Cryo-EM structure of AdhE in spirosome form

To examine the molecular architecture of AdhE, we purified full-length AdhE and fractionated it further via Superdex 200 gel-filtration chromatography wherein a broad elution profile was observed indicating the existence of various oligomeric states of the AdhE monomer (96.1 kDa) (Fig. 1a). To characterize the nature of these species, we selected fractions of presumed different molecular weights and examined them by negative stain electron microscopy (EM) (Fig. 1b). Fraction 1 contained a longer spirosome structure of length 25–100 nm and fraction 2 contained a shorter spirosome. Lastly, fraction 3 comprised relatively small particles, possibly tetrameric or dimeric AdhE. We analyzed the length distribution of AdhE in fraction 1 showing that the length varies from 15 to 120 nm without any dominant population of one specific length (Supplmentary Fig. 1). These data indicate that AdhE can be isolated in a wide range of oligomeric states. To determine the atomic structure of AdhE, we undertook cryo-EM imaging of AdhE. Cryo-grids were prepared by plunge-freezing AdhE from fractions 1 or 2, which are dominated by different AdhE oligomeric states. First, a total of 1255 micrographs of the sample from the fraction 2 were collected using a Titan Krios 300 keV microscope with a Falcon III direct detector in electron counting mode. 160,830 particles out of 251,604 particles picked were further processed using cisTEM 14 to generate a 3.5 Å resolution cryo-EM map (Fig. 1c, d, Supplementary Fig. 2, Supplementary Fig. 3 and Supplementary Table 1). 2D class averages show clear secondary structure features and indicate the existence of a helical structure (Fig. 1c). Most of the side chains were resolved in the cryo-EM map, and we built atomic models of six complete AdhE molecules and two ADH domains (Fig. 2a, b and Supplementary Fig. 3). These molecules are stacked upon each other to form a right-handed helix with a 70 Å helical pitch and 150 Å diameter (Fig. 2). The cryo-EM structure of AdhE shows that the ALDH and ADH domains in the AdhE monomer are separated by a linker (residues 441–448) (Fig. 3a). Together with a β-hairpin protruding from the ALDH domain, the linker makes a three-stranded β-sheet stabilizing connections between the two catalytic domains (Fig. 3a). The structure of the ALDH domain is similar to other known ALDHs and is composed of two lobes. Each lobe has a canonical Rossman fold15 formed by a β-sheet surrounded by helices forming a NADH+ binding cleft, as observed in other dehydrogenases16. The ADH domain also consists of two lobes with a Fe2+ and NADH+-binding pocket between them, which is similar to other ADH domains8. Two AdhE monomers form a dimer in a head-to-head arm-crossing fashion (Fig. 3b). The three-stranded β-sheet in the linker forms a continual β-sheet interaction with the β-sheet within the ALDH domain from the other molecule (Fig. 3b). Subsequently, two dimers (four AdhE molecules) form one helical pitch via the interaction of ADH domains in a tail-to-tail manner (Fig. 3c). With this configuration, six AdhE molecules and two ADH domains at the top and the bottom of the helical structure comprise about one-and-a-half helical turns in our cryo-EM structure. By repeating the helical unit, AdhEs form into a spirosome structure, which might lead to activation of its biochemical activity by clustering enzymes.

Fig. 1 Cryo-EM analysis of AdhE. a AdhE eluted across a broad molecular weight range in Superdex 200 gel-filtration. The void volume (V 0 ) and elution volume for a molecular weight marker are indicated above the elution profile, and the fractions examined by negative stain EM are indicated below the profile. An SDS-PAGE gel shows the purity of AdhE used as input for the gel-filtration. b Negative stain EM analysis of fractions 1, 2 and 3 showing that AdhE forms a range of higher order structures. Scale bar 50 nm. c A representative micrograph (left) and 2D class averages (right). d Cryo-EM maps of AdhE in two different orientations. The residual density at the top and the bottom in the left panel indicates the helical property of AdhE Full size image

