Poster abstracts
Poster number 5 submitted by Tyler Billings
Native mass spectrometry reveals ATP-bound populations of Rho termination factor
Tyler D Billings (The Ohio State Biochemistry Program), Kristie Baker (Department of Chemistry and Biochemistry, The Ohio State University), Philip Lacey (Department of Chemistry and Biochemistry, The Ohio State University), William Moeller (Department of Chemistry and Biochemistry, The Ohio State University), Vicki Wysocki (School of Chemistry & Biochemistry, Georgia Institute of Technology), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract:
Hexameric helicases are essential motor proteins that unwind nucleic acid duplexes by converting the chemical potential of nucleotide triphosphates into translational motion. Several hydrolytic mechanisms have been proposed according to structural, biochemical, and single-molecule studies, with the rotary model of ATP hydrolysis emerging as the most suited to explain rapid, directional translocation. In one formulation of the rotary model, a trio of sites bearing no substrate, ADP product, and ATP substrate undergo catalytic turnover in a defined order: hydrolysis of ATP substrate in one site is coupled with release of ADP product in the preceding site while free ATP enters the empty site. The resulting sequential hydrolysis is coupled to conformational changes in RNA-binding loops located in the central pore of the helicase.
A requirement of the rotary mechanism is that six otherwise identical sites must coordinate nucleotide binding, hydrolysis, and product release. Previous studies using conventional bulk binding assays have suggested that strong, negative homotropic cooperativity in ATP binding results in rings that are not fully saturated.1 A drawback of indirect signal measurements is that the signal could be distorted by the allosteric effects that result in the observed cooperativity. We have used native mass spectrometry (nMS) to observe and quantify ATP binding to E. coli Rho termination factor as a model hexameric helicase. Unlike in bulk binding assays, nMS can directly measure each liganded population, thereby removing the confounding effects of allostery.2 Recording nMS spectra of Rho as a function of varying ATP concentration allowed us to quantify populations of rings bound with differing numbers of ATP ligands. These populations can then be fit with mechanism-based thermodynamic models to learn about binding thermodynamics and the origin of the observed cooperativity. This information is crucial for understanding and testing models for ATP-driven translocation of Rho and related helicases.
References:
1. Skordalakes, E. and Berger, J. M. Structure of the Rho Transcription Terminator: Mechanism of mRNA Recognition and Helicase Loading. Cell 2003, 114 (1), 135–146.
2. Li, W., et al. Structural Basis of Nearest-Neighbor Cooperativity in the Ring-Shaped Gene Regulatory Protein TRAP from Protein Engineering and Cryo-EM. Proc. Natl. Acad. Sci. U.S.A. 2025, 122 (1), e2409030121.
Keywords: native mass spectrometry, thermodynamics, helicase