*4.2. The Transition-State Analogues ATP*/*ADP:Vi and Aluminium Fluorides (ADP:AlFx)*

In order to investigate the transition states of BmrA and DnaB, we used the solid-state NMR techniques already described above, and also complemented them by EPR (Figure 1). The conformation of DnaB in the presence of ADP:Vi was compared with DnaB apo (Figure 6A) and DnaB in the presence of ADP only (Figure 6B). We also studied the protein with ADP and DNA, in the presence or absence of vanadate (Figure 6C).

The <sup>13</sup>C-13C 2D DARR spectra of DnaB apo and DnaB:ADP:Vi display a few shifting resonances upon binding of the nucleotide (Figure 6A). However, the comparison of DnaB:ADP with and without vanadate shows that the NMR fingerprints of both samples are actually highly similar (Figure 6B), indicating that vanadate did not bind to the NBD and did not induce significant conformational changes. In contrast, when DNA is added to both samples, the NMR spectra of DnaB:ADP:Vi+DNA are different from the ones in the absence of DNA (DnaB:ADP:Vi and DnaB:ADP), with significant CSPs, but the most obvious CSPs are observed for the complex DnaB:ADP:DNA (Figure 6C, left panel). Since these two samples behave differently, a <sup>31</sup>P NMR spectrum was recorded to probe the bound ATP-mimics. The 1D <sup>31</sup>P-CP spectrum of DnaB:ADP:DNA displays two phosphorus peaks assigned to DNA (two DNA nucleotides bind to one DnaB monomer leading to two different phosphate binding environments [82]), and four peaks which can be assigned to bound ADP [88] (Figure 6C, right panel). Pα and Pβ correspond to the DnaB:ADP complex in the absence of DNA, and Pα' and Pβ' to the DnaB:ADP:DNA complex, indicating an insufficient DNA concentration to saturate the protein completely with DNA. However, the 1D <sup>31</sup>P-CP spectrum of DnaB:ADP:Vi:DNA (Figure 6C, right panel) shows only one population of ADP, with <sup>31</sup>P chemical-shift values similar to the DnaB:ADP complex, and a reduced intensity of the peaks assigned to the DNA. One can conclude from these spectra that the presence of vanadate actually inhibits the binding of DNA to DnaB.

γ

γ

− −

**Figure 6.** Comparison of the effect of vanadate on BmrA and DnaB. Alanine region from <sup>13</sup>C-13C-DARR spectra of DnaB:ADP:Vi overlaid with DnaB apo (**A**), DnaB:ADP (**B**). Alanine region from <sup>13</sup>C-13C-DARR (**C**, left panel) and 1D <sup>31</sup>P-CP (**C**, right panel) spectra of DnaB:ADP:Vi;DNA overlaid with DnaB:ADP:DNA. Alanine region from <sup>13</sup>C-13C-DARR spectra of BmrA:ADP:Vi overlaid with BmrA apo (**D**), BmrA-E504A:ATP (**E**) and BmrA:ADP (left panel) complemented with the 1D <sup>31</sup>P spectrum (**F**, right panel). The resonance peak from the lipids (0 ppm) was cut, the separation between the two spectra is indicated by a dashed line. The blue spectrum in (**A**) was adapted from Wiegand et al. 2019 and spectra (**D**) and (**E**) were adapted from Lacabanne et al. 2019 [82,92] (http://creativecommons.org/licenses/by/4.0/.). The red spectra in (**A**) and (**C**), as well as the spectra in (**F**) are original data.

For the ABC transporter BmrA, one must say beforehand that conformational changes are not observed in BmrA upon incubating with vanadate and ADP. The protein requires vanadate and ATP instead of ADP, since ATP hydrolysis is required to induce the conformational changes. The inorganic phosphate is then exchanged by a vanadate anion, and Vi and ADP remain bound. The comparison between the <sup>13</sup>C-13C DARR spectra of BmrA apo and BmrA:ATP:Vi shows both CSPs and new appearing peaks (Figure 6D). The presence of additional signals appearing in the protein spectra is indicative of a decrease of the flexibility of the corresponding protein residues [92]. When compared to the pre-hydrolytic state (obtained with BmrA-E504A:ATP), the new peaks appearing in the spectra overlay to a large extent (Figure 6E), indicating that these residues show a similar conformation. Some differences with respect to the pre-hydrolytic state can be observed, which can be associated to the addition of vanadate. To highlight the effect of vanadate, the spectrum of BmrA:ADP:Vi was compared to BmrA:ADP only (Figure 6F). This revealed the presence of new peaks, but only minor CSPs. The appearing peaks can serve as the fingerprint pattern that allows to distinguish the pre-hydrolytic and transition states, while the CSPs serve as the fingerprint pattern reflecting the kind of nucleotide bound.

A 1D <sup>31</sup>P-CP NMR experiment can yield complementary information about the bound ATP-mimics (Figure 6F, right panel). The <sup>31</sup>P spectrum of BmrA:ADP shows the presence of two populations of ADP (labeled Pα, Pβ and Pα', Pβ'), and the presence of vanadate induces <sup>31</sup>P chemical-shift changes for BmrA which were not observed for DnaB [12]. In case of BmrA:ADP:Vi, two populations of Pβ can be clearly distinguished and also for Pα, but less significantly (Pβ of ADP with vanadate has a different chemical shift than Pβ of ADP without vanadate). It is known that the trapping of one nucleotide during the transition state (in presence of vanadate) is possible while the second nucleotide can be poorly bound. This property has been observed for several ABC transporters (p-gp [109]; BmrA [99]; LmrA [110]; Maltose transporter [53]) suggesting an asymmetry of the NBDs [111].

