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Structures of the mycobacterial membrane protein MmpL3 reveal its mechanism of lipid transport

['Chih-Chia Su', 'Department Of Pharmacology', 'Case Western Reserve University School Of Medicine', 'Cleveland', 'Ohio', 'United States Of America', 'Philip A. Klenotic', 'Meng Cui', 'Department Of Pharmaceutical Sciences', 'Northeastern University School Of Pharmacy']

Date: 2021-08

The mycobacterial membrane protein large 3 (MmpL3) transporter is essential and required for shuttling the lipid trehalose monomycolate (TMM), a precursor of mycolic acid (MA)-containing trehalose dimycolate (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), in Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium smegmatis. However, the mechanism that MmpL3 uses to facilitate the transport of fatty acids and lipidic elements to the mycobacterial cell wall remains elusive. Here, we report 7 structures of the M. smegmatis MmpL3 transporter in its unbound state and in complex with trehalose 6-decanoate (T6D) or TMM using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. Combined with calculated results from molecular dynamics (MD) and target MD simulations, we reveal a lipid transport mechanism that involves a coupled movement of the periplasmic domain and transmembrane helices of the MmpL3 transporter that facilitates the shuttling of lipids to the mycobacterial cell wall.

Funding: This work was funded by the National Institues of Health R01AI145069 (to EWY) ( https://taggs.hhs.gov/Detail/AwardDetail?arg_AwardNum=R01AI145069&arg_ProgOfficeCode=104 ), in part, supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E. A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All relevant data are within the paper and its Supporting Information files. Atomic coordinates and structure factors have been deposited with accession codes 7K8A (PDB) and EMD-22724 (EMBD) for MmpL3-ND; 7K8B (PDB) and EMD-22725 (EMDB) for MmpL3-GDN; 7K8C (PDB) and EMD-22726 (EMDB) for MmpL3-TMM I; 7K8D (PDB) and EMD-22728 (EMDB) for MmpL3-TMM II; 7N6B (PDB) and EMD-24206 (EMDB) for MmpL3-TMM III; and 7K7M (PDB) for MmpL3773-T6D I and MmpL3773-T6D II.

Interestingly, it has been observed that M. tuberculosis mmpL3 is able to rescue the utility of the Mycobacterium smegmatis mmpl3 null mutant [ 3 ]. Both viability and lipid transport studies have shown that these 2 mmpL3 orthologs are functionally interchangeable. We recently determined X-ray structures of the M. smegmatis MmpL3 transporter, revealing a monomeric molecule that is structurally distinct from all known bacterial membrane proteins [ 14 ]. This structural information indeed agrees with the structure of MmpL3 in the presence of a variety of inhibitors [ 15 ]. The MmpL3 transporter contains 12 transmembrane helices and 2 periplasmic flexible loops, posing a plausible pathway for substrate transport. Using native mass spectrometry, we found that MmpL3 is a promiscuous membrane protein, capable of binding a variety of lipids, including TMM, phosphatidylethanolamine (PE), phosphatidylglycerol, and cardiolipin, all with dissociation constants within the micromolar range [ 14 ]. Our previous experimental data highlight a possible role for MmpL3 in shuttling different lipids across the membrane. To further elucidate the molecular mechanism of lipid translocation across the mycobacterial membrane via the MmpL3 transporter, we here present 7 structures of M. smegmatis MmpL3, either alone or bound with lipid moieties, using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. We also investigate the functional dynamics of this transporter using molecular dynamics (MD) simulation. Together, combined with new structural information, target MD simulation provides us with a pathway for lipid transport via the MmpL3 membrane protein.

