The Structural And Functional Research Of Spt4:Spt5 And Anti-ErbB2 Antibody E9

Gene expression, a complex process that involves transcription, translation, and post-translational modifications, is mediated by the activity of several multi-subunit protein complexes. The beginning of gene expression is transcription, transferring genetic information from DNA to RNA, which is mediated by RNA polymerase. Proteins regulating transcription are called transcription factors, which are not highly conserved in all three domains of life except for the Spt5/NusG family. Spt5 (NusG in bacteria) is the only RNA polymerase-associated factor known to be conserved in all three domains of life. In archaea and eukaryotes, Spt5 associates with Spt4, an elongation factor that is absent in bacteria, to form a functional heterodimeric complex. Previous studies suggest that the Spt4:Spt5 complex interacts directly with DNA at the double-stranded DNA exit tunnel of RNA polymerase to regulate gene transcription.In this study, the DNA-binding ability of Spt4:Spt5 from the archaeon Methanocaldococcus jannaschii was confirmed via nuclear magnetic resonance chemical shift perturbation and fluorescence polarization assays. Crystallographic analysis of the full-length MjSpt4:Spt5 revealed two distinct conformations of the C-terminal KOW domain of Spt5. A similar alkaline region found on the Spt4:Spt5 surface in both crystal forms was identified as a double-stranded DNA binding patch through mutagenesis-fluorescence polarization assays. Based on these structural and biochemical data, a Spt4:Spt5-DNA binding model was built.ErbB2 belongs to the tyrosine protein kinase family, epidermal growth factor receptor subfamily. The protein activates the tyrosine kinase activity and phosphorylate the intracellular C-terminal domain by dimerizating the extracellular domain. Phosphorylation of the ErbB2 C-terminal results in activating of the downstream signaling pathways which leads to cell replication or apoptosis. Abnormal activation of ErbB2 always lead to metastatic tumor, which makes ErbB2 an important target molecule in cancer therapy.Cellular and molecular immunology laboratory of University of Science and Technology of China has been committing in anti-ErbB2 antibody exploitation, and has developed a chimeric single-chain antibody, chA21. Recently, they finished the humanization of chA21 and enhanced its antigen affinity by antibody affinity maturation, to create a new single-chain antibody, E9. We crystalized and determined the structure of E9 scFv-ErbB2 ECD complex, and conformed the binding site of E9 scFv on ErbB2 ECD. Comparing with the model of chA21 scFv binding to ErbB2, we found that an ordered to disordered loop on the E9-ErbB2 interface is the reason of the affinity enhancement of E9. We further completed the E9-ErbB2 interaction model depending on our biochemical experiment data.