Structural and mechanistic analysis of a tripartite ATP-independent periplasmic TRAP transporter

Abstract

Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo.

Fig. 1. Cryo-EM structure of the TRAP transporter HiSiaQM.

a 3D reconstruction of HiSiaQM in complex with Mb3 in lipid nanodiscs (contour level 0.384). A map of the nanodisc was overlayed in transparent grey to show the relative position of the lipid bilayer. Selected secondary structure elements are indicated. The individual domains and their color code are identified in panel (b). b Topology diagram of HiSiaQM. c Cartoon representation of HiSiaQM with the same color scheme as in (a) and (b). Secondary structure elements are indicated.

Fig. 2. Comparison of the TRAP transporter HiSiaQM with elevator-type transporters.

a Superposition of HiSiaQM (colored coded as in Fig. 1) onto one unit of the dimeric VcINDY transporter in its C_i state (magenta, 5UL9). The outline of the two VcINDY monomers is drawn as a black line. Note that the Q-domain of HiSiaQM clearly crosses the “border” between the two VcINDY monomers. b Close-up view of the Q1–Q4 helices from (a). The magenta helices are part of the VcINDY stator domain. c The substrate-binding site of VcINDY aligned to HiSiaQM, showing the two Na⁺ ions and the citrate molecule in the VcINDY structure. d Side view of HiSiaQM in the C_i state from Fig. 1c (left) and a model of the outward open C_o state (right) that was constructed based on the C_o state of VcINDY^,–.

Fig. 3. Characterization of TRAP transporter-specific VHHs and inhibition of transport in vivo.

a A hierarchical clustering tree of nine HiSiaQM-specific VHHs, based on PBLAST e-values as the distance matrix for tree building. The binding affinities of the VHHs, determined from SPR experiments are given (x: no binding detected; nd: not determined, since no clear binding detected in size-exclusion chromatography). VHHs that bind to HiSiaQM mutually exclusively are grouped by yellow and violet boxes. The underlying data are described in detail in Supplementary Fig. 9. HiSiaQM was immobilized on the SPR chip in two different orientations as indicated. b Growth defect of cultures expressing different VHHs specific for either the QM-domains (VHH_QM) or the P-domain (VHH_P) in a sialic acid uptake assay. The VHHs were either localized in the cytosol or exported to the periplasm via a signal sequence, as indicated in the figure. An empty plasmid was used as a negative control. A VHH specific for a completely unrelated human protein (VHH_X) was used as an additional control. The growth of each culture was measured after 17 h. Data are presented as mean values ± SD of n ≥ 3 independent experiments. c Interface of HiSiaQM and Mb3, which derives from VHH_QM3. The color scheme is the same as in Fig. 1. Selected polar interactions and residues are highlighted. Source data are provided as a Source Data file.

Fig. 4. Constructing the tripartite PQM complex.

a Left: AlphaFold model of the tripartite complex between HiSiaQM and HiSiaP in the C_i state. Right: The outward open model of HiSiaQM (C_o state, Fig. 2d). Here, the P-domain was replaced by the open (substrate-free) structure of HiSiaP (2CEY) by aligning it onto the N-terminal lobe of the P-domain in the C_i state (left). b “Open book” view of the interface regions between QM and the N- (III) and C-lobe (IV) of the P-domain. The color code corresponds to the conservation of residues, calculated by Consurf. The highlighted residues are in regions I–V that are likely important for complex formation and were analyzed in the growth assay in Fig. 5.

Fig. 5. Validation of the tripartite P-QM complex.

a Structural context of the mutations in the AlphaFold model of the tripartite complex. The P-domain is colored red and the QM-domains are color-coded as in Fig. 1. Red spheres indicate mutants that showed a significant effect in the growth assay and are labelled. White spheres represent the remaining mutants. b Growth defect after 17 h (compared to a wildtype culture and normalized to the negative control) of TRAP transporter mutants in a sialic acid uptake assay. The positions of the mutants with respect to the tripartite complex are indicated by labels and the color code. Data are presented as mean values ± SD of n ≥ 3 independent experiments. Source data are provided as a Source Data file.

Fig. 6. Single-molecule interaction studies of HiSiaQM and HiSiaP on solid-supported bilayers (SSBs).

a HiSiaQM variants were integrated into DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine) SSBs and their interaction with single AF-647-labelled P-domain variants or AF-555-labelled VHH_QM3 was observed by TIRF microscopy. b SSB containing HiSiaQM visualised with AF-555 labelled VHH_QM3. c As a control, VHH_QM3-AF-555 was added to an SSB in the absence of HiSiaQM. No unspecific binding was observed. d–p Top row: first frame of an image sequence of a typical set of data. Bottom row: maximum intensity projections of the respective image sequence. The conditions are indicated below each panel. q Normalised interactions per second of P-domain variants with a SSB containing HiSiaQM variants. Unless otherwise stated, the P-domains were pre-incubated with 10 mM sialic acid (Neu5Ac). Statistical significance of the P-mutants and controls was assessed by applying a two-sided unpaired Student’s t-test with a 95% confidence interval (*p < 0.01, **p < 0.001). The full dots represent the average results of n = 3 independently prepared samples, respectively. The bars represent the mean value of the three averages. The distribution of the full dots indicates for every condition the reproducibility of the results. For each of these three samples, a fresh bilayer was prepared on a new coverslip and n = 30 individual measurements were performed. The underlying data is compiled in Supplementary Table 3. The scale bars equal 3 μm. r Sensorgrams showing the interaction of the P-domain with immobilised QM-domains (HiSiaQM K273C-biotin in DDM) in the absence (red) or presence (green) of sialic acid (5 mM). The green curve was fit with a 1:1 binding model (grey). Four independent experiments were performed. s Competition experiment between the P-domain and VHH_QM3 for the immobilized QM-domains. Green curve: a buffer injection followed by an injection of the P-domain. Orange curve: the chip surface was saturated with VHH_QM3 before the P-domain was added. Single experiment, supported by multiple other experiments in this study (e.g. panel (f), Fig. 3b, the cryo-EM structure). Source data are provided as a Source Data file.

Fig. 7. Proposed mechanism of TRAP transporters.

The schematic shows the components of the transport reaction in the different steps (numbers in circles) of the proposed transport mechanism, which are explained in the main text. The conformational state of the QM domains is annotated: C_i—inward open; C_o—outward open; C_o-S—outward open, substrate bound; C_i—S inward open, substrate bound. Neu5Ac—sialic acid (N-acetylneuraminic acid).