In order to gain additional insight into whether vanadate binding occurred or not, we performed EDNMR experiments. This approach can be used to detect the <sup>51</sup>V nucleus (I = 7/2) in proteins in which the Mg2<sup>+</sup> has been replaced by the EPR-active Mn2<sup>+</sup> metal ion [16,112] in the nucleotide-binding sites, as sketched in Figure 1. The experiment detects the hyperfine couplings of the unpaired electrons of Mn2<sup>+</sup> to the nuclei in the vicinity. We applied this both to the ABC transporter and the DnaB helicase. One should mention that it was shown by biochemical investigations for both proteins that upon substitution of Mg2<sup>+</sup> by Mn2+, their biological function is maintained [86,113]. Figure 7 shows the resulting EDNMR spectrum for the BmrA:ADP:Vi complex (shown in red) with an intense resonance for <sup>51</sup>V (for the echo-detected field-swept EPR spectra see Figure S2). In the absence of protein in the sample (black line) the spectrum only shows a <sup>51</sup>V peak with very low intensity assigned to vanadate in solution. Unresolved couplings to <sup>23</sup>Na would appear at very similar frequencies. We thus conclude that vanadate binds to the NBD in the case of BmrA.

For DnaB, no <sup>51</sup>V peak can be observed in the EDNMR spectrum, indicating that no vanadate is found in the vicinity of Mn2<sup>+</sup> (Figure 7B). The EDNMR spectrum indeed shows the same profile for DnaB in the presence of nucleotide with vanadate (red line) and without (black line). We can thus exclude the presence of vanadate in the NBD of the protein. However, as shown previously in Figure 6B, some spectral differences (mainly appearing peaks upon ADP:Vi incubation) can be noticed when DnaB:ADP:Vi was compared to DnaB:ADP. In other words, these experiments do not allow to exclude that vanadate might bind at another location than in the NBD.

We thus used a complementary experiment which can directly detect <sup>51</sup>V using solid-state NMR. <sup>51</sup>V has been intensively studied by solid-state NMR due to its rather small nuclear quadrupole moment and its high sensitivity [114–116]. Vanadate has also been studied in biological systems using solution-state NMR [117,118] and solid-state NMR [119]. Figure 7C shows the <sup>51</sup>V MAS spectrum of DnaB:ADP:Vi recorded at two different MAS spinning frequencies of 17 and 19 kHz. By measuring at two different MAS frequencies, the central transition (|−1/2> ↔ |+1/2>, to first order free from quadrupole interaction, can be distinguished from the spinning-sideband positions resulting from first-order quadrupolar interaction (a superposition of the remaining single-quantum transitions, marked by asterisks). The presence of the first order quadrupolar coupling sideband pattern already points to immobilized <sup>51</sup>V species. We can distinguish two resonances at around −600 ppm (−604 ppm and −618 ppm) and two further vanadate species bound to DnaB at −533 and −681 ppm (Figure 7C). To assign those resonances, a spectrum of the not immobilized (the supernatant) <sup>51</sup>V was recorded and assigned (Figure 7D). The resonances of the <sup>51</sup>V MAS spectrum can be assigned by comparison with the solution-state spectrum of the supernatant as follows: VO<sup>4</sup> <sup>3</sup><sup>−</sup> (V1), V2O<sup>7</sup> <sup>4</sup><sup>−</sup> (V2), V4O<sup>12</sup> <sup>4</sup><sup>−</sup> (V4) and V5O<sup>15</sup> <sup>5</sup><sup>−</sup> (V5) [120,121]. We can exclude that the peaks corresponding to the immobilized phase peaks result from the precipitation of vanadate, since with an initial orthovanadate concentration of 5 mM (0.92 g L−<sup>1</sup> ) at pH 6, we are two orders of magnitude below the solubility limit. The detected signal thus must stem from DnaB-bound vanadate, which might be related to the observation that addition of vanadate interferes with DNA binding to DnaB (Figure 6C).

To sum up, vanadate is a reasonable ATP-transition-state mimic for the ABC transporter BmrA. The transporter is trapped, most likely in its outward-facing state, when binding ADP:Vi. The transition state is characterized by a characteristic fingerprint in the NMR <sup>13</sup>C-13C DARR spectrum, and vanadate is indeed present in the vicinity of the metal ion. In contrast, ADP:Vi is not a suitable ATP-transition-state mimic for the helicase DnaB. Indeed, solid-state NMR and EPR experiments reveal that vanadate does not bind to the NBD together with the nucleotide. Instead, vanadate is bound elsewhere to DnaB, most likely in an unspecific manner. ADP:Vi strongly inhibits binding of DNA, suggesting that they share the same binding site on DnaB, and that vanadate outcompetes DNA.

−

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− − −

− ↔

− −

− − −

**Figure 7.** Localisation of the vanadate ion using NMR and EPR experiments. Background corrected 94 GHz EDNMR spectra of BmrA (**A**) and DnaB (**B**) incubated with Mn2<sup>+</sup> and ADP, with and without vanadate. For <sup>31</sup>P, a doublet due to the hyperfine coupling to <sup>31</sup>P is observed (~4 MHz), as well as a singlet not assigned so far [122]. <sup>51</sup>V spectra of DnaB:ADPMg:DNA:Vi recorded at a MAS frequency of 17 and 19 kHz (**C**). <sup>51</sup>V spectra of DnaB:ADPMg:DNA:Vi overlaid with the solution-state spectrum of ADPMg:Vi (**D**). Central transitions are indicated with dashed lines whereas spinning sidebands are indicated with a black star. All panels represent original data.