Fig. 2 AdhE forms a spirosome structure. a The cryo-EM structure of AdhE with six AdhE and two ADH domains fitted to the cryo-EM map. Each AdhE subunit is in a different colour. The structure is viewed from different angles (left, right and below). b The cryo-EM map with the refined model Full size image

Fig. 3 Hierarchical formation of AdhE spirosome from a monomer. a A full-length AdhE monomer is shown as a ribbon model. The N-terminal ALDH domain (royal blue) and C-terminal ADH domain (light purple) are linked by a short β-sheet composed of one β-strand from the linker and two β-strands from the ALDH domain. NAD+ and Fe2+ were modeled from other alcohol dehydrogenase structures (PDB IDs: 3MY7, 3ZDR). b AdhE forms a dimer by interacting in a head-to-head arm-crossing fashion. The short β-sheet in the linker forms a continual β-sheet with the β-sheet from the ALDH domain. c ADH domains from the AdhE dimer interact in a tail-to-tail manner and a total of four AdhE molecules make one helical turn Full size image

The spirosome topologically separtes ALDH and ADH activities

To further investigate the nature of the spirosome structure of AdhE, we collected cryo-EM micrographs from the sample containing longer spirosomes (fraction 1) with a Talos Artica 200 keV microscope using a Falcon III direct detector in an integration mode. The ends of the spirosome molecules were manually picked and subsequently picked with helical auto-picking using Relion17. A final 39,443 particle set was further processed according to the helical reconstruction process to generate 2D class-averages (Fig. 4a). 2D class-averages were selected and 3D classification was subsequently undertaken to generate an 11.2 Å cryo-EM structure of helical AdhE (Fig. 4b and Supplementary Fig. 4). Having the high-resolution cryo-EM structure of one-and-a-half helical turns, we were able to reconstitute a continual AdhE spirosome structure based on the cryo-EM structure resulting from helical reconstitution (Fig. 4b). In the spirosome structure, there are inter-helical interactions between ADH domains near the ADH catalytic site (Fig. 4b). Specifically, residues N492 and R488 from two ADH domains interact with each other, and Q821 interacts with the backbone of the loop between residues 816–821 from the other ADH domain. Interestingly, the NADH binding pocket is located near to the site of interaction between two ADH domains, suggesting that AdhE spirosome formation might affect its activity. In the spirosome structure, ALDH domains, as well as ADH domains from adjacent subunits, are clustered, which might render the substrate easily accessible to each activity (Fig. 4c). To further investigate the implication of the spirosome structure of AdhE, the plausible active sites of ALDH and ADH were highlighted on the helical structure. This practice reveals that ALDH active sites are located towards the outer surface of the helical structure while ADH active sites reside towards the inner surface. Thus, these two activities are topologically separated in the spirosome architecture (Fig. 4d). To further examine the properties of AdhE spirosome in solution, we undertook small angle X-ray scattering (SAXS). SAXS data for AdhE were obtained in batch mode without further fractionation by SEC. SAXS profiles were computed with the FoXS server18,19 for a series of models constructed from the continual spirosome structure (two of which are shown in Fig. 4e). This analysis indicates that while the experimental data derive from a polydisperse AdhE sample, the overall shape of the SAXS curve is broadly consistent with that computed for a spirosome model and is better described by the profile computed for the 24-mer model (the largest tested) than for the 12-mer or any lesser oligomers. This analysis is also consistent with the average length of spirosomes (460 Å), which corresponds to a 24 mer comprising 7 pitches (1 pitch = 70 Å). Overall, these combined data show that AdhE forms into a spirosome structure by which the activity of AdhE might be activated.

