Poster abstracts
Poster number 29 submitted by Rodrigo Muzquiz
Negative cooperativity in nucleotide binding to a AAA+ ATPase hexamer
Rodrigo Muzquiz (Presenting Author), Kelly Karch (Department of Chemistry and Biochemistry, OSU), Vycki Wysocki (Department of Chemistry and Biochemistry, OSU), Mark Foster (Department of Chemistry and Biochemistry, OSU )
Abstract:
Hexameric helicases are motor proteins that play important physiological roles in cellular replication, chromosome packaging, and transcription termination3. The Escherichia coli (E.coli) Rho factor is an essential motor protein involved in regulating protein expression1. This ring-shaped protein binds nascent mRNA in its central pore and fuels its translocation by ATP hydrolysis. This allows it to move down the substrate to contact the RNA polymerase, thereby terminating transcription1,3. The process of ATP and RNA binding lead to a conformational change of an open ring (lock-washer) to a closed ring. Currently, it is not understood how ATP and RNA binding are coupled on a single subunit and whether the ATP or RNA bound to one subunit affects the affinity for these ligands on neighboring subunits. We have implemented native mass spectrometry (nMS) in tandem with surface induced dissociation (SID) to probe the allosteric and structural properties of Rho. nMS allows us to see the populations of each ATP bound state of Rho rather than just the solution averages. ATP titration experiments of apo-Rho have shown that there is negative cooperativity in binding. These experiments have also shown that Rho exists in variable oligomeric states (dimer, trimer, etc.) during electrospray process that are not normally observed in solution under these same conditions. nMS experiments of other AAA+ ATPases have observed similar features with variability in spraying under the same conditions. From this we can fit the data to a statistical thermodynamic model to quantify the cooperative coupling free energies between subunits. Our next steps we plan to optimize spraying conditions and nucleotide analogs to reduce the variability in oligomeric conformations observed so that the data fitting can yield more accurate parameters. We also want to look at how the presence of RNA shifts the populations of ATP bound states of Rho. By using SID we can also compare the binding energies of the subunits for the apo and holo state of Rho, where different oligomeric conformations will be present at higher dissociation energies.
References:
1. Mitra P.; Ghosh G.; Hafeezunnisa M.; Sen R. Rho protein: roles and mechanisms. Annu Rev Microbiol. 2017, 8 (71) 687. DOI: 10.1146/annurev-micro-030117-020432
2. Yu Y.; Liu H.; Yu Z.; Witkowska H.E.; Cheng Y. Stoichiometry of nucleotide binding to proteasome AAA+ ATPase hexamer established by native mass spectrometry. Mol Cell Proteomics. 2020, 19 (12): 1997. DOI: 10.1074/mcp.RA120.002067
3. Patel, S. S., & Picha, K. M. (2000). Structure and Function of Hexameric Helicases. Annual Review of Biochemistry, 69(1), 651–697. https://doi.org/10.1146/annurev.biochem.69.1.651
Keywords: native mass spectrometry, cooperativity, transcription termination