Poster abstracts
Poster number 28 submitted by Rodrigo Muzquiz
Allosteric Coupling in an ATP-drive Motor Protein
Rodrigo Muzquiz (OSBP), Kristie Baker (Department of Chemistry and Biochemistry OSU), Philip Lacey (Department of Chemistry and Biochemistry OSU), Vicki 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 via RNA binding loops. This facilitates movement along the ssRNA substrate to contact stalled RNA polymerase thereby terminating transcription. Conserved residues in these loops make RNA contacts via sidechain orientations that correspond to different ATP-bound states1,3. The process of ATP and RNA binding to Rho leads to a global conformational change from an open ring (lock-washer) to a closed ring structure. Sequential ATP hydrolysis to ADP around the ring coordinates translocation by a mechanism that is not understood. Therefore, understanding how ATP alters the conformation of RNA-binding loops 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 negative cooperativity in binding to the six sites on Rho. From these titration experiments we expect to quantify thermodynamic coupling between ATP binding sites. These coupling free energies are expected to describe how the conformation of the RNA-binding loops is perturbed by nearest-neighbor interactions of ATP-bound protomers. By repeating these experiments in the presence of an RNA substrate we expect to reveal how this coupling allows for coordinated ATP hydrolysis and translocation by 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