Computer Modelling of the Interaction Between CeRme-8 J Domain and CeHsp70-1

Stephen F Austin State University, Baker Pattillo Student Center, Student Center Theatre and Twilight Ballroom

Abstract: Rme-81 is a J domain-containing plasma membrane protein that is required for endocytosis in various cells2. The J domain is a characteristic structural motif found mainly in heat shock protein 40 (Hsp40 or DnaJ) and other proteins such as Rme-83. Within the J domain is a tripeptide, the HPD motif, that is required by the J-domain protein to interact with and stimulate the ATPase activity of Hsp70, a major cellular chaperone4. The Rme-8 protein in C. elegans, CeRme-8, has not been identified with a particular Hsp70 partner. CeHsp70-1 is the only cytosolic Hsp70 in C. elegans, therefore, we hypothesize that CeHsp70-1 is the binding partner for the J domain of CeRme-8. To test this hypothesis, we employed computer modelling to predict the interaction between CeRme-8 and CeHsp70-1 using the known DnaK-DnaJ protein complex as a template. In addition, we report the successful cloning and expression of the J domain of CeRme-8. AlphaFold, SwissModel, and Phyre2 modelling programs revealed that CeRme-8 possesses a J domain that contains the canonical HPD tripeptide motif. ClusPro docking program predicted similar binding interface to the DnaK-DnaJ complex, along with several nonconforming models. Complementary DNA of the J domain of CeRme-8 was cloned into the pGEX-Tev-KG plasmid, in-frame with the gene for glutathione-S-transferase (GST), to yield a GST-CeRme-8 fusion protein. IPTG-induced expression of the expected 37- kilodalton fusion protein was confirmed by both SDS-PAGE and western blotting using antibody against GST. Future work will involve purifying and testing the effect of the J domain protein on the ATPase activity of CeHsp70-1.