STRUCTURAL EVALUATION AND INTERACTION STUDIES OF THE Sorghum bicolor RPAP3: DIFFERENCES BETWEEN YEAST AND HUMAN ORTHOLOGUES

The R2TP complex is a molecular machinery initially identified in yeast that regulates several cellular processes. In yeast, it consists of four components: Rvb1, Rvb2, Tah1 and Pih1. The Tah1 has a homolog in humans that is named RPAP3 (RNA polymerase II-associated protein 3), which is six times longer than the yeast Tah1. RPAP3 spans two TPR domains, which binds the conserved MEEVD C-terminal tail of Hsp90, and an additional RPAP3 domain, which is absent in yeast. Given these differences and the biological importance of these proteins, it becomes necessary to broaden our understanding of Tah1/RPAP3 to other organisms, particularly in plants that are less studied. The tertiary structure of SbRPAP3 was predicted by computational modeling tools such as RoseTTaFold and AlphaFold2 and compared with high-resolution structures of orthologs available on Protein Data Bank (PDB). The superimposed structures of SbRPAP3 with yeast and human present significant similarity in their structural characteristics. pET28a-TEV-His-SbRPAP3, pET28a-His-SsHsp90 and pET28a-His-SsHsp70 were expressed into BL21(DE3) and pCOLADuet-1-His-HsRUVBL1-RUVBL2 into pRARE E. coli strains. The recombinant proteins were found in the soluble fraction and were purified by the combination of 2 steps: Nickel Affinity and Size Exclusion Chromatographies (SEC). SbRPAP3’s His-tag was efficiently removed by TEV protease and its conformational characterization was done by Circular Dichroism (CD), Tryptophan fluorescence and SEC coupled to Multi-angle and Quasi-Elastic Light Scattering (SEC-MALS-QELS). Protein-protein interaction assays were performed by combining Analytical SEC (a-SEC), SEC-MALS-QELS and pull-down experiments (confirmed by Western blot). Recombinant SbRPAP3 had purity of about 95% and was folded with a secondary structure content rich in α-helices. Intrinsic tryptophan fluorescence emission analysis showed a redshift in the spectra when SbRPAP3 was exposed to urea as a denaturing agent, suggesting that at least one of the Trp residues is predominantly embedded within a nonpolar region of the protein in its native state and become solvent-exposed upon unfolding. SbRPAP3 had an elongated monomeric structure with hydrodynamic radius of 38 ± 0.1 Å and diffusion coefficient of 7.5 ± 0.3 x 10-7 cm2 s-1 as other RPAP3 orthologues. Moreover, SbRPAP3 interacted with molecular chaperones Hsp90 and Hsp70 from sugarcane as well as with human RUVBL1/2. In conclusion, the characterization of a novel RPAP3 protein in monocot plants can provide valuable insights that contributes to the assembly and stability of protein complexes, thereby shedding light on plant-specific regulatory processes and advancements in biotechnological applications. The authors are grateful by the support of foment agencies CNPq, CAPES and FAPESP.