The unique architecture of the mycobacterial cell wall plays a predominant role in M. tuberculosis pathogenesis. This complex cell wall layer provides a strong barrier to protect M. tuberculosis against the host immune response as well as clinically relevant antibiotics. The outer membrane of M. tuberculosis is distinguished by the hallmark lipid mycolic acid (MA), a specific 2-alkyl-3-hydroxyl long-chain fatty acid. MA is shuttled across the inner membrane as trehalose monomycolate (TMM), which is then either incorporated into trehalose dimycolate (TDM) or covalently linked to the arabinogalactan-peptidoglycan layer as mycolyl arabinogalactan peptidoglycan (mAGP). Recently, it has been demonstrated that mycobacterial membrane protein large (MmpL) transporters are critical for mycobacterial physiology and pathogenesis by shuttling fatty acids and lipid components to the mycobacterial cell wall. Of the 13 M. tuberculosis MmpLs, only MmpL3 is shown to be capable of exporting TMMs [ 3 , 4 ]. A detailed analysis suggested that MmpL3 is a TMM flippase [ 5 ] that is absolutely essential for transporting TMMs across the cell membrane. Because of the requirement of MmpL3 for mycobacterial cell wall biogenesis, this membrane protein has been a target of several potent anti-TB agents [ 6 – 13 ].

Tuberculosis (TB) is an airborne disease caused by the bacterium Mycobacterium tuberculosis. It is the leading cause of death from a single infectious agent, exceeding both malaria and HIV [ 1 , 2 ]. The emergence and spread of multidrug-resistant TB (MDR-TB) presents an increasingly difficult therapeutic challenge, thus a serious threat to our global public health. The lethality of TB combined with its multidrug-resistant capacity has already transformed this disease into a worldwide problem.

Results

Cryo-EM structure of MmpL3 reconstituted in nanodiscs We expressed the full-length M. smegmatis MmpL3 transporter by cloning mmpL3 into the Escherichia coli expression vector pET15b, with a 6xHis tag at the carboxyl terminus to generate pET15bΩmmpL3. The MmpL3 membrane protein was overproduced in E. coli BL21(DE3)ΔacrB cells. We delipidated and purified this membrane protein using a Ni2+-affinity column. We then reconstituted the delipidated, purified MmpL3 transporter into lipidic nanodiscs. This MmpL3-nanodisc (MmpL3-ND) sample was further purified using a Superose 6 column to separate the MmpL3-ND complex from empty nanodiscs. The structure of MmpL3-ND was solved using single-particle cryo-EM. The reconstituted sample led to a cryo-EM map at a nominal resolution of 3.65 Å (Fig 1A, S1 Table and S1 Fig). Density modification [16] allowed us to improve the resolution of the map to 3.00 Å. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Structures of the M. smegmatis MmpL3 transporter. (a) Side view of the surface representation of MmpL3-ND. The narrowest region of the channel created by the MmpL3 membrane protein, measured between the Cα atoms of residues S423 and N524 (red triangles), is 8.6 Å. (b) Ribbon diagram of MmpL3-ND viewed in the membrane plane. (c) Side view of the surface representation of MmpL3-GDN. The narrowest region of the channel created by the MmpL3 membrane protein, measured between the Cα atoms of residues S423 and N524 (red triangles), is 11.5 Å. (d) Ribbon diagram of MmpL3-GDN viewed in the membrane plane. (e) Superimposition of the structures of MmpL3-ND and MmpL3-GDN. This superimposition suggests that the major difference between these 2 structures is the location of PD2, which rotates by approximately 6° in a rigid body fashion (red arrow) when compared the MmpL3-GDN structure to that of MmpL3-ND. MmpL3, mycobacterial membrane protein large 3; MmpL3-GDN, MmpL3-glycol-diosgenin; MmpL3-ND, MmpL3-nanodisc. https://doi.org/10.1371/journal.pbio.3001370.g001 The cryo-EM structure of MmpL3-ND indicates a monomeric molecule that spans the inner membrane and protrudes into the periplasm (Fig 1B). Like the X-ray structure of the detergent-solubilized MmpL3 protein, the carboxyl terminal end of MmpL3 is absent in the cryo-EM structure. MmpL3 consists of 12 transmembrane helices (TMs 1 to 12) and 2 periplasmic subdomains (PDs 1 and 2). Overall, the conformation of the cryo-EM structure of MmpL3-ND is comparable to that of the X-ray structure of MmpL3 773 -PE, although there are some conformational differences between these 2 structures. Superimposition of 710 Cα atoms of these 2 structural models gives rise to a root mean square deviation (RMSD) of 1.5 Å (S2 Fig). The conformation of MmpL3-ND is distinct in that the gap between subdomains PD1 and PD2 is larger when compared with that of MmpL3 773 -PE. The volume of the periplasmic central cavity formed between PD1 and PD2 was measured to be 1,794 Å3 in MmpL3-ND. The corresponding volume of this cavity in the MmpL3 773 -PE structure is 946 Å3, only half of the volume observed in the cryo-EM structure. These volumes were computed using the program CASTp 3.0 [17]. Based on the structural information, the increased volume of the periplasmic central cavity of MmpL3-ND is in part caused by the increased separation of PD1 and PD2. However, the expansion of this central volume is also related to the change in conformation of several transmembrane helices, which accommodates for the rearrangement of the periplasmic domain. It is observed that TM7, TM9, TM10, TM11, and TM12 slightly shift toward the cytoplasm by approximately 2 to 3 Å (S2 Fig). This shift, in turn, helps expand the capacity of the periplasmic central cavity.

