Correlating dynamics with function in the eukaryotic exoribonuclease enzymes Xrn1 and Xrn2

• Applicant: Professor Dr. Remco Sprangers
• Subject Area: Structural Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 501637118

Eukaryotic 5′→3′ exoribonucleases are large (> 100 kDa) enzymes that remove nucleotides from the 5’ end of single stranded RNA in a processive manner, thereby releasing mononucleotides. Eukaryotic organisms contain two closely related 5′→3′ exoribonucleases: the cytoplasmic Xrn1 enzyme (also called PACMAN) that is involved in the degradation of mRNA and the nuclear Xrn2 enzyme (also called Rat1) that plays a role in the degradation and processing of a variety of RNA species. The few known static structures of Xrn1 and Xrn2 reveal that the active site is only accessible for single-stranded RNA. Differences between the determined structures, however, suggest that the processive translocation of the substrate towards the active site is associated with conformational changes in the enzymes. The aim of this proposal is to address how these and other dynamics processes in Xrn1 and Xrn2 correlate with function. Experimentally, we will make extensive use of methyl TROSY and 19F NMR spectroscopic methods to directly quantify motions in Xrn1 and Xrn2 with a high spatial resolution. In preliminary work, we have shown that these approaches yield high quality data that can provide detailed insights into populations and exchange rates of the associated conformational states. Our extensive NMR studies will thus provide an extensive and unique picture of the conformantional dynamics of these enzymes. Changes of dynamics in the presence of inhibitors, substrates and binding partners (e.g. DcpS and Rai1 that modulate the activity of Xrn1, respectively Xrn2) or in the absence of specific domains will enable insights into the enzymatic mechanisms that regulate catalytic activity. In addition, we will design Xrn1 and Xrn2 enzymes that display altered dynamics and determine how this influences the catalytic turnover rates. Combined, these data will allow us to establish a (potentially) direct functional link between enzyme dynamics and catalytic activity. In brief, our proposed work goes well beyond the static structural information that is available for Xrn1 and Xrn2. In particular, we will gain unique insights into dynamic processes that are important for catalytic activity and the regulation thereof. These findings will have general implications for our understanding of enzymes, as data that link motions with function are sparse. In addition, our NMR work is at the forefront of what is currently technically possible.

• DFG Programme: Research Grants