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Bonnie Su, Bryan Dunyak, Kathleen Molnar, Leah Makley; IPV-containing peptides stabilize mutant αB-crystallin oligomers through conformational rearrangement. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5692.
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Age-related damage and/or mutations of the lens chaperone protein αB-crystallin (cryAB) lead to its destabilization and aggregation, which culminates in cataract formation. A hydrophobic binding site between strands β4 and β8 in cryAB permits oligomer assembly via interactions of cryAB homodimers. We found that binding of exogenous peptides at this site can stabilize the protein and suppress aggregation in vitro. We hypothesized that stabilization arises from allosteric rearrangement of the cryAB multimeric complex through engagement of this binding site. Using in vitro techniques, we sought to understand how peptide sequence, affinity, and conformational dynamics promote stabilization of mutant cryAB oligomers.
We designed a library of over 500 peptides based on naturally-occurring IXI- or IPV-motifs and measured their affinity to cryAB using fluorescence polarization (FP) and microscale thermophoresis (MST). We assessed the ability of high affinity peptides to prevent cryAB aggregation using a destabilized mutant of cryAB (R120G) in a turbidity aggregation assay. To understand peptide-induced conformational rearrangement upon binding, we characterized the interaction with heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) spectroscopy and size exclusion chromatography with multi-angle light scattering (SEC-MALS).
In the screen we identified multiple peptides with single-digit micromolar affinity for cryAB. One peptide, p11, bound cryAB with a Kd of 5.7 ± 1.8μM and showed promising activity in disaggregating mutant cryAB (EC50 = 25.9 ± 1.1μM). Another peptide, p8, bound cryAB with high affinity (Kd = 4.6 ± 2.2μM) but was significantly less effective at disaggregating cryAB (EC50 >300μM). NMR spectra of p11 bound to cryAB exhibited widespread chemical shift perturbations and peak broadening, suggestive of allosteric rearrangement. These effects were not observed with p8 in the NMR.
Exogenous peptides can bind the cryAB β4-β8 groove with high affinity. However, only a subset are competent to stabilize mutant cryAB and prevent or reverse aggregation. This suggests stabilization due to conformational rearrangement is not solely affinity driven. Peptide binding-induced allostery at this site may be a general mechanism for interactions of cryAB with certain proteins, allowing for manipulation of its oligomeric architecture and function.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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