Inter-Ligand-STD NMR (IL-STD NMR): an additional tool to detect adjacent binding

Even without being quantitative, the model proposed for the ternary complex in Figure 4.22 agrees with the ILOE results. This gave us ground to approach the system by IL-STD NMR, as introduced in Chapter 3, Section 3.4. The ternary complex at a 1 to 40 protein to ligand ratio was analysed by STD NMR, first at the standard irradiation frequency of 0.60 ppm (aliphatic region) and then at 7.27 ppm, on resonance with the protons Hc and Hd of 3NPG.

By simple visual analysis of the STD difference spectra acquired at 0.60 ppm and 7.27 ppm, an overall decrease of the STD signals when irradiating at the aromatic region (7.27 ppm) rather than at the aliphatic region (0.60 ppm) was evident. This evidence is easily explained by the generally lower occurrence of aromatic residues relative to aliphatic residues, meaning that a lower number of directly irradiated protein protons is irradiated at 7.27 ppm than at 0.60 ppm. This translate into less overall available magnetization and lower STD intensities upon irradiation in the aromatic region. An expected increase was observed for the STD intensities of the peak coming from Hc and Hd (7.27 ppm, directly irradiated) and for the aromatic signals of Ligand 33, as an effect

141 | P a g e of direct irradiation (these resonate within 0.15 ppm high-field from it, respectively 7.21 ppm, 7.16 ppm, 7.12 ppm). Also, the intensity of the Hb proton (7.69 ppm) increased when irradiating the adjacent Hc,d, due to intra-ligand NOE.

Interestingly, we observed an increase in the STD intensity of proton Htriazole of Ligand

33 (7.67 ppm) upon irradiation of proton Hc and Hd of 3NPG (7.27 ppm) (Figure 4.23).

This STD increase cannot be due to direct irradiation (the signal is 0.40 ppm apart from the source of irradiation) or intra-ligand NOE (as the directly irradiated aromatic protons of Ligand 33 are far in space from the triazole); hence, the signal increment can only be explained by the inter-ligand transfer of magnetization between Hc and d and Htriazole, which is only possible if those protons from the two ligands established a close contact in the bound state. This is in perfect agreement with the ILOE data (Figure 4.12). As argued in Chapter 3, Section 3.4, the risk of direct irradiation is the main limitation of this approach. For example, the inter-ligand correlation Hc and Hd/Htriazole observed by ILOE could be detected by IL-STD only via irradiation on the Hc and Hd signal of 3NPG. This is because the irradiation of Htriazole would have implied the direct irradiation of the 3PNG, due to the proximity of Htriazole to Hb of 3NPG, thus leading to spurious results.

Figure 4.23. IL-STD difference spectra of the ternary complex 3NPG/CTB/Ligand 33 with

irradiation at 0.60 ppm (in black) and at 7.27 ppm, on resonance with Hc and Hd of 3NPG (in red). The spectra are zoomed on the single protons for space optimization purposes. The assignment of all peaks is given and the peak of Htriazole is squared in turqouise and magnified, showing increased intensity when irradiating at 7.27 ppm, whereas all the other protons (with

142 | P a g e the exceptions discussed in the main text) decrease. A saturation time of 2 s and line broadening factor of 1 Hz were employed.

To exclude any possible artefacts, acquisition of control experiments under the same experimental conditions on the two binary complexes was necessary. The control experiment carried out on the binary complexes Ligand 33/CTB showed that, in the absence of 3NPG, the intensity of the triazole proton (as well as the rest of protons of

Ligand 33) decreased when irradiating at 7.27 ppm, ruling out that the observed IL-STD

was due to direct irradiation. An increase was observed for the aromatic protons between 7.24 and 7.04 ppm, which experience direct irradiation as indicated above (Figure 4.24a).

Figure 4.24. IL-STD control experiments on (a) 3NPG and (b) Ligand 33 in a complex with CTB.

Difference spectra with irradiation frequency of 0.60 ppm are shown in black, and difference spectra with direct irradiation at 7.27 ppm are shown in red. Same experimental conditions as for the spectra in Figure 4.23.

The control experiment on the binary complex 3NPG/CTB is shown in Figure 4.24b. Expectedly, all the STD intensities decreased when irradiated at 7.27 ppm, except for

143 | P a g e the directly irradiated protons Hc and Hd and the Hb, which received direct intra-ligand NOE from the adjacent Hc and Hd.