Fig. 4 Helical reconstruction of AdhE spirosome. a A representative micrograph for helical reconstruction (left) and 2D class averages from the helical reconstruction in Relion (right). b The high-resolution structures of 12 AdhE molecules were placed on the helical cryo-EM structure (right). Two-and-a-half helical turns of AdhE are shown with the atomic model. The red square indicates the region of the inter-helical interactions between ADH domains. Residues (R488, N492, and Q821) involved in inter-helical interactions are shown in stick representation. c ALDH and ADH domains are coloured yellow and blue respectively, revealing domain clustering (side view on left and top view on right). d The locations of NAD+ cofactors modeled are shown in space-fill representation, revealing that the ALDH (red) and ADH (blue) catalytic pockets are topologically separated (side view on left and top view on right). e Surface representation of 12 (green) and 24 (red) AdhE molecules in spirosome formation (right) for which SAXS profiles (left, green and red lines, respectively), computed with the FoXS server18,19, fit the experimental SAXS data (left, black line) with \({\boldsymbol{\chi }}^2\) values of 48.7 and 7.12, respectively Full size image

The spirosome undergoes structural changes with NADH

AdhE spirosomes in bacterial lysate have previously been observed to occupy either an ‘open’ or a ‘closed’ conformation, depending on the presence or absence of cofactors (Fe2+ and NAD+)20. Work by Kessler and colleagues20 showed via negative stain EM that, in the presence of 5 mM NAD+ and 0.3 mM FeSO 4 , AdhE spirosomes relax to a looser helical assembly, changing length from 40–120 nm to 60–220 nm, and diameter from 15 ± 2 nm to 13.5 ± 1 nm. Here, we confirm a change in spirosome conformation in the presence of cofactors using negative stain EM and SAXS (Fig. 5). Compared with the negative stain image of AdhE in the absence of the cofactors, which is well fitted by our cryo-EM structure (Supplementary Fig. 5), the spirosome structure of AdhE in the presence of NAD+ and FeSO 4 seems to be extended along the long axis resulting in a narrower width and longer pitch than AdhE without cofactors (Fig. 5a), suggesting that spirosomes in an extended conformation are formed upon addition of the cofactors.

Fig. 5 AdhE spirosomes change conformation in the presence of cofactors. a Negative staining of AdhE in the absence (Apo) and the presence of NAD+ and FeSO 4 . The yellow triangles indicate the positions of one pitch of the spirosome. The box plot below shows the distribution of spirosome pitch sizes in the absence (- orange, average = 77.2 ± 12.1 Å) and the presence (+ blue, average = 103.6 ± 13.1 Å) of the cofactors (n = 100, ***p value: 4.79 × 10–25). The box includes the inter-quartile range from Q1 to Q2 and the x in the box indicates the average. The upper and lower dots indicate the maximum and minimum value respectively. b SAXS data acquired in batch mode for AdhE fraction 1 in the presence (blue) and absence (orange) of cofactors reveal conformational changes evidenced by a shift in a conserved feature from q = 0.086 Å−1 (indicated by 1 in the inset) for Apo-AdhE to q = 0.075 Å−1 (2 in the inset) for AdhE + cofactors, which translates to 73.0 and 83.7 Å in real space Full size image

To observe the global conformational change in the entire population of spirosome species in each sample in the presence and absence of cofactors in solution, SAXS data for AdhE in its apo form and with different combinations of cofactors (NADH + FeSO 4 , NADH, NAD+ + FeSO 4 and NAD+) were acquired in batch mode without fractionation by SEC. A dramatic shift in the reciprocal space position of the feature characteristic of AdhE spirosomes from q = 0.086 Å−1 for apo- AdhE to q = 0.075 Å−1 is observed upon the addition of all cofactor combinations (Fig. 5b and Supplementary Fig. 6). This feature is consistent with the helical pitch of the cryo-EM structure of spirosomes and translates to a relaxation in real-space from 73.0 to 83.7 Å upon cofactor addition. These data suggest that the flexibility of the spirosome structure might be implicated in its activity.

