Differences in solution dynamics between lens β-crystallin homodimers and heterodimers probed by hydrogen–deuterium exchange and deamidation

Abstract

Background Lens transparency is due to the ordered arrangement of the major structural proteins, called crystallins. βB2 crystallin in the lens of the eye readily forms dimers with other β-crystallin subunits, but the resulting heterodimer structures are not known and were investigated in this study. Methods Structures of βA3 and βB2 crystallin homodimers and the βA3/βB2 crystallin heterodimers were probed by measuring changes in solvent accessibility using hydrogen–deuterium exchange with mass spectrometry. We further mimicked deamidation in βB2 and probed the effect on the βA3/βB2 heterodimer. Results were confirmed with chemical crosslinking and NMR. Results Both βA3 and βB2 had significantly decreased deuterium levels in the heterodimer compared to their respective homodimers, suggesting that they had less solvent accessibility and were more compact in the heterodimer. The compact structure of βB2 was supported by the identification of chemical crosslinks between lysines in βB2 within the heterodimer that were inconsistent with βB2's extended homodimeric structure. The compact structure of βA3 was supported by an overall decrease in mobility of βA3 in the heterodimer detected by NMR. In βB2, peptides 70–84 and 121–134 were exposed in the homodimer, but buried in the heterodimer with ≥50% decreases in deuterium levels. Homologous peptides in βA3, 97–109 and 134–149, had 25–50% decreases in deuterium levels in the heterodimer. These peptides are probable sites of interaction between βB2 and βA3 and are located at the predicted interface between subunits with bent linkers. Deamidation at Q184 in βB2 at this predicted interface led to a less compact βB2 in the heterodimer. The more compact structure of the βA3/βB2 heterodimer was also more heat stable than either of the homodimers. Conclusions The major structural proteins in the lens, the β-crystallins, are not static, but dynamic in solution, with differences in accessibility between the homo-and heterodimers. This structural flexibility, particularly of βB2, may facilitate formation of different size higher-ordered structures found in the transparent lens. General significance Understanding complex hetero-oligomer interactions between β-crystallins in normal lens and how these interactions change during aging is fundamental to understanding the cause of cataracts.

Chemical Properties Determine Solubility and Stability in βγ‐Crystallins of the Eye Lens

“…βB2‐ [145] and βA3‐crystallins [146] are similarly destabilized by single or double deamidation, although their overall structures remain largely native‐like. Deamidations also impact the dynamics of β‐crystallins in the context of homo‐ and heterodimers: enhanced flexibility, especially for βB2, might promote the formation of larger oligomers that could lead to cataract [147] …”

“…Deamidations also impact the dynamics of βcrystallins in the context of homo-and heterodimers: enhanced flexibility, especially for βB2, might promote the formation of larger oligomers that could lead to cataract. [147] Deamidation also causes destabilization and altered intermolecular interactions in γ-crystallins. Although the structures of the N76D and N143D variants of HγS are similar to HγS-WT, these variants exhibit increased attractive forces to other proteins.…”

Cataract-associated deamidations on the surface of γS-crystallin increase protein unfolding and flexibility at distant regions

“…For NMR data collection, gS WT, N14D, and N76D were expressed in MJ9 minimal media at 37°C with 15 Heat-induced aggregation of gS-crystallin and deamidated mimics. gS WT was compared to deamidated mimics by measuring changes in their turbidity during heating at 70 °C using previously published methods (5,22,30,33,34). This temperature was chosen based on the midpoint of thermal aggregation at 65°C previously reported (26) Nuclear magnetic resonance of gS-crystallin and deamidated mimics.…”

Cumulative deamidations of the major lens protein γS‐crystallin increase its aggregation during unfolding and oxidation

“…12 The γ-crystallins are primarily monomeric with their domains linked by a connecting peptide that is bent whereas in β-crystallins the connecting peptide is either bent or extended leading to oligomer formation with hydrophobic interactions stabilizing the interface between the domains. [13][14][15][16] The β/γ-crystallins comprise approximately two-thirds of the protein content of the human lens by mass and are extensively modified. 17,18 Due to the absence of protein turnover, the lens is an incubator that accumulates multiple crystallin modifications over the individual's lifetime.…”

“…In γS‐crystallin (γS), the predominant γ‐crystallin produced postnatally, each domain comprises two Greek key motifs, that is, a total of eight β‐strands (Figure 1). 12 The γ‐crystallins are primarily monomeric with their domains linked by a connecting peptide that is bent whereas in β‐crystallins the connecting peptide is either bent or extended leading to oligomer formation with hydrophobic interactions stabilizing the interface between the domains 13–16 …”