Quantitatively, these data can be better analysed by determining the IL-STD factor for each proton of the ligands constituting the ternary complex, and by comparing them with the IL-STD factor obtained in the binary complexes used as control samples (Figure 4.25). The equation for IL-STD calculation is described in Sub-section 3.4.2 (Equation 3.6). In our case, N = 1, therefore the equation simplifies to:

ΔIL − STD_{𝑖,𝑡𝑒𝑟𝑛𝑎𝑟𝑦} = 𝑆𝑇𝐷 𝑜𝑛−𝑙𝑖𝑔𝑎𝑛𝑑,𝑖 𝑆𝑇𝐷 _{𝑜𝑓𝑓 −𝑙𝑖𝑔𝑎𝑛𝑑,𝑖}− 1

By definition, the IL-STD factor will only be positive for protons receiving inter-ligand saturation from the irradiated proton of the adjacent fragment (if we are able to exclude the protons receiving extra saturation by intra-ligand NOE or direct irradiation excluded from the analysis).

Figure 4.25. ΔIL-STD histograms of the 3NPG/CTB/Ligand 33 ternary complex, and the two

control complexes Ligand 33/CTB and 3NPG/CTB (and controls), represented individually for

Ligand 33 (a) or 3NPG (b). The values relative to the Htriazole of Ligand 33 are squared in

turquoise. The bar chart is cut at 3.0. Raw and processed data in Table 4.2.

In fact, protons presenting positive ΔIL-STD in both the experiment and the control are subject to either intra-ligand NOE or direct irradiation (this is easily determined by looking at the spectra), while protons presenting negative values in both the experiment and the control are too far in space from the irradiated proton(s) to be affected. By looking at the histogram representation in Figure 4.25a we can easily appreciate how the Htriazole from Ligand 33 is the only proton with a positive IL-STD only in the ternary complex experiment (and a negative ΔIL-STD in the control experiment). The IL-STD data are reported in Table 4.2.

144 | P a g e Ligand 33/CTB/3NPG Ligand 33/CTB 1_{H δ Atom ID} STD% off ligand STD% on ligand ΔIL-STD STD% off ligand STD% on ligand ΔIL-STD 2.24 Me 11.46 5.02 -0.56 10.91 4.54 -0.58 3.48 H-2'' 4.19 3.69 -0.12 5.62 2.28 -0.59 3.79 H-3'' 4.95 4.26 -0.14 5.99 2.33 -0.61 4.23 H-4''b 2.9 2.41 -0.17 3.28 1.67 -0.49 5.03 CH2(Bn) 6.97 3.29 -0.53 6.14 3.45 -0.44 6.36 H-3''' 10.61 5.68 -0.46 10.7 5.73 -0.46 7.12 H para 9.16 42 3.59 11.57 52 3.49 7.16 H meta 11.13 54 3.85 10.5 57 4.43 7.21 H orto 9.25 65 6.03 10.15 73 6.19 7.68 Htriazol 7.83 9.71 0.24 5.84 3.48 -0.40 3NPG/CTB 5.50 H1 12.27 1.42 -0.88 12.51 13.43 0.11 3.73 H2 16.6 12.27 -0.26 18.49 11.92 -0.38 3.76 H3 18.13 11.69 -0.36 19.79 11.69 -0.42 3.81 H4 16.77 10.5 -0.37 17.44 11.24 -0.32 3.73 H5 17.1 11.02 -0.36 18.31 11.24 -0.38 3.40 H6/H6' 12.27 7.68 -0.37 15.81 6.7 -0.55 7.27 H cd 6.9 99 13.35 7.68 94.31 11.65 7.69 Hb 12.63 36.96 1.93 13.27 36.25 1.68 7.75 Ha 21.61 15.36 -0.29 23.59 15.81 -0.34

Table 4.2. Raw STD intensities for the ternary complex (3NPG/CTB/Ligand 33) and the two

controls. The STDoff ligand at 0.60 ppm and STDon ligand at 7.27 ppm, as well as the calculated ΔIL-STD

are shown. The ΔIL-STD relative to Htriazole are highlighted in red.

4.2.7 Relevance of the discovery of a novel binding subsite and the IL-STD approach from