Acetyllysine Structure-Function Analysis by 13C Direct-Detect NMR Spectroscopy

PROJECT SUMMARY Protein post-translational modifications are ubiquitous events that regulate the core functions of the cell. This project focuses on lysine acetylation, which was first characterized in the context of chromatin structure and transcription. Recent proteomic data demonstrate that lysine acetylation is ubiquitous among both nuclear and cytosolic proteins. However, the mechanisms whereby lysine acetylation regulates function are still poorly understood. To overcome this knowledge gap, the PI’s laboratory has recently established protocols for transfer of 13C-acetyl groups to recombinant proteins and developed nuclear magnetic resonance (NMR) experiments that provide one resonance per acetyllysine sidechain without introducing sterically bulky chemical modifications that would impair investigation of downstream interactions. The current proposal seeks to generalize this 13C- enhanced NMR strategy to broaden the enzyme/substrate scope, enable structure determination for complexes, and demonstrate the utility of the method for non-histone proteins. This plan aligns with the PI’s track record of technology development in 13C direct-detect biomolecular NMR, which is used to probe the biophysics of disordered proteins and the complexes they form. In this context, the current project’s first specific aim is to develop a broad platform for production and chemical shift characterization of acetyllysine. To demonstrate breadth while using well-described systems for proof-of-concept, the approach will target Ada2/Gcn5, p300, and MOF to represent the three well annotated families of nuclear lysine acetyltransferases, using histone H3 and H4 as substrates. To enable chemical shift assignment, NMR pulse sequences will be developed that correlate acetyllysine resonances with backbone chemical shifts that define position in the primary structure of proteins. The second specific aim is to design NMR experiments that will enable structure determination of complexes with bound acetyllysine. The approach will be to develop a set of 3D 1H- and 13C- detect nuclear Overhauser and exchange (NOESY) spectroscopy techniques based on established isotope filtering platforms. Utility will be demonstrated by solving structures of histone tails in complex with Gcn5 and Brd4 bromodomains, for which high resolution crystal structures are available as gold-standards for assessment. The third specific aim is to demonstrate the applicability of the new technology beyond histone peptides using reconstituted nucleosomes and the transactivation domain of FoxO1 as biomedical examples. FoxO1 acetylation, which is catalyzed by p300, read by Brd4, and reversed by Sirt6 serves as the capstone. The new technology proposed here will enable applications including authentication of in vitro derived acetylation patterns against those known from proteomics, investigation of novel binding modes as new reader proteins are discovered, and determination of solution NMR structures based on isotope filtered NOE measurement. The generality of the developed technology will provide sustained benefits for the transcription and signaling communities, potentially driving translation of biological toward the clinic.