Poster abstracts

Poster number 76 submitted by Jeff Levengood

Examination of the disordered C-terminal domain of a host regulator of HIV-1 splicing

Jeffrey D. Levengood (Chemistry, Case Western Reserve University), Christopher Morgan (Chemistry, Case Western Reserve University), Blanton S. Tolbert (Chemistry, Case Western Reserve University)

Abstract:
Alternative splicing of the HIV-1 genome is necessary for translation of the complete viral proteome. Host proteins, such as hnRNP A1, are used to regulate splicing at the various donor and acceptor sites along the viral genome. The multi-faceted hnRNP A1 is composed of three domains, two structurally identical RRM domains that form the nucleic acid binding protein UP1, and a glycine rich C-terminal domain that is primarily involved in protein-protein interactions.
A crystal structure of UP1 was solved with a rAGU trinucleotide bound to the protein. This structure revealed that while only RRM1 binds the RNA, RRM2 is responsible for positioning several residues involved in the binding of the substrate. This mode of binding opens the possibility for allosteric regulation of hnRNP A1, presenting a need to study the structural properties of the C-terminus and its potential interactions with RRM2.
A preliminary view of the structure of full length hnRNP A1 has been obtained through SAXS-scored structural modeling. This model revealed the C-terminal domain to be a long, unstructured, disordered polypeptide chain extending away from the UP1 domain. In this model, the C-terminus appears to be independent of UP1 as there are no interactions between the two. The C-terminus is further being studied through the use of NMR with amino acid selective labeling. Mutations to mimic modification of the C-terminus are also being carried out. Finally, the binding of small molecules and peptides to the C-terminus is being examined through the use of both Isothermal Titration Calorimetry (ITC) and NMR.

References:
Levengood, J.D., Rollins, C., Mishler, C. H. J., Johnson, C.A., Miner, G., Rajan, P., Znosko, B.M., and Tolbert, B.S. (2012) Solution Structure of the HIV-1 Exon Splicing Silencer 3. J. Mol. Biol. 415:680-698.

Rollins, C. Levengood, J.D., Rife, B., Salemi, M., and Tolbert, B.S. (2014) Thermodynamic and phylogenetic insights into hnRNP A1 recognition of the HIV-1 exon splicing silencer 3 element. Biochemistry 53(13):2172-84.

Morgan, C.E., Meagher, J.L., Levengood, J.D., Delproposto, J., Rollins, C., Stuckey, J.A., and Tolbert, B.S. (2015) The first crystal structure of the UP1 domain of hnRNP A1 bound to RNA reveals a new look for an old RNA binding protein. J. Mol. Biol.

Keywords: RRM, Splicing, HIV