Cryo-EM structure of MmpL3 embedded in detergent micelles Overall, the cryo-EM structure of MmpL3 embedded in nanodiscs is in good agreement with that of the X-ray structure of MmpL3 773 -PE, although these 2 structures represent different transient states of the membrane protein. It is uncertain if the cryo-EM structure of this membrane protein would be different in a detergent environment. To answer this question, we delipidated and purified MmpL3 in a solution containing 0.01% glycol-diosgenin (GDN) detergent. We then determined the cryo-EM structure of MmpL3 surrounded by GDN detergent micelles at a nominal resolution of 2.94 Å (MmpL3-GDN) (Fig 1C, S1 Table and S3 Fig). The density modification program [16] allowed us to improve the resolution of the cryo-EM map to 2.40 Å (Fig 1D). The structures of MmpL3-GDN and MmpL3-ND largely resemble each other. However, there are conformational differences between the two. Superimposition of these 2 structures gives rise to an RMSD of 1.4 Å (for 710 Cα atoms), suggesting that these conformations are distinct from each other (Fig 1E). The calculated volume of the periplasmic central cavity of GDN solubilized MmpL3 using CASTp 3.0 [17] is 1,716 Å3, which is similar, albeit smaller, to that of the protein embedded in nanodiscs. A detailed inspection on these 2 structures suggests that there is a relative rotational motion involved between the periplasmic subdomains PD1 and PD2 of the MmpL3 membrane protein, where PD2 is found to rotate by 6° in relation to PD1 of MmpL3-GDN when compared with the structure of MmpL3-ND (Fig 1E).

Cryo-EM structures of MmpL3 in the presence of TMM Since cryo-EM structures of the transporter surrounded by nanodiscs or detergent micelles are in good agreement with each other, we decided to determine a cryo-EM structure of MmpL3 embedded in nanodiscs in the presence of TMM lipids. Surprisingly, we found the existence of 2 distinct conformations of single-particle images in the sample. These images led us to resolve 2 different cryo-EM structures of MmpL3 at nominal resolutions of 4.26 Å and 4.33 Å (MmpL3-TMM I and MmpL3-TMM II), respectively (Fig 2A and 2B, S1 Table and S4 Fig). Additional density modification [16] permitted us to improve the resolution of these 2 structures to 3.30 Å (Fig 2C and 2D). The conformations of the 2 structures are markedly different from each other (Fig 2E). They are also distinct from those of MmpL3-ND and MmpL3-G6D. Pairwise superimpositions of the structures of MmpL3-TMM I and MmpL3-TMM II to that of MmpL3-ND result in RMSD values of 1.7 Å and 2.3 Å (for 710 Cα atoms each), respectively. Unfortunately, we did not observe any bound TMM molecules within these 2 MmpL3 structures. In MmpL3-TMM I, the volume of the periplasmic cavity was measured to be 1,303 Å3, which is 491 Å3 smaller than that of apo MmpL3-ND and 357 Å3 larger than that of MmpL3 773 -PE. However, the calculation of the volume of this periplasmic central cavity in MmpL3-TMM II gave rise to 2,212 Å3, which is approximately 420 Å3 and 1,266 Å3 larger than those of MmpL3-ND and MmpL3 773 -PE. These volumes were computed using CASTp 3.0 [17]. A comparison of the 3 nanodisc-embedded structures of MmpL3 suggests that there is a drastic conformational change of the transmembrane helices in addition to the rigid body movement of subdomain PD2. This change can be interpreted as a shift in the relative distance between TM2 and TM8. This change also alters the relative positions of several TM helices, including TMs 7 to 12. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. Structures of MmpL3-TMM I and MmpL3-TMM II. (a) Side view of the surface representation of MmpL3-TMM I. The narrowest region of the channel created by the MmpL3 membrane protein, measured between the Cα atoms of residues S423 and N524 (red triangles), is 10.5 Å. (b) Side view of the surface representation of MmpL3-GDN. The narrowest region of the channel created by the MmpL3 membrane protein, measured between the Cα atoms of residues S423 and N524 (red triangles), is 12.0 Å. (c) Ribbon diagram of MmpL3-TMM I viewed in the membrane plane. (d) Ribbon diagram of MmpL3-TMM II viewed in the membrane plane. (e) Superimposition of the structures of MmpL3-TMM I and MmpL3-TMM II. This superimposition suggests that there is a drastic change in conformation of the transmembrane helices, including TMs 7 and 10, in addition to the rigid body movement of subdomain PD2. MmpL3, mycobacterial membrane protein large 3; MmpL3-GDN, MmpL3-glycol-diosgenin; MmpL3-TMM, MmpL3-trehalose monomycolate; TM, transmembrane. https://doi.org/10.1371/journal.pbio.3001370.g002