The spirosome structure is required for AdhE activity

Next, we asked whether spirosome structure has implications for AdhE activity. To design a mutant disrupting the helical formation, the interface between AdhE molecules was examined. F670 in the ADH domain is inserted into a hydrophobic pocket formed by F462, I460, and I712 of the other ADH domain and holds the ADH-ADH domains together (Fig. 6a). To disrupt the AdhE self-association, F670 was mutated to several amino acids: Val (V), Ala (A), and Glu (E) (Supplementary Fig. 7). All mutants eluted much later in gel-filtration indicating that oligomerisation was disrupted in all the mutants (Fig. 6b). The gel-filtration profiles of all mutants showed a symmetric single peak and the mutants behaved well during purification indicating that the mutations did not disrupt the global structure of AdhE (Fig. 6b and Supplementary Fig. 7). To examine if the spirosome structure was disrupted in these mutants, they were examined by negative stain EM, analytical ultracentrifugation (AUC) and SAXS. Compared with the wild-type (WT), the spirosome structure was not observed in negative stain EM for any of the mutants (Fig. 6c), suggesting that the hydrophobic interaction mediated by F670 is critical for spirosome formation. Analysis of sedimentation velocity (SV)-AUC data for AdhE F670A (F670A), AdhE F670V (F670V) and AdhE F670E (F670E) demonstrates that large species remain in all three mutant samples but that, in comparison with AdhE from Yersinia pestis (AdhE YP ), the population becomes dominated by two lower s species (peaks ‘1’ and ‘2’ in Fig. 6d) at ≈ 5.1 S and 7.9 S. The smallest species in AdhE YP has s 20,w = 7.6 S (peak ‘3’ in Fig. 6d). s 20,w was calculated using SOMO21 for the coordinates of monomeric AdhE extracted from the high-resolution cryo-EM structure giving 5.1 S, in perfect agreement with that observed for peak ‘1’ in Fig. 6d, confirming that WT AdhE YP. is devoid of monomer.

Fig. 6 The helical organization of AdhE is critical for its activity. a The interface between ADH domains. Yellow, purple, blue and green colour indicates four AdhE molecules comprising one helical pitch. F670 inserted into a hydrophobic pocket is shown in a stick model and electrostatic surface representation (second from left). The detailed interaction around F670 with the hydrophobic pocket formed by I460, F462 and I712 (second from right and orthogonal view, right). b Gel-filtration profiles of WT AdhE (WT) and the mutants: AdhE F670A (F670A), AdhE F670V (F670V), and AdhE F670E (F670E). c Negative stain EM analysis of WT AdhE and the mutants reveals spirosome disruption. d c(s) analysis of SV data for AdhE F670A (F670A), AdhE F670V (F670V) and AdhE F670E (F670E) and AdhE expressed from Yersinia pestis (AdhE YP ). Spirosome disruption by the F670 mutation is evident in the s 20,w range ≈ 4–10 S. The distributions for all three F670 mutants include a peak (1) with \({\boldsymbol{s}}_{20},_{\boldsymbol{w}}\) = 5.1 S that is absent from AdhE YP . However, peak ‘2’ (\({\boldsymbol{s}}_{20},_{\boldsymbol{w}}\) = 7.9 S), observed in all F670 mutants, and peak ‘3’ (\({\boldsymbol{s}}_{20},_{\boldsymbol{w}}\) ≈ 7.6 S) for AdhE YP are almost overlapping and consistent with \({\boldsymbol{s}}_{20},_{\boldsymbol{w}}\) computed for dimeric AdhE. Primary data and quality of the fits to the data are in Supplementary Fig. 8. e Cartoon representation of an AdhE dimer extracted from the cryo-EM structure (blue) superimposed (r.m.s.d. = 16.22 Å) on the SREFLEX output model (red) (right) which fits the experimental SAXS data (left, black line) with a \({\boldsymbol{\chi }}^2\) value of 1.18 (red line). f Enzymatic assay of AdhE WT and its mutants. For the forward reaction (left), AdhE was incubated with acetyl-CoA and NADH and the consumption of NADH was monitored. For reverse reaction (right), AdhE was incubated with ethanol, CoASH, and NAD+ and the amount of NADH generated was monitored. The error bars show standard deviation (n = 3). g A scheme of the reaction Full size image