3.2.1 Binding epitope anomalies and “on-resonance scanning”
Serendipitously, during our studies of the interactions of the Carbohydrate Binding Module domain of RgNanH (CBM40) with Neu5Acα6Lac (6’SL, Figure 3.4a,b), we obtained the first results that indicated that relevant structural information can indeed be encrypted in the spin diffusion inhomogeneity. CBMs are small carbohydrate recognising domains associated to glycosidases. We were analysing the ligand specificity of CBM40 (21 kDa) through STD NMR investigation of a library of 15 sialic acid-containing ligands, leading to the structural elucidation of key features of their binding to CBM40 (whole study reported in Chapter 5, Section 5.3)97. The binding pocket of CBM40 is quite
shallow (Figure 3.4b) and the main element of recognition for all the ligands is the non- reducing sialic acid capping (Figure 3.4a). The binding of CBM40 to 6’SL (and to all the other ligands studied) can be considered weak (transient), with 𝐾𝐷 in the mM range, as quantified by ITC.
Figure 3.4. (a) Chemical structure of Neu5Acα6Lac (6’SL). (b) Crystal structure of the complex
6’SL/CBM40 (PDB ID: 6ER497): the binding site is shallow and the Neu5Ac is the main recognition
element. Ile95 is coloured in turquoise and indicated by an arrow (see Figure 3.5c for more details). (c) STD NMR experiment at 0.60 ppm and 2 s saturation time: reference spectra (x 1) in red and difference spectra (x 4) in black. (d) STD NMR experiment at 6.77 ppm and 2 s saturation time: same colouring scheme and scaling as (c). The signals coming from the sialic acid (main
88 | P a g e element of recognition) are labelled. The relative ratio betwee n the signal from the methyl group and the rest of the sialic acid signals is visibly higher when irradiating at 0.60 ppm.
The binding of 6’SL to CBM40 could be detected by STD NMR, with STD (%) signals for the sialic acid protons in the range of 10% when CBM40 was irradiated at a frequency of 0.60 ppm (where most of aliphatic resonances are found) and 2 s saturation time. STD signals from the lactose moiety where weaker as this part of the ligand is loosely bound. Interestingly, the methyl group of the sialic acid moiety showed a significantly higher STD % (60%, Figure 3.4c). The over 5-fold difference observed between the strongest STD signals of the methyl group and the signal of the rest of the protons lead us to name this effect the “STD incremental effect”. However, when the same sample was irradiated at 6.77 ppm (in the aromatic region of the protein) the result was a stark drop in the STD intensity of the methyl group, relative to the rest of the signals (Figure 3.4d). The same behaviour of the STD intensity of the methyl group in STD NMR experiments at two different frequencies (0.60 ppm and 6.77 ppm) was observed for all the other sialic-acid containing binders (as reported in Chapter 5, Section 5.3).
To exclude the likelihood of direct ligand irradiation during irradiation at the aliphatic region, i.e. at 0.60 ppm (the methyl group resonates at 1.74 ppm), STD NMR experiments at 2 s saturation time were performed at increasing irradiation frequencies with 0.20 ppm increments. This meant scanning the spectral region from -0.20 ppm to 1.40 ppm, in what we call “on-resonance scanning” (Figure 3.5a). The STD intensities for the methyl protons and for H6 of the sialic acid (as a reporter signal) were recorded, showing a very interesting trend: after an increase of the STD signal of the methyl group from 60% to 80% moving from 0.60 ppm to 0.80 ppm, there was a drop between 1.00 ppm and 1.20 ppm. The signal then slightly increased at 1.40 ppm, where we were only 0.40 ppm away from the methyl resonance frequency itself and direct irradiation was certainly playing a role. On the contrary, the control peak H6 stayed almost steady along the spectral window (Figure 3.5a). This suggests that the Neu5Ac methyl group reaches a maximum STD intensity between 0.80 ppm and 1.00 ppm.
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Figure 3.5. (a) “On-resonance scanning” from -0.2 ppm to 1.4 ppm for the CBM40/6’SL complex.
For each experiment, the absolute STD intensity is recorded at 2 s saturation time. (b) Predicted distribution of protein protons resonating at each 0.20 ppm interval, as obtained from ShiftX2 on the crystal structure of CBM40 at the experimental conditions. (c) Detail of the crystal structure showing the relative orientation of the sialic acid of 6’SL and Ile95 (PDB ID: 6ER497).