X-ray structures of the MmpL3 773 -T6D complexes In addition to elucidating the structures of MmpL3 via cryo-EM, we crystallized the delipidated, purified MmpL3 773 transporter, which contains residues 1 to 773, in the presence of trehalose 6-decanoate (T6D). T6D is an ideal ligand to mimic the TMM lipid, as it contains a trehalose moiety and is structurally similar to TMM. Crystals of MmpL3 773 bound with T6D diffracted X-rays to a resolution of 3.34 Å (Fig 3A–3D, S2 Table and S5 Fig). Surprisingly, the data indicate that each asymmetric unit of the crystal contains 2 independent MmpL3 773 monomers, where the conformations (MmpL3 773 -T6D I and MmpL3 773 -T6D II) are different from each other (Fig 3E). These 2 structures are also similar but distinct from that of MmpL3 773 -PE. Pairwise superimpositions of the structure of MmpL3 773 -PE to those of MmpL3 773 -T6D I and MmpL3 773 -T6D II give rise to RMSD values of 1.4 Å and 1.5 Å (for 710 Cα atoms each), respectively. Interestingly, the structures of MmpL3 773 -T6D I and MmpL3 773 -T6D II are quite different both in the periplasmic and transmembrane domains. Particularly, it is observed that TM9 of MmpL3 773 -T6D II tilts away from the innermost surface of the cytoplasmic membrane when compared with that of MmpL3 773 -T6D I. Consistent with the structure of MmpL3 773 -PE, each MmpL3 773 -T6D structure is found to create 2 substrate binding sites that are situated at cavities formed by TM7-TM10 in the transmembrane region and between PD1 and PD2 of the periplasmic domain, respectively. Each binding site is occupied by a T6D molecule. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Structures of MmpL3-T6D I and MmpL3-T6D II. (a) Side view of the surface representation of MmpL3-T6D I. The narrowest region of the channel within MmpL3, as measured between the Cα atoms of residues S423 and N524 (red triangles), is 9.8 Å. The 2 bound T6D molecules (T6D 1 and T6D 2 ; insert) are in blue and red spheres, respectively. (b) Side view of the surface representation of MmpL3-T6D II. The narrowest region of the channel within MmpL3, as measured between the Cα atoms of residues S423 and N524 (red triangles), is 9.5 Å. The 2 bound T6D molecules (T6D 1 and T6D 2 ; insert) are in green and red spheres. (c) Ribbon diagram of MmpL3-T6D I viewed in the membrane plane. The F o —F c electron density maps of the 2 bound T6D molecules are contoured at 3σ (insert). The bound T6D 1 and T6D 2 molecules are shown as sticks (blue, carbon; red, oxygen). (d) Ribbon diagram of MmpL3-T6D II viewed in the membrane plane. The F o —F c electron density maps of the 2 bound T6D molecules are contoured at 3σ (insert). The bound T6D 1 and T6D 2 molecules are shown as sticks (green, carbon; red, oxygen). (e) Superimposition of the structures of MmpL3-T6D I and