Poster number 9 submitted by Aparna Biswas
Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit
Aparna R. Biswas (Biology, Saint Louis University), Yu-Fong Peng, Poonam Roshan (Biology, Saint Louis University), Jonah Elliff (Department of Immunology, The University of Iowa, Department of Biological Sciences, Marquette University), Sahiti Kuppa, Nicola Pozzi, Edwin Antony (Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine ), Brian Bothner (Department of Chemistry and Biochemistry, Montana State University), Sofia Origanti (Biology, Saint Louis University)
Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its regulation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6, we have uncovered the critical interface entailing 8 key residues in the C-tail of RPL23 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provides a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to RPL23 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting a key residue in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Further, we are establishing a system to study the domain dynamics of SBDS, an eIF6 release factor, by using non-canonical amino acids and bio-orthogonal chemistry to site-specifically label individual domains. Determining the mechanism of eIF6 release from 60S and establishing the key interfaces that regulate eIF6 will provide a therapeutic framework for targeting eIF6 in cancers and SDS.
Keywords: eIF6-uL14 interaction, eIF6 somatic mutations, Shwachman-Diamond Syndrome