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Raquel L Lieberman, Shannon E Hill, Elaine Nguyen, Rebecca K Donegan; Molecular architecture of myocilin and structural defects imparted by non-synonymous mutations in its coiled coil domain. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4906.
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Myocilin has been associated with primary open angle and steroid-induced glaucoma for ~20 years. Studies to date have generally focused on disease pathogenesis of mutant myocilins found within the C-terminal olfactomedin domain, which account for ~90% of documented variants and cause amyloid-like protein aggregation. Little is known about the N-terminal coiled coil region in which the remaining ~10% of mutations are located. Coiled coils are known to confer oligomeric states to proteins. We hypothesized that glaucoma-associated mutations in the coiled coil alter the quaternary structure of myocilin, instead of causing aggregation. The purpose of this study was to structurally characterize the myocilin N-terminal domain and assess effects of glaucoma-associated mutations in molecular detail.
A variety of N-terminal domain constructs were defined and prepared recombinantly, including Cys-->Ser and 7 missense variants documented in genetic studies. Proteins were characterized by a combination of biochemical and biophysical techniques including non-reducing gel electrophoresis, size exclusion chromatography (SEC), circular dichroism melts, and transmission electron microscopy (TEM). Molecular structures were determined by a combination of synchrotron radiation methods including SEC-small-angle X-ray scattering (SEC-SAXS) and X-ray crystallography.
The three Cys residues in the N-terminal domain serve to thermally stabilize the coiled-coil quaternary structure, a defined oligomeric entity whose kinked molecular envelope is defined by SAXS and was confirmed by TEM. The high resolution crystal structure of the C-terminal ~10 kDa of the coiled coil reveals features that are unique when compared to canonical coiled-coils like myosin. Glaucoma-associated variants like L95P exhibit detectable structural defects, but do not result in a protein prone to aggregation.
The extended myocilin architecture is a unique stable entity. In contrast to earlier suggestions, properly folded myocilin is not a disulfide-linked polymer. Misfolding introduced by glaucoma-associated mutations in the coiled-coil region does not result in aggregation but nevertheless impairs trafficking. This work broadens our appreciation for protein misfolding in the pathogenesis of myocilin-associated glaucoma.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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