doi:10.1038/nature17991 Supplementary Discussion Structural comparison with E. coli EmrE The DMT superfamily includes a wide variety of transporters with 4-10 TM segments 1. Since the subfamilies of the DMT superfamily share very low sequence similarity, their evolutional relationship was elusive. The present crystal structure of YddG provides insight into the molecular evolution of the DMT superfamily. The small multidrug resistance (SMR) family is a subfamily of the DMT superfamily, and its members form homo/hetero-dimeric transporters with 4-TM protomers 2. Previous bioinformatics analyses suggested that this SMR family is the progenitor of the other DMT proteins 3,4, and that the insertion of a new TM segment between TM1 and TM2 of the SMR transporters generated the 5-TM DMT proteins, whose duplication finally formed the 10-TM DMT proteins. However, the insertion of one TM segment in the middle of the membrane protein topology seems to be unlikely, because it requires the inversion of the other TM segments, which could change the overall protein folding topology, and thus possibly affect the function of the transporter. A comparison between the crystal structures of SnYddG and E. coli EmrE 2,5, one of the best characterized SMR transporters, provides insight into a possible evolutional relationship between the 4-TM and 10-TM DMT proteins (Extended Data Fig. 4). As shown in Extended Data Fig. 4a-c, the superimposition of the TM helices of SnYddG and the EmrE dimer (PDB ID: 2I68) revealed good structural alignment (RMSD: 2.9 Å over 127 Cα atoms). The central cavity of YddG coincides well with the tetraphenylphosphonium (TPP) binding site of EmrE, observed in the previous crystal structures 2,5. TM3, TM4, TM5, and TM1 of YddG correspond to TM2, TM3, TM4, and TM1 (TM1 of another molecule) of the EmrE dimer, respectively, while EmrE lacks the TM segments corresponding to TM2 and TM7 of YddG (Extended Data Fig. 4a-c). WWW.NATURE.COM/NATURE 1
This comparison suggests that the DMT superfamily transporters with the 5+5 TM topology were evolutionarily formed by i) the insertion of a new TM segment between TM1 and TM2, and ii) the swapping of the two TM1 segments of both protomers (Extended Data Fig. 4d). After the insertion of the new TM segment between TM1 and TM2, TM1 cannot be docked into its original site, since the insertion of the new TM resulted in the inversion of the insertion direction of TM1 (Extended Data Fig. 4d, middle panel). However, if the up- and down-stream loop regions of the new TM segment are long enough, TM1 can be docked into the site where the TM1 of another protomer was originally docked. This swapping of TM1 between the protomers allows the complete maintenance of the same inter-tm interactions, and thus preserves the overall integrity of the membrane protein fold, after the insertion of the new TM segment into the middle of the membrane protein fold (Extended Data Fig. 4d, middle panel). Furthermore, this insertion of the TM segment enabled the fusion of two inverted protomers (Extended Data Fig. 4d, right panel), which finally generated the enormous variety of DMT transporters with complex topologies, including YddG, from the simple inverted homodimer of the SMR transporters. Overall, the present structure of SnYddG not only exemplifies the evolutionary model of the dual-topology membrane proteins 6, but also provides insights into the molecular evolution of membrane proteins with novel topologies, generated through the insertion and swapping of TM segments. It should also be noted here that this insertion and swapping mechanism is only possible in homodimeric membrane proteins with dual topology. Evolutionary covariation analysis of YddG To predict the possible interactions between the TM helices of YddG, we performed an evolutionary covariation analysis, using the program EVcoupling 7. As the input, 59,114 sequences of the DMT protein homologs of SnYddG were used for the EVcoupling calculation WWW.NATURE.COM/NATURE 2
