The Structural Biology Research Of E. Coli TrmB And Anti-ErbB2 Antibody ChA21

The maturation of tRNAs is a complex process involving numerous post-transcriptional modifications.N7-methylguanosine modification at position 46 (m7G46) in the variable loop is one of the few modifications that introduce a positive charge to the base,and is conserved in almost all of classⅠtRNAs in bacteria, eukaryotes and some archaea.When lacking m7G46 in combination with other non-essential modifications,the yeast cellular tRNAs are sent to the rapid tRNA degradation(RTD) pathway.Here,we determined the crystal structure of E.coli tRNA(m7G46) methyltransferase(EcTrmB) at high resolution.Similar to its homo- or heterodimeric homologues,EcTrmB also adopts a modified Rossmann fold.But,the function-essential insertion of EcTrmB has a conformation different to all of its homologues.In EcTrmB,the C-terminal part of this insertion separates from the S-adenosyl-L-methionine(SAM) binding site for several angstroms,which destroys the binding pocket for base G46.It seems that a structural rearrangement of the insertion of EcTrmB is required to form the G46 pocket during catalysis.In contrast, in homodimeric TrmBs,the steric hindrance from the other subunit prevents the C-terminal part of the insertion separating from the SAM-binding site.Residues 202-206 and 223-227 of EcTrmB form two protruding loops at the dimeric interface of other homodimeric TrmBs.These protruding loops would cause many conflicts if EcTrmB formed a homodimer,which provides the structural insight to why monomeric TrmBs cannot form dimers.ErbB2(Her2/neu/p185) is a key member of epidermal growth factor receptor (EGFR) family,which has become an attractive target for therapeutic intervention. Prof.Jing Liu and her colleagues at USTC previously developed an anti-ErbB2 chimeric antibody chA21 that could specifically inhibit the growth of ErbB2-overexpressing cancer cells in vitro and in vivo.Herein,the antibody-antigen complex consists of the single-chain variable fragment(scFv) of chA21 and an N-terminal fragment(residues 1-192,named EPⅠ) of ErbB2 extracellular domain was crystallized.The complex structure shows that the complementarity determining regions(CDR) of chA21 form a vast pocket to bind to three loops at the backside of ErbB2 ECD dimerization interface.The antibody-antigen interface contains 17 specific hydrogen bonds and 172 van der Waals contacts. chA21 was also found to have a strong bivalency-dependent effect of on down-regulating ErbB2.We proposed a model that chA21 could capture two ErbB2 molecules belonging to separate homo- or hetero- dimers and cross-link these dimers to form a large complex.The large complex contains the ErbB2 could be internalized and degradated efficiently.Transcription in archaea employs a eukaryotic-type transcription apparatus but uses bacterial-type transcription factors.NusG is one of the few archaeal transcription factors whose orthologs are essential in both bacteria and eukaryotes.Archaeal NusG is composed of only an NusG N-terminal(NGN) domain and a KOW domain,which is similar to bacterial NusG but not to the eukaryotic ortholog,Spt5.However, archaeal NusG was confirmed recently to form a complex with rpoE" that was similar to the Spt5-Spt4 complex.In this work,we report the crystal structure of NGN domain from the archaea Methanocaldococcus jannaschii(MjNGN).MjNGN folds to an a-β-a sandwich without the appendant domain of bacterial NGNs,and forms a unique homodimer in crystal and solution.MjNGN alone was found to be sufficient for rpoE" binding and an MjNGN-rpoE" model has been constructed by rigid docking and validated by mutagenesis analysis.Furthermore,we have determined the crystal structure of MjNusG-rpoE" complex very recently.Futher structural and biochemical analysis of this complex is still under way.