Abstract
Nonsynonymous gene mutations can be beneficial, neutral, or detrimental to the stability, structure, and biological function of the encoded protein, but the effects of these mutations are often not readily predictable. For example, the β-propeller olfactomedin domain of myocilin (mOLF) exhibits a complex interrelationship among structure(s), stability, and aggregation. Numerous mutations within mOLF are linked to glaucoma; the resulting variants are less stable, aggregation-prone, and sequestered intracellularly, causing cytotoxicity. Here, we report the first stable mOLF variants carrying substitutions in the calcium-binding site that exhibit solution characteristics indistinguishable from those of glaucoma variants. Crystal structures of these stable variants at 1.8-2.0-Å resolution revealed features that we could not predict by molecular dynamics simulations, including loss of loop structure, helix unwinding, and a blade shift. Double mutants that combined a stabilizing substitution and a selected glaucoma-causing single-point mutant rescued in vitro folding and stability defects. In the context of full-length myocilin, secretion of stable single variants was indistinguishable from that of the WT protein, and the double mutants were secreted to varying extents. In summary, our finding that mOLF can tolerate particular substitutions that render the protein stable despite a conformational switch emphasizes the complexities in differentiating between benign and glaucoma-causing variants and provides new insight into the possible biological function of myocilin.
Highlights
Why has nature elected to employ an aggregation-prone, disease-associated mOLF in the trabecular meshwork (TM) of the human eye where mutations, sustained UV exposure, and other environmental and mechanical stressors (17, 24 –26) likely render myocilin susceptible to aggregation when a more stable variant like cOLF could, in theory, be resistant to these stressors and avoid disease [23]? Upon inspection of the major distinguishing structural feature in mOLF compared with cOLF, namely, the presence of a central heptacoordinate calcium ion bound in the hydrophilic central pore (Fig. 1, A and B), we considered the counterintuitive suggestion that the missing ionic interaction endows cOLF with higher stability
In line with the nearly 40 individual mutations we introduced previously into mOLF, which had either a neutral or detrimental effect on thermal stability [8, 10, 11, 27], our knowledge of the effect of the calcium-binding site on mOLF stability and structure (Fig. 1, A and B) to date has been limited to the effect of the glaucomatous mOLF(D380A) variant, which abolished Ca2ϩ binding at the expense of ϳ7 °C stability (melting temperature (Tm) ϭ 46.6 °C) [27]
We noticed that calcium ligands Asp-380 and Asn-428 in mOLF are largely conserved across the olfactomedin protein family, whereas Asp-478 exhibits larger variation (Fig. S2) [8]
Summary
Calcium-ablating mOLF variants at position 380, but not at 478, are thermally compromised. In line with the nearly 40 individual mutations we introduced previously into mOLF, which had either a neutral or detrimental effect on thermal stability [8, 10, 11, 27], our knowledge of the effect of the calcium-binding site on mOLF stability and structure (Fig. 1, A and B) to date has been limited to the effect of the glaucomatous mOLF(D380A) variant, which abolished Ca2ϩ binding at the expense of ϳ7 °C stability (melting temperature (Tm) ϭ 46.6 °C) [27]. In line with the results for D380A [27], changing Asp-380 to either Ser or Asn abolished calcium affinity, decreased thermal stability, and reduced soluble expression levels. Substitutions at Asp-478 abolished calcium binding as expected but conferred ϳ5–7 °C higher thermal stability than WT mOLF. The combination of the stabilizing D478S variant with selected glaucoma variants D380A, Y437H, and P370L rescues their impaired stability (Table 1). mOLF(D380A/D478S) and mOLF(D380A/D478N) are ϳ10 °C more stable than
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