They all rock: A systematic comparison of conformational movements in LeuT-fold transporters

SUMMARY Many membrane transporters share the LeuT fold—two five-helix repeats inverted across the membrane plane. Despite hundreds of structures, whether distinct conformational mechanisms are supported by the LeuT fold has not been systematically determined. After annotating published LeuT-fold structures, we analyzed distance difference matrices (DDMs) for nine proteins with multiple available conformations. We identified rigid bodies and relative movements of transmembrane helices (TMs) during distinct steps of the transport cycle. In all transporters the bundle (first two TMs of each repeat) rotates relative to the hash (third and fourth TMs). Motions of the arms (fifth TM) to close or open the intracellular and outer vestibules are common, as is a TM1a swing, with notable variations in the opening-closing motions of the outer vestibule. Our analyses suggest that LeuT-fold transporters layer distinct motions on a common bundle-hash rock and demonstrate that systematic analyses can provide new insights into large structural datasets.

LeuT-fold transporters with structures in multiple conformations, except BetP, previous analyses have noted that the bundle tilts relative to the hash, closing the outer vestibule and opening the inner vestibule (Table S1).During this movement, the angle between the bundle helices and TM4 of the hash decreases 5 .In LeuT, SERT, Mhp1, and LAT1, the entire bundle moves toward the hash in the outer vestibule and away from the hash in the inner vestibule 3,[6][7][8] .In contrast, TM6a is the only bundle helix that has been reported to move independently during the outwardto-inward transition, in MntH 9 .In BetP, the outward-to-inward transition has been described as the outer portion of TM3 moving closer to and the inner portion of TM9 moving away from the bundle instead of the bundle moving relative to the hash 10 .Similarly, in KCC1 rocking of TM3 and TM8, as well as movement of TM4 and TM9, allow for alternating access 11 .The bundle can move either like a rigid body, like in Mhp1 where the Iop structure superimposes almost exactly with a model based on a screw transformation of the bundle from the Oop structure 12 or with independent helix movements, such as in MntH 9 .
The outward-to-inward transition also involves movement of TM5 away from the inner hash to open the inner vestibule, as seen in SERT, LeuT, Mhp1, and MntH (Table S1).TM5 movement is also seen in the Ioc→Iop comparisons for SGLT and LeuT, and in Oop→Ioc for MntH (Table S1).The TM5 movement is greater in Mhp1 than in SGLT and LeuT (Table S1).The movement of TM5 away from the inner vestibule in SERT was proposed to allow Na + release 3 .

The I oc →I op transition involves swinging of TM1a
Finally, the Ioc→Iop transition involves an independent swinging movement of TM1a up and away from the bundle in LeuT, SGLT, SERT, and MntH (Table S1) 3,5,6,13 .Deletion of TM1a interfered with transport in the Deinococcus radiodurans homolog of MntH 13 but did not completely abrogate transport in the Staphylococcus capitis homolog 14 .In site-directed spin labelling experiments with LeuT, TM1a did not show as large a magnitude of movement as expected from the structural comparison of the Ooc→Iop structures, leading to the suggestion that the structural data may overestimate this movement 6 .Conformations were assigned through a combination of clustering of similar structures and information gathering from the relevant publications.For KCC1, the RMSD matrices were constructed based on alignment of its transporter domain (omitting the C-terminal cytosolic domain), i.e., up to residue 655.In the case of MntH, PDB IDs in black correspond to Deinococcus radiodurans (Dra)MntH, and PDB IDs in grey to Staphylococcus capitis or Eremococcus coleocola MntH.This figure includes the four clustered hb-DDMs not illustrated Figure 3.The helix labels are colored blue for the hash, yellow for the bundle, and green for the arms.The rigid bodies are highlighted with colored boxes on each matrix, with blue boxes for rigid bodies containing the hash, and yellow boxes for rigid bodies containing the bundle.Only in BetP do the four hash helices (TMs 3, 4, 8, and 9) not all cluster together, with TM8 excluded from the hash cluster and clustering instead with the bundle helices.
Figure S1.RMSD matrices for LeuT-fold transporters with structures in more than one conformation.
Figure S2.RMSD matrices for LeuT-fold transporters with multiple structures assigned to the same conformation.Conformations were assigned from information gathering from the relevant publications and visual confirmation.For the CCC-family transporters, the RMSD matrices were constructed based on alignment of their transporter domains (omitting their C-terminal cytosolic domains), i.e., up to residue 660 (KCC2 and KCC4), or 675 (KCC3 and NKCC1).In the case of ApcT, PDB ID codes in grey correspond to Geobacillus kaustophilus ApcT, and those in black to Methanocaldococcus jannaschii.
Figure S5.Intrahelix root-mean-square distance difference (RMSDD) values validate the selection of transmembrane helix ranges.The distributions of RMSDDs for each of the 14 helices with itself (intrahelical distance differences) for all twenty-two comparisons are shown on the left, with violin plots on the right representing all intrahelical distance differences or interhelical RMSDDs.No individual helix has a distribution of intrahelical RMSDDs consistent with the interhelical RMSDDs, which would suggest that it should be subdivided.The two largest outliers are TM10a during the Ioc→Iop transition of SGLT and TM4 during the Oop→Iop transition of SERT.

Table S1 . Conformational movements of described in literature. Unless
otherwise noted, the outward to inward transition is 60 described.61 10inner vestibule in Iop is narrower than in other transporters; negatively charged membrane lipid restricts the movement of the bundle

Table S2 . Literature-based identification of the helices or sidechains that occlude the binding site in each occluded structure.
Occlusion can be mediated by entire helices or by a single sidechain.

Table S7 . Angles between predicted membrane planes of transporter structures in O op and I op conformations when aligning by distinct structural element
27opt a monomeric or dimeric state under native conditions27.Monomer was used because PPM3.0 cannot fit both protomers of the dimeric O op structure (PDB: 7TTI) into a planar membrane.