Calcium-ligand variants of the myocilin olfactomedin propeller selected from invertebrate phyla reveal cross-talk with N-terminal blade and surface helices

Abstract

Olfactomedins are a family of modular proteins found in multicellular organisms that all contain five-bladed β-propeller olfactomedin (OLF) domains. In support of differential functions for the OLF propeller, the available crystal structures reveal that only some OLF domains harbor an internal calcium-binding site with ligands derived from a triad of residues. For the myocilin OLF domain (myoc-OLF), ablation of the ion-binding site (triad Asp, Asn, Asp) by altering the coordinating residues affects the stability and overall structure, in one case leading to misfolding and glaucoma. Bioinformatics analysis reveals a variety of triads with possible ion-binding characteristics lurking in OLF domains in invertebrate chordates such as Arthropoda (Asp–Glu–Ser), Nematoda (Asp–Asp–His) and Echinodermata (Asp–Glu–Lys). To test ion binding and to extend the observed connection between ion binding and distal structural rearrangements, consensus triads from these phyla were installed in the myoc-OLF. All three protein variants exhibit wild-type-like or better stability, but their calcium-binding properties differ, concomitant with new structural deviations from wild-type myoc-OLF. Taken together, the results indicate that calcium binding is not intrinsically destabilizing to myoc-OLF or required to observe a well ordered side helix, and that ion binding is a differential feature that may underlie the largely elusive biological function of OLF propellers.

Common and rare myocilin variants: Predicting glaucoma pathogenicity based on genetics, clinical, and laboratory misfolding data

“…A starting point for evaluating the consequences of OLF mutations in the absence of other data would be based on their likelihood of affecting one of the aforementioned main structural features of the OLF domain. In general, though not always (Hill, Cho, et al, 2019; Hill, Kwon, et al, 2019), structure‐based physicochemical intuition provides a rationale for a deleterious prediction. For example, modifications to surface loops lacking discrete intermolecular interactions with other residues in the domain are likely to tolerate mutations more readily than changes internal to the structure where the substitution interferes with the native packing and intermolecular interactions.…”

“…Based on this observation, the substitution of residues found on the interior of the OLF propeller would be expected to be poorly tolerated. Unexpectedly, we increased OLF stability by introducing single or double‐point variants at relatively conserved internal positions corresponding to calcium ligands, which should not have been tolerated (Hill, Cho, et al, 2019; Hill, Kwon, et al, 2019). One variant we did not study in the lab but has clinically been found only in control subjects, N428S, may be similarly stabilizing, or perhaps additional functional insights will reveal a loss of function relevant to glaucoma.…”

Competition between inside-out unfolding and pathogenic aggregation in an amyloid-forming β-propeller

“…The side helix that sits between blades E and A, which likewise remains unassigned, may also be somewhat unwound based on our structures of metal ligand variants (Supplementary Fig. 11 ) but this can happen without introducing thermal destabilization characteristic of aggregation-prone disease variants 27 , 42 . Overall, we deduce that perturbations yielding destabilized, aggregation-prone OLF are distinct from what is observed for stable variants that do not aggregate.…”

Quantitative differentiation of benign and misfolded glaucoma-causing myocilin variants on the basis of protein thermal stability

“…Previously, we established that disease variants within OLF were thermally destabilized ( Burns et al, 2011 ) and exhibited non-native structural signatures in solution ( Hill et al, 2014 ); the other structural domain of myocilin, a coiled coil, does not aggregate and has a T m ∼15°C higher than OLF (67°C; Hill et al, 2017 ). However, OLF can tolerate particular variations ( Hill et al, 2019a ; Hill et al, 2019b ), including some that lead to pronounced structural deviations ( Hill et al, 2019b ), without pathogenic signatures. For example, the D478S point mutation is thermally tolerated even though it ablates calcium binding, shifts an entire β-propeller blade by 0.5 Å and disorders the side helix ( Fig.…”