The turquoise surface encloses the directly irradiated protein protons and the ligand methyl group object of the analysis.
We identified the two methyl groups of Ile95 at the binding pocket of CBM40 as having predicted resonance frequencies (by ShiftX2) at 0.84 ppm and 0.58 ppm. Ile95 is pointing the two methyl protons towards the ligand methyl group in the bound state (Figure 3.5c), and thus, potentially direct irradiation of those Ile95 protons along the scanning were responsible for the behaviour of the Neu5Ac methyl group.
This data helps ruling out that the “STD incremental effect” observed for the methyl group of Neu5Ac is given by direct irradiation (further evidence to this argument is provided in Chapter 5, Sub-section 5.3.2). More importantly, this data gave ground to formulate Hypothesis A: arguably, as the isoleucine methyl protons are being directly irradiated, thus they can transfer an enlarged amount of saturation to the Neu5Ac’s methyl group, very close in space in the bound state, in comparison to other frequencies, hence boosting its STD intensity (Figure 3.5c). In this case, central irradiation of the isoleucine seemed to be achieved at 0.80 ppm in agreement with the ShiftX2 prediction (at 0.60 ppm the effect is also appreciable).
To our best knowledge, the data shown in Figure 3.5a is the first reported evidence that selective irradiation of residues lining the binding pocket and contacting the ligand can help identify which part of the ligand is closer to those amino acids.
90 | P a g e This was a preliminary result of paramount importance for us, as it paved the way to carry out differential frequency irradiation experiments, which, applied to the optimal systems, lead to the formulation of DiffErential EPitope mapping STD NMR (DEEP-STD NMR), object of Section 3.3.
3.2.2 Further considerations on the “STD incremental effect” observed on the 6’SL/CBM40 complex
The “STD incremental effect” observed on the methyl group of 6’SL as bound to CBM40 can be considered as an anomalous STD outcome. To fully justify such a large effect, a few additional considerations seem necessary before moving to more standard situations (where 5-fold STD differences between the first and the second strong signal are rarely observed). First, it must be considered that CBM40 is a relatively small protein (21 kDa) whose 1D 1H NMR peaks look not as broad as larger-sized proteins, due to its
shorter τc (estimated to be circa 20 ns). For comparison, Figure 3.6 shows the 1D 1H NMR
spectra of a larger protein (GH33, τc estimated to 50 ns, Figure 3.6a), whose peaks look
more like a so-called “protein envelope” (Figure 3.6b). Arguably, sharper protein peaks allow more intense direct irradiation and stronger saturation transfer, magnifying the effect of inhomogeneous spin diffusion for small proteins as compared as large proteins, as sketched in Figure 3.6c,d. The chemical shift dispersion of the protein protons under the effect of T2 broadening have already been reported by Kang et al. to affect strongly
the STD outcome (for a large antibody they had to correct CORCEMA-ST calculations to account for line broadening)98. Furthermore, the low affinity, the shallow geometry of
the subsite and most likely a very fast kinetics of interaction intensify the intensity gap between the saturation received by the methyl group, which is the only one buried in the hydrophobic pocket (containing Ile95, Tyr116 and Tyr210), and the other Neu5Ac protons. Low affinity typically involves short residence time, which could explain the fact that the rest of the Neu5Ac protons receive a considerably lower saturation. This effect is partially cancelled out when irradiation at 6.77 ppm is performed, where a more homogeneous protein saturation is achieved, so there are no “hot spots” in the binding pocket close to ligand protons (like the “hot” methyl groups of Ile95 when irradiating at 0.60 ppm).
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Figure 3.6. Left panel: 1H NMR spectral region (0.10 ppm‐1.40 ppm) of the proteins GH33 (52
kDa) (a) and CBM40 (21 kDa) (b) Fourier transformed with the same line broadening factor of the window function. Due to its smaller size (and τc), the peaks of CBM40 look sharper and less
“envelope‐like”. Right panel: cartoon representing the T2 broadening effect on the protein
signals. Sharp signals receive much more magnetization when primarily irradiated (and thus transfer it more efficiently to the close ligand protons). Ideally isolating a single protein signal, irradiation pulse of the same width will saturate a much higher percentage of the sharp signal
(c) than on the broad one (d).