V44M and V44A cause large CSP at the ε-cleavage site T48

Isotopically labelled APPTM WT V44M were reconstituted in dodecylphosphocholine (DPC) micelles as described in Chen et al.32 and their complete assignments have been achieved with triple resonance experiments and deposited in BioMagResBank (BMRB entry 18,648 and 18,649). The residues are numbered according to Aβ (Supplementary Fig. S1). Figure 1a shows the 15N–1H HSQC spectra of WT and V44M APPTM, with narrow dispersion typical of TM helices. The two spectra have similar peak patterns, suggesting that the V44M mutation does not change the overall fold of APPTM. Large chemical shift perturbations (CSPs) are observed at and near the site of the mutation (residues 44–48), as expected (Fig. 1b). While the largest 15N CSP is at V44M, the largest 1H CSP occurs at T48 (Fig. 1b), whose amide proton chemical shift decreases from 8.14 p.p.m. in WT to 7.93 p.p.m. in the mutant. The ~0.2 p.p.m. CSP indicates significant changes in magnetic environment of T48 amide proton, most likely due to changes in hydrogen bonding. We also have compared the backbone amide chemical shifts of V44A, another FAD mutant, with WT (Fig. 1b). While the largest 15N CSP is at the site of mutation V44A, the largest 1H CSP again occurs at T48 (Fig. 1b). As T48 is the initial recognition site of γ-secretase for the Aβ42 production line (Supplementary Fig. S1)14, such changes in the local conformations of the ε-cleavage site may have important implications for the Aβ42/Aβ40 ratio.

Figure 1: FAD mutations within APPTM of V44M and V44A cause large changes in amide proton chemical shift of T48. (a) 15N-H HSQC of APPTM WT (blue peaks), overlaid with that of V44M (red peaks). Residues M-2 and A-1 are non-APPTM residues N-terminal to APPTM due to cloning. (b) CSP caused by V44M (▲ joined by solid lines) and V44A (● joined by dashed lines) in amide proton (red) and nitrogen (black). Residues are numbered according to Aβ for easy correlation with Aβ production (Supplementary Fig. S1). In both FAD mutants, largest proton CSP occurs at T48. While the proton CSP pattern is similar to V44M, V44A causes larger nitrogen CSP at residues 44–46. Average backbone nitrogen chemical shifts of V, M, A are 121.08, 120.07 and 123.22 p.p.m., respectively, which mirrors the larger and opposite change in 15N CSP at residue 44 in V44A compared with V44M. Full size image

V44M changes local conformations of ε-cleavage sites

To further investigate the detailed structural changes brought about by the V44M mutation, we solved the solution structures of both APPTM WT and V44M dimers, which were based on unambiguous intermolecular NOEs (Fig. 2) and RDCs from two different alignment media (Table 1). The dimerization of APPTM in DPC micelles was confirmed by analytical ultracentrifugation (Supplementary Fig. S2) and supported by numerous previous studies24,25,26,27,28. Unambiguous intermolecular NOEs were determined by a number of chimera NMR samples with selective isotopical labelling (Fig. 2). Because only one set of NMR resonances are observed (Fig. 1a), the structure was calculated as a symmetric dimer. On average, 25.4 (WT) and 25.9 (V44M) constraints per residue were obtained, including 9 intermolecular NOEs for WT and 11 intermolecular NOEs for V44M. An ensemble of 20 structures were calculated from XPLOR-NIH for WT and V44M (for stereo images of superimposed NMR structures, see Supplementary Figs S3 and S4), with backbone and heavy atom pairwise RMSDs at 0.79 and 1.28 Å (WT) and 0.81 and 1.31 Å (V44M), respectively (Table 1). The coordinates and constraints have been deposited in the protein data bank as 2LZ3 (WT) and 2LZ4 (V44M).

Figure 2: Unambiguous determination of intermolecular NOEs. Reciprocal intermolecular NOE peaks between V39HN and V39HG1 (a), and between V46HN and V46HG2 (b), detected in a 1:1 mixed sample of uniformly 13C labelled APPTM and uniformly 15N labelled APPTM, yielding unambiguous intermolecular NOEs between 13C-H and 15N-H. The left panel is the strip from 13C-selected 15N-NOESY, while the right panel is the strip from 15N-selected 13C-NOESY. (c) V40HN-V39G1 intermolecular NOE detected in a 1:1 mixed sample of uniformly 15N-labelled and perdeuterated V44M, and natural abundance V44M, by 3D 15N-NOESY, giving rise to the intermolecular NOEs between 15N-H and aliphatic protons. (d) Intermolecular NOEs detected by filtered 13C-NOESY in a 1:1 mixed sample of 13C,15N-APPTM and unlabelled APPTM. A new intermolecular NOEs M44E-V49G1 appears in the V44M mutant. Full size image

Table 1 NMR and refinement statistics for APPTM structures. Full size table

Both WT and V44M form right-handed dimers, as in the classical TM dimer of glycophorin A (GpA) (Fig. 3)33. The crossing angle between the two helices is 22° for WT and 33° for V44M. For both APPTM WT and V44M, the GXXXA motif (residues 38–42) mediates the dimer interface (Fig. 3a). In contrast, the GXXXG motifs in APPTM do not have a direct role in dimerization. In addition, the side chains of M35, V39, I45, V46 and L49 provide key hydrophobic interactions at the dimer interface for the WT molecule. There is no hydrogen bond or salt bridge across the dimer interface.