(Extended Data Fig. 7a). The results correctly predicted the interaction sites between the TM helices observed in the present crystal structure, including the interactions in the intracellular gate, which is composed of the intracellular tips of the TM6-TM7 and TM8-TM9a hairpins (Extended Data Fig. 7a,b). Furthermore, the results predicted the evolutionarily correlated sites, where no direct interactions are observed in the present outward-open crystal structure. At the extracellular entrance of the central cavity, which is formed by the extracellular tips of the TM1-TM2 and TM3-TM4a hairpins, we detected evolutionarily correlated residues with high EC scores. This result suggests that this site is evolutionarily conserved, to allow the interactions between them (Extended Data Fig. 7a,b). Interestingly, this site corresponds to the putative extracellular gate, which sequesters the central cavity from the extracellular space, in our model structure of the inward-facing state. Next, we predicted the overall structure of SnYddG by the program EVfold_membrane 8, using these EC sites. The highest ranking predicted structure was calculated using the 170 EC sites. Surprisingly, the overall folding and the TM topology of the predicted structure coincided well with those of the crystal structure (Extended Data Fig. 7c). The predicted structure is reminiscent of the occluded state, in which the intracellular and extracellular gates are both closed. Thus, the results of the evolutionary covariation analysis strongly support our model of the inward-facing state and the formation of the putative extracellular gate. Possible transport model of YddG The superimposition of the N- and C-halves provides further insights into the structural changes that occur during the transport cycle. The structural differences between the N- and C-halves mainly exist in TM1, TM3, and TM4 in the N-half, and TM6, TM8, and TM9 in the C-half, in the degrees of the bending and tilting of these TM helices (Fig. 2c). Thus, the inter-conversion of the two different conformations of these TM helices probably occurs during the structural transition between the inward- and outward-facing states. Especially, the structural WWW.NATURE.COM/NATURE 3
comparison of TM4 and TM9 suggests the bending of these TM segments, with the conserved intra-membrane loops between TM4a and TM4b, and between TM9a and TM9b, serving as hinges. In contrast, the other TM segments (i.e., TM2 and TM5 in the N-terminal half) superimpose well on TM7 and TM10 in the C-terminal half (Fig. 2c). This suggests that these TM segments maintain their structures during the transport cycle. TM2, TM5, TM7 and TM10 may function as a scaffold to stabilize the overall structure. This notion led us to propose a model of the structural changes occurring during the transport cycle of YddG (Fig. 4c and Supplementary Video). Starting from the outward-open state, the straightening of TM4 and TM9 occurs to form the interactions between TM4a and TM9b, which in turn lead to the movement of the extracellular half of TM3. These structural changes in the TM helices probably involve the kinks of TM3 around Gly71, Gly73, Gly74, and Gly77 (Extended Data Fig. 8a), as well as those of TM4 and TM9 around the intra-membrane loops. The Ala mutations of these Gly residues affected the transport activity, suggesting their importance in the transport mechanism (Extended Data Fig. 8a). The structural changes result in the packing of the TM3-TM4a hairpin against the extracellular sides of TM1 and TM9b, which closes the extracellular entrance of the central cavity, leading to the occluded state (Fig. 4c). Upon the structural change to the inward-open state, TM4b and TM9a move away from each other, thereby creating part of the intracellular entrance of the central cavity. Along with this structural change, the bending of TM8 around Gly217, Gly219, Pro220, and Gly222 shifts the tip of the TM8-TM9a hairpin away from TM6 and TM7 (Extended Data Fig. 8b), which opens the intracellular gate to form the inward-open structure (Fig. 4c). The Ala mutations of these Gly/Pro residues affected the transport activity, suggesting their importance in the transport mechanism (Extended Data Fig. 8b). Molecular dynamics simulations of SnYddG To support the structural change mechanism suggested by the present crystal structure, we WWW.NATURE.COM/NATURE 4
performed molecular dynamics (MD) simulations, based on the present crystal structure. In the equilibrium simulation without biasing forces, no large structural change was observed during 500 ns, suggesting that the outward-facing structure is stable in the lipid bilayer (Extended Data Fig. 9a). This result is also consistent with the slow transport rate of SnYddG, observed in the liposome-based assay (Fig. 1a). Next, to facilitate the structural change, we performed the 1-μs MD simulation with biasing forces. In the first 500-ns simulation, a harmonic restraint was applied between the center of the mass of the Cα atoms of TM4a and TM9b (i.e. Pro91 Ala98 and Ala246 Leu253), and the equilibrium distance of the restraint was gradually decreased from 15 Å to 9 Å, to move these two regions towards each other (Extended Data Fig. 9b). During the simulation, the TM1-TM2 hairpin and TM3 approached to each other and new interactions were formed between them, although no biased force was