can form an entrance for siderophore transport. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Structural comparison of the MmpL4 and MmpL5 transporters. (A) Channel formation in each MmpL transporter. The MmpL4 protomer (green) forms a channel (orange) spanning the outer leaflet of the inner membrane up through the periplasmic domain. Residues Y417, Y767, Q771, and W835 (magenta sticks) create the narrowest region of this MmpL4 channel. Similarly, the MmpL5 protomer (pink) forms a channel (orange) spanning the outer leaflet of the inner membrane up through the periplasmic domain. Residues Y417, Y767, Q771, and W835 (cyan sticks) create the narrowest region of this MmpL5 channel. (B) Superimposition of the structures of the MmpL4 and MmpL5 transporters. This superimposition indicates that the major conformational difference between MmpL4 and MmpL5 is the location of TM8. It appears that the section of TM8 located at the outermost surface of the cytoplasmic membrane of MmpL5 shifts into the core by as much as 3.5 Å in relation to the position of TM8 in MmpL4. This rigid-body shift may help control the opening and closing of both channels formed within the MmpL4 and MmpL5 transporters. MmpL, mycobacterial membrane protein large. https://doi.org/10.1371/journal.pbio.3002874.g003 We used the same approach, utilizing the program CAVER (https//www.caver.cz/), to search for channel formation in the MmpL5 transporter. Distinct from MmpL4, the structure of MmpL5 indicates that this transporter only possesses one channel that spans the outer leaflet of the transmembrane domain up through the periplasmic domain (Fig 3A). However, the midway point of this channel is much narrower, suggesting that the structure of MmpL5 may depict a different intermediate state with respect to that of MmpL4. In comparing the structures of MmpL4 and MmpL5, it can be interpreted that TM8 is critically important for the opening and closing of the transporter’s channel (Fig 3B). In MmpL5, the section of TM8 located at the outermost surface of the cytoplasmic membrane shifts into the core of the membrane protein by as much as 3.5 Å in relation to the position of TM8 in MmpL4 (Fig 3B). This rigid-body shift may help control the opening and closing of the channels formed within the MmpL4 and MmpL5 transporters. In MmpL5, the narrowest region of the channel is surrounded by residues Y417, Y767, Q771, and W835, residues that may be important for controlling the width of the MmpL5 channel (Fig 3A). The corresponding 4 residues in MmpL4 (Y417, Y767, Q771, and W835) also create the narrowest region of its channel (Fig 3A). Therefore, in both instances, these residues appear critical for controlling the opening and closing of the channel, thus regulating substrate export.

The MmpL4 and MmpL5 proton-relay networks According to the protein sequence alignment with MmpL3, it is speculated that both MmpL4 and MmpL5 are proton motive force (PMF)-dependent transporters that function via a proton/substrate antiport mechanism. Within the transmembrane domain of MmpL3, the conserved residues D256, Y257, D645, and Y646 are involved in forming the proton-relay network for energy coupling [25,27] (S7 Fig). Interestingly, these proton-relay residues are conserved between MmpL3 and MmpL4. The corresponding residues in MmpL4 are D278, Y279, D851, and Y852 (S7 Fig). Based on the structural information of MmpL4, residue D278 forms a hydrogen bond with Y852, presumably enabling the transfer of protons to facilitate substrate transport. Within the transmembrane domain of the MmpL5 transporter, the conserved residues that make up the proton-relay network are also residues D278, Y279, D851, and Y852 (S7 Fig). Similar to MmpL4, residue D278 and Y852 of MmpL5 interact with each other and create a hydrogen bond, presumably relaying protons for energy coupling.