Figure 3: NMR structures and dimer interfaces of WT and V44M. The interface residues are colored green in WT structure (a) and magneta in V44M structure (b). Interfacial residues unique to WT is labelled in green letters while those unique to V44M is labelled in magenta. GXXXA is the dimerization motif in both structures, but the interface is shifted towards the C-terminus in V44M. (c) Overlay of the ribbon representation of structures of WT (red) and V44M (blue). V44M conserves the overall dimer fold, with an RMSD between the average structures of WT and V44M of 2.4 Å. Full size image

The dimer fold and interface are generally retained in the V44M mutant but the dimerization pattern is altered significantly (Fig. 3b). The GXXXA motif and the side chains of M35, V39 and V46 still mediate dimerization; however, there is a registry shift in hydrophobic side-chain packing towards the C-terminus. I45 and L49 are no longer present at the dimer interface in V44M while T43 and V50 provide new hydrophobic packing for V44M dimerization. While T48 is not at the dimer interface in either structure, V44M mutation relocates L49 away from the dimer interface.

The large CSP in the T48 amide proton led us to more closely examine the helical hydrogen bonds. Overall, the V44M mutation strengthens hydrogen bonding at the N-terminal half of the helix while weakening those at the C-terminal half (Fig. 4). The helical hydrogen bonds involving the ε-cleavage site T48 and L49 amides experience large changes. In the 20 NMR structures, the average distance between the T48 amide proton and the V44 carbonyl oxygen is 2.2±0.2 Å and 3.23±0.14 Å in the WT and V44M mutant, respectively. The average distance between the L49 amide proton and the I45 carbonyl oxygen is 2.3±0.2 Å and 2.8±0.4 Å in WT and V44M, respectively. Thus the helical hydrogen bond involving both T48 and L49 amide protons are both weakened and the effect is much more pronounced for T48 than for L49.

Figure 4: Average length of helical hydrogen bonds in APPTM WT and V44M. The average distances between the donor amide proton (residue i+4) and the acceptor carbonyl (residue i) in the TM helix in the 20 NMR structures and their standard deviations are plotted versus residue number of the donor amide. The error bars here are standard deviations of hydrogen bond lengths derived from 20 NMR structures. Overall, hydrogen bonds at the N-terminal half become shorter while hydrogen bonds at the C-terminal half become longer in V44M, suggesting that the V44M mutation tightens up the N-terminal half helix while loosening up the C-terminal half helix. In particular, the helical hydrogen bond involving T48 amide is significantly lengthened, consistent with its decreased amide chemical shift in V44M. Full size image

V44M and V44A enhance k ex of T48 more than L49

To further probe helical stability and dynamics with residue-specific resolution, we have carried out hydrogen–deuterium (HD) exchange measurements at 298 K (Fig. 5a). Helical core residues between I41 and I47 had no decrease in their signal intensity in HSQC spectra after 30 h in D 2 O, due to very slow exchange with water, in both WT and V44M (Fig. 5a and Supplementary Fig. S5). Residues V36–V40 display decreasing exchange rates towards the centre of the helix while exchange rates of residues T48–V50 increase towards the C-terminus in both WT and V44M. T48 has an ~4-fold enhancement in HD exchange rate, increasing from 0.009 per h in WT to 0.034 per h in V44M (Figs 4 and 5a), while L49 has an ~2-fold increase (0.028 per h in WT versus 0.05 per h in V44M) (Fig. 5a). Consequently, both initial ε-cleavage sites are destabilized by V44M, but the effect is more significant for T48 than for L49. Titration with paramagnetic relaxation enhancement (PRE) probes demonstrated similar micelle embedment around the ε-cleavage sites in WT and V44M (Supplementary Fig. S6), suggesting the observed changes in HD exchange rates are not due to the differences in micelle embedment.

Figure 5: HD exchange shows that FAD mutants V44M and V44A destabilize the local conformations of T48 more than L49. (a) HD exchange rates for slowly exchanging residues in WT APPTM, V44M and V44A are plotted. Terminal residues (K28-M35 and L52-K55) exchange with D 2 O completely within 1 h (k ex >5 per h) and their rates are not plotted. Exchange rates for T48 are significantly enhanced by V44A and V44M mutations while the increases for L49 are to a lesser extent. Increased k ex values at A44 and T43 are likely due to the decrease in side-chain size from a valine to an alanine in V44A. The error bars are standard deviations that are derived from a Monte Carlo simulation assuming Gaussian distribution of random error in peak heights. (b) Novel mechanism of how FAD mutation V44M and V44A increase Aβ42/Aβ40 ratio. V44M and V44A open up T48 for the initial ε-cleavage by γ-secretase. The change in Aβ product line preference towards Aβ42 by FAD mutations increases Aβ42/Aβ40 ratio. Full size image