Loosening of Side-Chain Packing Associated with Perturbations in Peripheral Dynamics Induced by the D76N Mutation of β2-Microglobulin Revealed by Pressure-NMR and Molecular Dynamic Simulations

β2-Microglobulin (β2m) is the causative protein of dialysis-related amyloidosis, and its D76N variant is less stable and more prone to aggregation. Since their crystal structures are indistinguishable from each other, enhanced amyloidogenicity induced by the mutation may be attributed to changes in the structural dynamics of the molecule. We examined pressure and mutation effects on the β2m molecule by NMR and MD simulations, and found that the mutation induced the loosening of the inter-sheet packing of β2m, which is relevant to destabilization and subsequent amyloidogenicity. On the other hand, this loosening was coupled with perturbed dynamics at some peripheral regions. The key result for this conclusion was that both the mutation and pressure induced similar reductions in the mobility of these residues, suggesting that there is a common mechanism underlying the suppression of inherent fluctuations in the β2m molecule. Analyses of data obtained under high pressure conditions suggested that the network of dynamically correlated residues included not only the mutation site, but also distal residues, such as those of the C- and D-strands. Reductions in these local dynamics correlated with the loosening of inter-sheet packing.

. Displacement of C  atoms between several crystal structures of β2m.
The regions of residues 4-11, 21-40, 46-48, and 61-94 were aligned optimally and the distances between the same C  atoms were then calculated. The PDB data examined were 2yxf (separate monomer, pH 7.0), 1lds (separate monomer, pH 5.7), 4fxl (D76N, pH 7.0), and 1hsb (X-ray, complexed state in MHC class 1). Selection of experimental pH conditions for wild-type and D76N β2ms using tryptophan fluorescence pH-dependent spectral changes in the tryptophan fluorescence of wild-type and D76N β2ms were investigated ( Figure S2A). The sample for measurement contained 0.05 mg ml -1 protein, 2 mM sodium phosphate, 2 mM sodium acetate, and 100 mM NaCl at 25°C. Excitation and emission wavelengths were 280 nm with a slit-width of 5 nm and 300-400 nm with a slit-width of 5 nm, respectively. The pH of 3 mL of the sample solution in a 1×1 cm quartz cell was lowered by gradually adding 1 N HCl solution and spectra were obtained with a FP-6500 spectrofluorometer (JASCO Inc., Japan) at the respective pH points. The starting pH value was 6.7. Red and blue lines in Figure S2A indicate the spectra obtained at pH 6.7 and 2.5, respectively. The spectra obtained were quantified using the center of the spectral mass, <>, which expresses the average energy of each spectrum calculated from the following equation:[1] where i and Fi are the wavelength and fluorescence intensity at i. pH-dependent <> values ( Figure S2B) were analyzed with a sigmoidal function to obtain the midpoint pH value of unfolding. The midpoint pH values of unfolding were 4.11 ± 0.04 and 4.54 ± 0.03 for the wild type and D76N, respectively. Figure S3. Comparisons of Δδapp values obtained for WT at pH 5.0 and 5.5, and D76N at pH 5.5. We prepared a wild-type β2m solution in 20 mM NaAc (pH 5.5).
Then, measured the HSQC spectrum of the sample. Panel A shows the Δδapp pattern between WT at pH 5.0 and pH 5.5. Panel B shows the Δδapp pattern between WT and D76N at pH 5.5. Panel C (identical to the Figure 2B in the main text) shows the Δδapp pattern between WT at pH 5.0 and D76N at pH 5.5. It was found that no significant Δδapp values were observed for all residues in panel A.
On the other hand, the Δδapp patterns in panel B and C were similar to each other.
These observations indicate that the Δδapp pattern in Figure 2B reflects the conformational changes upon the mutation and does not include effects from the pH difference. The data process of pressure-induced Δδ data using the PCA-based method We performed high-pressure NMR measurements for the wild type ( Figure S4A).
We conducted the singular value decomposition (SVD) of the pressuredependent chemical shift data to obtain nine PCs and corresponding contribution ratios. The cumulative contribution ratio of the first two PCs was 0.81 ( Figure   S4C). Furthermore, the score plots, or variations in the fractions of each PC, of the 1st and 2nd PCs for all proteins were found to change smoothly in a pressuredependent manner, whereas those of the 3rd PC showed abrupt changes at certain pressure points ( Figure S4E). Therefore, we speculated that the first two PCs contained information on pressure-dependent structural changes, whereas the 3rd and higher PCs reflected the measurement noises of the chemical shift values. Figure S4G (identical to Figure 3B in the main text) shows a plot of PC1 and PC2. Although the major spectral change is in the direction of PC1, a significant contribution of PC2 was observed, which indicates that the spectral change includes contributions from two certain transitions, presumably (i) mechanical compression and (ii) thermodynamic transition. [2,3] The spectral change at lower pressure regions is often attributed to mechanical compression in the case of a structured protein. Thus, we set the direction along which the initial spectral change occurred as (i) mechanical compression. On the other hand, (ii) thermodynamic transition generally occurs in a certain pressure range. Thus, the direction of the spectral change observed in the middle pressure region is considered to represent thermodynamic transition (ii) and we assumed that its contributions may be orthogonal to the direction corresponding to (i) mechanical compression. Based on the directions identified, we calculated the Δ patterns corresponding to the directions of vectors (i) and (ii) ( Figure 3D and E in the main text). The same measurements and analyses were performed on D76N ( Figure   S4B). The cumulative contribution ratio of the first two PCs was 0.77 and we similarly interpreted the data obtained for D76N ( Figure S4D,F). Therefore, we also discuss the data containing the first two PCs for D76N ( Figure S4H and Figure 3C in the main text). The Δδ patterns obtained for (i) and (ii) on D76N are shown in Figure 3F and G in the main text, respectively.