Docking of mycobactin onto MmpL4 and MmpL5 MmpL3 binds TMM in the pocket formed by TM7-10 and the cavity located between PD1 and PD2. We observed that MmpL4 also possesses a similar pocket and cavity that potentially generate 2 siderophore-binding sites. Several hydrophobic and polar residues, including L407, L410, Y413, F334, A774, N775, L778, I779, V811, I812, G815, F818, W835, L838, A839, and V842, surround the pocket formed by TM7-10 of the MmpL4 transporter. Many of these residues belong to TM8, TM9, and TM10, and these lining residues may constitute a binding site for siderophores. Interestingly, it appears that TM8 and TM9 coordinate to form a V-shape feature to create part of this pocket. At the periplasmic domain of MmpL4, a mixture of hydrophobic, polar, and charged residues line the wall of the periplasmic central cavity sandwiched between PD1 and PD2. These residues include M66, M77, N90, S92, Q159, P191, L194, S195, Q198, F263, Y333, N418, D419, R420, Y433, A443, N446, P447, D727, P728, M729, T763, T766, F767, H834, and M836 and may constitute a second siderophore-binding site. In view of this central cavity, residues of the 2 extended loops that run across subdomains PD1 and PD2 (residues 83–92 and residues 437–448) form the front and back sides of this cavity in relation to the orientation of Fig 1. Residues 61–72 of the loop connecting TM1 and PD2, and residues 416–426 of the elongated loop connecting TM7 and PD1 form the bottom of this large cavity. Additionally, the periplasmic loop of PD1 (residues 155–162), the periplasmic loop of PD2 (residues 724–731), the N-terminal end of TM2 (residues 191–195), and the N-terminal end of TM7 (residues 416–426) surround different sides to secure the formation of this cavity. It should be noted that TM8 participates in forming both the pocket at the membrane region and the periplasmic central cavity. Therefore, TM8 may be required for substrate recognition and possibly substrate transport. To predict how MmpL4 recognizes and contacts siderophore substrates, we used AutoDock Vina [36] to computationally dock the mycobactin (Mbt) ligand onto MmpL4. The docking predictions suggest that MmpL4 forms 2 siderophore-binding sites. The locations of these bound Mbt compounds overlap with the pocket and cavity constituted within the transmembrane and periplasmic regions of the transporter (Fig 4A). The predicted Mbt binding affinities are −7.1 and −5.4 kcal/mol for the transmembrane and periplasmic binding sites of MmpL4, respectively. These binding affinities roughly correspond to dissociation constants (K D s) of 6.1 and 92 μm. These values are in the same range as those found in MmpL3, where MmpL3 binds TMM and phosphatidylethanolamine (PE) lipids with measured K D s of 3.7 and 19.5 μm, respectively [25]. Interestingly, these binding strengths are also comparable with those of many other RND-type multidrug efflux pumps, where these pumps bind toxic compounds and antibiotics within the micromolar range [37–40]. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Docking of Mbt and targeted MD simulation. (A) The predicted Mbt binding sites of MmpL4. The docked Mbt molecules are shown as yellow sticks (at the binding site located in the outer leaflet of the cytoplasmic membrane) and magenta sticks (at the binding site located in periplasmic domain). Residues predicted to be responsible for Mbt binding at both binding sites are in cyan sticks. (B) The predicted Mbt binding sites of MmpL5. The docked Mbt molecules are shown as yellow sticks (at the binding site located in the outer leaflet of the cytoplasmic membrane) and green sticks (at the binding site located in periplasmic domain). Residues predicted to be responsible for Mbt binding at both binding sites are shown as cyan sticks. (C) Targeted MD simulations showing different time points for MmpL4–Mbt interactions (0 ns to 1.2 ns). (D) Targeted MD simulations showing different time points for MmpL5-Mbt interactions (0 ns to 3.0 ns). Mbt, mycobactin; MD, molecular dynamics; MmpL, mycobacterial membrane protein large. https://doi.org/10.1371/journal.pbio.3002874.g004 We also analyzed how MmpL5 potentially interacts with Mbt. The transmembrane binding pocket of MmpL5 consists of several residues, including Y413, Y414, T415, T416, Y417, L729, T730, E731, T734, Y767, Q771, A774, D775, L838, and P839. Again, many of these residues originate from TM8, TM9, and TM10. The periplasmic central binding cavity of MmpL5 contains various residues, such as A67, T74, I77, S90, S91, S92, M94, Q129, Q152, Y154, Q159, T194, D418, R420, Y433, M445, N446, L449, R719, T721, T766, and Y767, which are hydrophobic, polar, or charged in nature (Fig 4B). This cavity is surrounded by several loops as well as the N-terminal residues of TM2 and the N-terminal residues of TM8, similar to what is observed in the MmpL4 periplasmic cavity. Autodock Vina suggests that MmpL5 binds Mbt in the transmembrane pocket and periplasmic cavity with affinities of −7.9 and −8.0 kcal/mol, which correspond to K D s of 1.6 and 1.3 μm, respectively. Again, these strengths are in the same range as those found in MmpL3–lipid interactions [25]. We also used Vina [36] to predict potential substrate binding sites for carboxymycobactin (Cmb) and bedaquiline (Bed) in the MmpL4 and MmpL5 transporters. For MmpL4, the results indicate that MmpL4 possesses 1 Cmb binding site with a predicted binding affinity of −7.0 kcal/mol. This site is located at the outer leaflet of the transmembrane domain and is surrounded by TMs 7–10 (S8A Fig). MmpL4 also possesses 1 Bed binding site with a predicted binding affinity of −7.1 kcal/mol, although increased Bed resistance due to MmpL4 has not been reported. This site coincides with that of the Cmb binding site (S8B Fig). In the case of MmpL5, this transporter appears to contain 2 Cmb binding sites and the locations of these 2 sites are similar to those found for the Mbt binding sites, at the outer leaflet of the transmembrane domain and at the periplasmic domain. The predicted binding affinities for these 2 sites are −6.9 kcal/mol and −7.4 kcal/mol, respectively (S8C Fig). However, MmpL5 only contains 1 Bed binding site with a predicted binding affinity of −8.4 kcal/mol. This site is located at the outer leaflet of the transmembrane domain and is surrounded by TMs 7–10 (S8D Fig).

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