MmpL3-T6D II. This superimposition suggests that there is a drastic change in conformation of subdomain PD2 when compared between these 2 structures. In addition, the locations of the 2 bound T6Ds are quite distinct. MmpL3, mycobacterial membrane protein large 3; MmpL3-T6D, MmpL3-trehalose 6-decanoate; T6D, trehalose 6-decanoate. https://doi.org/10.1371/journal.pbio.3001370.g003 A large density corresponding to the first bound T6D molecule (T6D 1 ) was observed in the pocket surrounded by TMs 7 to 10 in each structure. In the MmpL3-T6D I structure, residues S423, L424, Q554, F561, L564, A568, and I636 are within 4.5 Å of the bound T6D 1 , performing hydrophobic or polar interactions with this ligand (Fig 3C). In addition, N524 and H558 are situated approximately 5.3 Å away from the trehalose headgroup, interacting with this bound T6D 1 molecule via polar interaction. Interestingly, at least half of these contacting residues belong to TM8, suggesting that TM8 may be an important transmembrane helix for recognizing and shuttling TMM to the periplasm. In the MmpL3-T6D II structure, residues that are found to be important for T6D 1 binding are S423, L424, Q554, F561, L564, and I636, in which the composition of these residues are very similar to that of MmpL3-T6D I (Fig 3D). The second large density corresponding to the other bound T6D (T6D 2 ) was observed in the central periplasmic cavity between PD1 and PD2. For MmpL3-T6D I, this bound T6D 2 molecule is surrounded by residues Q40, S41, F43, R63, D166, L171, L178, and T549, which are within 4.5 Å of bound T6D 2 , performing electrostatic or polar interactions to bind the ligand (Fig 3C). Similarly, the MmpL3 transporter utilizes residues Q40, S41, F43, Y44, D58, R63, L171, L178, N524, and T549 to contact and participate in electrostatic interactions with the trehalose headgroup of T6D 2 in the MmpL3-T6D II (Fig 3D). One obvious distinction between these 2 structures are the exact locations of their bound T6Ds. The 2 bound T6Ds in the MmpL3-T6D II structure seemingly shift upward and move away from the surface of the membrane by 3.2 Å (at the transmembrane binding site) and 4.2 Å (at the periplasmic binding site) in comparison with the corresponding bound T6Ds in the MmpL3-T6D I structure (Fig 3E). It appears that these 2 structures capture different transient states of substrate transport mediated by MmpL3. Interestingly, these 2 structures suggest that the interactions between MmpL3 and T6D seem to shift from a more hydrophobic nature in the transmembrane region to a more electrostatic nature at the periplasmic domain. It is likely that the phosphate headgroups of the outer leaflet surface of the inner membrane provide electrostatic interactions to the bound T6D molecule in the substrate binding site of the transmembrane region.

