3.2. Oligomer interface
Protein oligomerization occurs in biological systems, with about 30-35% of total proteins involved (Gabizon and Friedler 2014; Kumari and Yadav 2019). Thus, the ability to regulate protein oligomerization by introducing mutations in oligomerization interfaces or engaging a specific modulator should prove useful for studying the biological role of protein oligomerization of interest (Kumari and Yadav 2019). On the one hand, there is a variety of circumstances in which a process of protein folding is incomplete or interrupted, eventually leading to formation of pathological or pathogenic protein oligomers (Figure 3B). For instance, the development of cognitive disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) is typically accompanied by the formation and accumulation of amyloid fibrils in the patient’s brain (Araki et al., 2019; Brown et al., 2020; Salahuddin et al., 2021). In this sense, artificial or natural modulators capable of inhibiting or reversing aberrant protein oligomerizations would be of great prophylactic or therapeutic measures to treat relevant diseases (Galzitskaya 2019; Lee et al., 2017). Above all, understanding the underlying mechanisms of protein misfolding is one of the primary steps for the development of novel therapeutic strategies. In the study using HDMS by Stephens et al., the unstructured amyloid protein, α-synuclein (aSyn) involved in PD, exhibited the highest solvent protection at the C-terminus, indicative of a fold at the C-terminal domain that had been presumed to play a role in modulating aggregation but without structural evidences (Stephens et al., 2018).
Aggregation of tau into the paired helical filament (PHF) is a characteristic feature of AD, and the way Cys-mediated disulfide bond is formed, either intermolecularly or intramolecularly, serves as a critical element in tau fibrillation. To understand a molecular feature of aggregation-resistance of tau conformer, Jebarupa et al. synthesized the intramolecular Cys cross-linked tau monomer by oxidation and analyzed the rearranged conformational dynamics by HDMS. As a result, they found that, due to induced intramolecular H-bonding, the oxidized tau exhibited increased conformational rigidity and reduced accessibility in the core of the oligo-inducing interface that otherwise would have made an intermolecular H-bonded β-sheet formation and subsequently tau fibrillation (Jebarupa et al., 2019). Other than the disulfide bond, aggregation propensity of tau was investigated in the context of its extent of phosphorylation by Zhu et al. Time-resolved electrospray ionization (TRESI) mass spectrometry in combination with hydrogen/deuterium exchange (TRESI-HDX) is responsive to dynamic, temporary, and weak hydrogen bond interactions, as well as solvent accessibility, both of which are influenced by residual structure — biases in their native conformational ensembles of intrinsically disordered proteins. Authors used TRESI-HDX to characterize the native structural ensembles of a full-length tau, one of the main amyloidogenic species in AD, offering a detailed picture of the conformational changes that occur upon hyperphosphorylation by a kinase GSK-3β (Zhu et al., 2015). Increased deuterium uptake of the hexapeptide motif (H2) of hyperphosphorylated tau sampled appropriately such that aggregates were not significantly populated at this time pointed to a dominant role for H2 in GSK-3β-mediated increases in tau amyloidogenic propensity, consistent with the conclusion of a previously reported loss-of-function mutagenesis study (von Bergen et al., 2000).
Aggregation-prone apolipoprotein E4 (ApoE4) is a major risk factor for AD and cardiovascular diseases. Huang et al. coupled HDMS to gas-phase electron-transfer dissociation fragmentation (Zehl et al., 2008) to achieve single amino acid resolution and specify residues responsible for self-association of ApoE4. Despite the lack of a determined crystal structure as a reference due to a high tendency of ApoE4 to aggregate, the method could tune the analytic concentrations appropriately and identify 15 residues in the C-terminal domain deemed critically situated in ApoE4 oligomerization interface (Huang et al., 2011).
Amyloid fibrils formed by β-2-microglobulin (β2m) are an inevitable symptom of kidney failure-induced dialysis in patients’ joints. Borotto et al. used HDMS to reveal a few structural insights into metal-induced amyloid development of β2m. They reported that the Cu(II) binding to Asp59 is required for the formation of amyloid-competent dimers, as well as cis-trans isomerization of the His31-Pro32 amide bond essential for the formation of the amyloidogenic conformer. In contrast, Ni(II) only binds to His31 and does not cause structural changes favorable for producing oligomers or amyloids. Interestingly, the dimer formation was observed in response to Zn(II) binding in a similar fashion with Cu(II)-induced β2m dimerization, but did not lead to the pathway to the amyloid. Focused investigation into the dimer interfaces by HDMS found out the Zn(II)-induced dimer interface quite different from the Cu(II)-induced dimer interface in terms of stability and local interfacial areas involved (Borotto et al., 2017). Abovementioned and related studies are summarized in Table 2.