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Cassandra L Barnes, Bruce Knutson, Michael Cosgrove, Peter D Calvert; Amphibian Arrestin 1 forms dimers using a novel interaction interface. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3907 – A0109.
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© ARVO (1962-2015); The Authors (2016-present)
Photoreceptor arrestin 1 (Arr1) from mammalian species self-associates into dimers and tetramers. We have shown that amphibian Arr1 oligomerization is limited to dimers – higher order oligomers are not detected at the highest experimental concentrations examined, nor projected to form at millimolar, physiological concentrations. We thus examined if the mechanism of amphibian Arr1 dimer formation differed from that of mammalian.
Recombinant Arr1s from the African clawed frog, Xenopus laevis (xArr1), and the tiger salamander, Ambystoma tigrinum (salArr1), were expressed in E. coli, purified, and subjected to analytical ultracentrifugation (AUC). AUC results were analyzed in SEDFIT, plotted in GUSSI and fitted in MATLAB to determine oligomer formation and dissociation constants. 3D structure of xArr1 was estimated using Modeler in Chimera, using bovine Arr1 crystal (PDB: 1CF1 chain A) as template. Regions of greatest sequence divergence among Arr1s were found using MegAlign. Lysine crosslinking - Mass Spectrometry and Patchdock were used to identify potential dimer interfaces and create 3D models of xArr1 dimers. Small angle x-ray scattering (SAXS) was performed and analyzed using Raw to estimate dimer shape. xArr1 dimer structure was predicted by visualizing SAXS envelopes and comparing to 3D models from Patchdock using Chimera.
Arr1 from X. lavis and A. tigrinim self-associate in a concentration-dependent manner, however, oligomerization of both species was limited to dimers. AUC analysis of recombinant and endogenous bovine Arr1 supports previously published results that are interpreted to show the formation of tetramers at physiological concentrations. SAXS envelopes of xArr1 are consistent with dimer formation, without indication of larger oligomers. Inositol-hexakisphosphate (IP6) inhibits xArr1 self-association, but not as effectively as for mammalian Arr1. Mutations in xArr1 residues analogous to key residues in mammalian Arr1 that are essential for their tetramerization and in which mutations increase the Kd of dimerization tenfold, had no impact on the affinity of xArr1 dimers. Homology modeling and circular dichroism spectra suggest a similar fold for xArr1 and bovine Arr1.
Together, these results suggest that amphibian Arr1s have a similar structure to that of mammals but use a dimerization interface that differs from those found in mammalian Arr1s.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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