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.