Poster abstracts
Poster number 16 submitted by Rodrigo Muzquiz
Allosteric Coupling in an ATP-Driven Motor Protein
Rodrigo Muzquiz (OSBP), Kelly Karch (Department of Chemistry and Biochemistry, OSU), Vycki Wysocki (Department of Chemistry and Biochemistry, OSU), Mark Foster (Department of Chemistry and Biochemistry, OSU ), Kristie Baker (Department of Chemistry and Biochemistry, OSU), Philip Lacey (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 via RNA binding loops. This facilitates movement along the ssRNA substrate to contact stalled RNA polymerase thereby terminating transcription. Functionally conserved residues in these loops make RNA contacts whose sidechain orientation correspond to different ATP-bound states1,3. The process of ATP and RNA binding leads to a conformational change from an open ring (lock-washer) to a closed ring. ATP hydrolysis occurs sequentially around the ring; however, it is not known how these protomers communicate to coordinate translocation. Therefore, understanding how the state of an ATP-bound site alters the conformation of an RNA-binding loop is crucial to characterizing AAA+ ATPase translocation. 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 quantify the populations of each ATP-bound state of Rho rather than simply the solution ensemble. 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 the electrospray process that are not normally observed in solution under these same conditions. From these titration experiments we can fit the data to a statistical thermodynamic model to quantify coupling free energies. These coupling free energies would describe how the conformation of the RNA-binding loops is perturbed by nearest-neighbor interactions of ATP-bound protomers. In 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 seek to understand how the presence of RNA shifts the populations of ATP-bound states of Rho.
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: Allostery, ATPase, Mass-Spectrometry