applied to these helices (Extended Data Fig. 9b). These interactions are quite similar to those observed in the putative extracellular gate in the inward-facing model structure (Fig. 4b; right panel). The final snapshot of the 500-ns simulation represented the occluded-like structure (Extended Data Fig. 9b), and is superimposable on the model structure produced by EVfold_membrane (Extended Data Fig. 7c), with 3.0 Å RMSD over 254 Cα atoms. In the next 500-ns simulation, a similar restraint was applied between TM4b and TM9a (i.e., Trp101 Phe108 and Val237 Ser244), and the equilibrium distance was gradually increased from 9 Å to 15 Å, to move these two regions apart from each other (Extended Data Fig. 9c). During the simulation, the TM6-TM7 hairpin and TM8 were separated from each other and the intracellular gate was opened, although no biased force was applied to these helices (Extended Data Fig. 9c). In addition, the interactions between the TM1-TM2 hairpin and TM3, as well as those between TM4a and TM9b, were stably maintained during the 500-ns simulation with no biasing forces to these regions (Extended Data Fig. 9c). The final snapshot of the simulation is similar to the model structure of the inward-open state (Extended Data Fig. 9c). Together, the results of the MD simulation support the inward-facing model structure and the structural change WWW.NATURE.COM/NATURE 5
mechanism of YddG. Common transport mechanisms of DMT proteins The structural similarity between EmrE and YddG suggests the transport mechanisms of EmrE and other DMT transporters. TM3 of EmrE contains the GXG motif, which is conserved among the SMR transporters, at the middle of the TM segment (Extended Data Fig. 3c). The structural superimposition indicated that this region corresponds to the intra-membrane loops in TM4 and TM9 of YddG (C and C in Extended Data Fig. 4a-c), which are probably important for the transport mechanism. Thus, TM3 of EmrE may also be kinked in a similar manner to TM4 and TM9 of YddG, and the GXG motif probably serves as a hinge that facilitates its bending motion during the transport cycle (Fig. 4c). Moreover, the structural similarity suggested that the two-helix hairpin structure of TM2 and TM3 of EmrE undergoes a similar motion to that of the TM3-TM4 hairpin of YddG (Fig. 4c). This motion opens and closes the intra- and extracellular gates, thereby providing a mechanism for alternating the access to the central cavity. In contrast, TM4 of EmrE may function as a scaffold to support the structural changes of the other TM segments, in a similar manner to TM5 and TM10 of YddG. Therefore, the symmetric inter-conversion of the two different conformations of the protomers leads to the alternating access to the substrate binding site, which finally drives drug extrusion using the proton electrochemical gradient across the membrane. These key motifs for the transport mechanism; i.e., the hinge region in the middle of TM4 and TM9 and the two-helix hairpin structures of TM3-TM4 and TM8-TM9, are also conserved in other DMT transporters 9. Therefore, it is likely that the proposed transport model is widely shared among the other DMT superfamily proteins. WWW.NATURE.COM/NATURE 6
References 1. Jack, D. L., Yang, N. M. & Saier, M. H. The drug/metabolite transporter superfamily. Eur. J. Biochem. 268, 3620 39 (2001). 2. Chen, Y.-J. et al. X-ray structure of EmrE supports dual topology model. Proc. Natl. Acad. Sci. U. S. A. 104, 18999 19004 (2007). 3. Västermark, Å., Almén, M. S., Simmen, M. W., Fredriksson, R. & Schiöth, H. B. Functional specialization in nucleotide sugar transporters occurred through differentiation of the gene cluster EamA (DUF6) before the radiation of Viridiplantae. BMC Evol. Biol. 11, 123 (2011). 4. Bay, D. C. & Turner, R. J. Diversity and evolution of the small multidrug resistance protein family. BMC Evol. Biol. 9, 140 (2009). 5. Ubarretxena-Belandia, I., Baldwin, J. M., Schuldiner, S. & Tate, C. G. Three-dimensional structure of the bacterial multidrug transporter EmrE shows it is an asymmetric homodimer. EMBO J. 22, 6175 6181 (2003). 6. Rapp, M., Seppälä, S., Granseth, E. & von Heijne, G. Emulating membrane protein evolution by rational design. Science 315, 1282 1284 (2007). 7. Marks, D. S. et al. Protein 3D structure computed from evolutionary sequence variation. PLoS One 6, 2 5 (2011). 8. Hopf, T. A. et al. Three-Dimensional Structures of Membrane Proteins from Genomic Sequencing. Cell 149, 1607 1621 (2012). 9. Martin, R. E. & Kirk, K. The malaria parasite s chloroquine resistance transporter is a member of the drug/metabolite transporter superfamily. Mol. Biol. Evol. 21, 1938 1949 (2004). WWW.NATURE.COM/NATURE 7