Poster abstracts

Poster number 150 submitted by Jeff Levengood

Utilization of high-pressure NMR for the study of allosteric regulation of hnRNP A1

Jeffrey D. Levengood (Department of Chemistry, Case Western Reserve University), Julien Roche (Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University), Sebla Kutluay (Department of Molecular Biology, Washington University School of Medicine), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University)

The hnRNP A1 protein is involved in numerous processes of nascent RNA transcripts. These roles include, but are not limited to, translational control, splicing regulation, and mRNA stabilization. The protein performs its functions by binding RNA at a high affinity site before recruiting other proteins to aid in its RNA processing activities.

hnRNP A1 is composed of three domains, two structurally identical RNA recognition motifs (RRMs) that form the N-terminal protein UP1, and a disordered, glycine rich C-terminal domain (G-CTD). The UP1 domain is responsible for binding the nucleic acid substrates while the G-CTD assembles the multi-protein complexes the protein utilizes to carry out its various functions. It is also a low-complexity domain (LCD) capable of mediating liquid-liquid phase separation (LLPS).

A crystal structure of UP1 bound to a trinucleotide showed RRM1 to be the primary domain for binding. The structure presents the opportunity for allosteric regulation if RNA binding at RRM1 causes conformational changes that can alter the conformations of the other two domains.

High-pressure NMR spectroscopy is a powerful technique for examining the biophysical nature of proteins. Application of pressure to a protein increases solvent density and pushes the protein towards a lower molar volume, causing folded proteins to unfold. For disordered domains, pressure has been shown to have just minor effects on NMR parameters.

Applying pressure to UP1 revealed the protein to be remarkably stable. Variants of UP1 in which the interdomain interactions between the two RRMs are disrupted were much less stable than the wild type protein. Similar studies with full length hnRNP A1 revealed UP1 to be unaffected by the presence of the LCD. The application of pressure to the LCD caused it to adopt an elongated conformation, in contrast to its resting compact nature. This conformational change is not effected by the presence of the UP1 domain.

Levengood, J.D., and Tolbert, B.S. (2019) Idiosyncrasies of hnRNP A1-recognition: Can binding mode influence function. Semin Cell Develop Biol. 86:150-161.

Levengood, J.D., Peterson, J., Tolbert, B.S., and Roche, J. (2021) Thermodynamic stability of hnRNP A1 low complexity domain revealed by high-pressure NMR. Proteins 89(7):781-791.

Keywords: RRM, NMR, Splicing