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

Abstract

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.

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.

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.

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 (T6D₁ and T6D₂; 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 (T6D₁ and T6D₂; 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 T6D₁ and T6D₂ 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 T6D₁ and T6D₂ 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.

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 (TMM₁ and TMM₂) 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 TMM₁, 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 TMM₂ are colored green. MmpL3, mycobacterial membrane protein large 3; MmpL3-TMM, MmpL3-trehalose monomycolate.

Fig 5. Channel created by the MmpL3 transporter.

This figure indicates that the structures of MmpL3-ND, MmpL3-T6D I, MmpL3-T6D II, MmpL3-TMM I, and MmpL3-TMM III form truncated channels, where S423 and N524 are responsible to close these channels. However, the structures of MmpL3-GDN and MmpL3-TMM II represent the fully open channel conformations. MmpL3, mycobacterial membrane protein large 3; MmpL3-GDN, MmpL3-glycol-diosgenin; MmpL3-ND, MmpL3-nanodisc; MmpL3-TMM, MmpL3-trehalose monomycolate; MmpL3-T6D, MmpL3-trehalose 6-decanoate.

Fig 6. Conformational flexibility of the MmpL3 transporter.

(a) Superimposition of the 7 structures of MmpL3. The structures indicate that PD2 is able to perform a rigid body rotational motion, transitioning from one conformational state to the other. The transmembrane helices, particularly TM7–TM12, are also found to shift their locations, accompanying the rotational motion of PD2. (b) MD simulations of the MmpL3 transporter. Consistent with the 7 structures of MmpL3, the first eigenvector based on PCA suggests that the major motion of MmpL3 is the rigid body rotation of the PD2 subdomain. (c and d) The 2 flexible loops 423–426 and 523–525 create the narrowest region of the channel. These residues may be important for controlling the (c) closing and (d) opening of this channel by coupling with the dynamic motion of the periplasmic subdomain PD2. Residues S423, L424, R523, N524, and D525 are indicated as green sticks. MD, molecular dynamics; MmpL3, mycobacterial membrane protein large 3; PCA, principal component analysis.

Fig 7. Target MD simulations of the MmpL3 transporter.

The calculations depict snapshots (0, 1.26, 2.27, and 3.11 ns) of T6D shuttling from the outer leaflet of the inner membrane, between TMs 7–10, to the periplasmic central cavity, between PDs 1–2, of MmpL3. MD, molecular dynamics; MmpL3, mycobacterial membrane protein large 3; T6D, trehalose 6-decanoate.

Fig 8. Proposed mechanism for TMM translocation via MmpL3.

This schematic diagram indicates that the MmpL3 transporter is capable of flipping a TMM molecule from the inner leaflet to outer leaflet of the cytoplasmic membrane. The transporter will pick up this TMM molecule from the outer leaflet of the membrane, allowing the TMM molecule to pass through the channel formed by MmpL3 and arrive at the periplasmic lipid binding site. The orientation of the bound TMM molecule in the periplasmic domain of MmpL3 is antiparallel to that of bound TMM at the outer leaflet of the transmembrane region. MmpL3, mycobacterial membrane protein large 3; TMM, trehalose monomycolate.