Cryo-EM structure of the MmpL3-TMM complex After obtaining crystal structures of the MmpL3 773 -T6D complexes, we rationalized that TMMs should be bound at locations similar to the T6D binding sites. We then repeated the cryo-EM experiment of MmpL3 with a 2-fold increase in the concentration of TMM in the nanodiscs, with the expectation that we could identify the TMM binding sites within the transporter. We reconstituted the delipidated, purified MmpL3 membrane protein into these nanodiscs and collected single-particle cryo-EM images. The three-dimensional reconstitution of MmpL3 led to a 2.66 Å-resolution cryo-EM map. Density modification [16] enabled us to further improve the resolution, resulting in resolving the MmpL3-TMM complex structure (MmpL3-TMM III) to a resolution of 2.20 Å (Fig 4A–4D, S1 Table and S6 Fig). Pairwise superimpositions of 710 Cα atoms of the structure of MmpL3-TMM III to those of the above 6 MmpL3 structures give rise to RMSD values ranging from 0.6 Å to 2.4 Å. These superimpositions also indicate that the conformation of MmpL3-TMM III is nearly identical to that of MmpL3 773 -T6D II (RMSD = 0.6 Å), suggesting that these 2 MmpL3 structures represent a very similar transient state of the transporter. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Structures of the MmpL3-TMM complex. (a) Side view of the surface representation of MmpL3-TMM III. The narrowest region of the channel created by the MmpL3 membrane protein, measured between the Cα atoms of residues S423 and N524 (red triangles), is 11.0 Å. The 2 bound TMM lipids (TMM 1 and TMM 2 ) are in orange and red spheres, respectively. (b) Ribbon diagram of MmpL3-TMM III viewed in the membrane plane. The 2 bound TMM molecules are in orange sticks. The binding residues I416, L419, L422, S423, L424, I557, F561, L564, A568, I572, V573, T576, I590, A593, L594, A597, L598, L600, M604, I632, I636, and W640 for TMM 1 , as well as the binding residues Q40, Y44, D64, T66, S67, V70, V109, K113, A114, V122, M125, F134, S136, L171, L174, A175, L178, S300, I427, E429, Q442, F445, F452, R453, T454, E455, T488, P490, K499, Q517, and T549 for TMM 2 are colored green. MmpL3, mycobacterial membrane protein large 3; MmpL3-TMM, MmpL3-trehalose monomycolate. https://doi.org/10.1371/journal.pbio.3001370.g004 Based on the MmpL3-TMM III structure, we identified 2 extra densities, which indicate that there are 2 TMM molecules bound by the MmpL3 transporter (Fig 4A and 4B). As postulated, the locations of these 2 bound TMM molecules coincide with those of the T6D binding sites. The first bound TMM molecule (TMM 1 ) was observed within the pocket at the outer leaflet of the cytoplasmic membrane. This pocket is created by TMs 7 to 10 of the transmembrane region. The second bound TMM lipid (TMM 2 ) was found to sandwich between PD1 and PD2 of the periplasmic domain. At the outer leaflet of the cytoplasmic membrane, TMM 1 is bound in such a way that the trehalose headgroup is located at the surface of the membrane, presumably interacting with the phosphate headgroups of phospholipids, leaving the elongated hydrophobic tail of carbon chains contacting the hydrocarbon chains of phospholipids within the membrane. The length of the elongated tail of TMM is quite substantial such that it almost spans the entire cytoplasmic membrane of the bacterium. Within 4.5 Å of bound TMM 1 , the MmpL3 residues I416, L419, L422, S423, L424, I557, F561, L564, A568, I572, V573, T576, I590, A593, L594, A597, L598, L600, M604, I632, I636, and W640 are responsible for the binding (Fig 4B). In addition, the backbone oxygen of L422 forms a hydrogen bond with TMM 1 to further stabilize the binding. It should be noted that most of these amino acids are incorporated into TM8 and TM9, suggesting the importance of these 2 TMs for recognizing TMM. As mentioned, the bound TMM 2 molecule was found at the central cavity formed by subdomains PD1 and PD2 of the periplasmic domain of MmpL3. The 2 extended loops, which run across PD1 and PD2, are engaged in housing this lipid molecule. Surprisingly, the orientation of this bound TMM 2 molecule is more or less antiparallel to that of bound TMM 1 found in the transmembrane region. Therefore, the hydrophobic carbon tail of bound TMM 2 at the periplasmic domain was observed to point toward the cell wall skeleton. The binding of this TMM 2 lipid is extensive. Within 4.5 Å of bound TMM 2 , there are at least 31 residues engaged in binding this lipid. These residues are Q40, Y44, D64, T66, S67, V70, V109, K113, A114, V122, M125, F134, S136, L171, L174, A175, L178, S300, I427, E429, Q442, F445, F452, R453, T454, E455, T488, P490, K499, Q517, and T549, which provide electrostatic and hydrophobic interactions to bind this TMM 2 molecule (Fig 4B). Particularly, E429 and R453 also form hydrogen bonds with TMM 2 to strengthen the binding.

[END]

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