| Part I. Structural insights into the membrane binding ability of Ubig/Coq3familyUbiquinone (coenzyme Q) is a phospholipid, locating in the membrane fraction of prokaryotes and the mitochondrial inner membrane of eukaryotes. The most important function of ubiquinone in bacteria or higher eukaryotes is that it works in the respiratory chain by transferring electrons to synthesize ATP. Apart from this, ubiquinone is also closely associated with many diseases in Homo sapiens, such as cancer, neurodegenerative disorders and Parkinson’s disease.Ubig/Coq3family proteins are a class of S-adenosylmethionine-dependent O-methyltransferase, which catalyze two O-methylation reactions for ubiquinone biosynthesis from prokaryotes to eukaryotes. In the process of ubiquinone biosynthsis, firstly, UbiA/Coq2family proteins catalyze the condensation of isoprenylpyrophosphate (IPP) with the aromatic p-hydroxybenzoate (PHB), and then the benzoquinone ring are decorated, including C-methylation, decarboxylation, O-methylation, and hydroxylation. As we know, UbiA/Coq2catalyze the prenyl transfer reaction within lipid bilayers and releases a prenylated quinone product directly into the membrane. However, the mechanism by which Ubig/Coq3family anchor to the membrane is still elusive.Here, we firstly show that Ubig/Coq3family are a class of membrane anchoring proteins, directly associating with the membrane fraction of prokaryotes and the mitochondrial inner membrane of eukaryotes. Compared with the class I SAM-MTases, Ubig possesses a unique insertion sequence shaping a membrane interaction patch, which could recognize specific acid phospholipids, PG and CL. Interestingly, the residues surrounding this patch are highly conserved from prokaryotes to eukaryotes, and mutation of the corresponding residues in human Coq3sharply cripples the binding ability toward mitochondrial inner membrane. These data indicate that the Ubig/Coq3family proteins may adopt a similar membrane interaction model. We propose that this membrane lipid anchoring ability will help to recognize substrates located in the lipid bilayer, and is essential for Ubig/Coq3family to function in vivo. In addition, we map the SAM/SAH binding site of Ubig, and find a structurally conserved region that is in close proximity to the methyl donor SAM. The growth phenotype rescue results confirm that this region is important for catalysis. Overall, we firstly show that Ubig/Coq3family could directly bind to the membrane lipid, and unmask the molecular mechanism of the membrane recognition. Our results suggest that the O-methylation reaction catalyzed by Ubig/Coq3is membrane associated in vivo. Part II. Structural insights into the neutralization mechanism of mAb6C2against ricinRIPs, a class of N-glycosidases, could be divided into two families. The first type of RIPs are monomers, possessing the N-glycosidase activity with an approximate molecular weight of32kDa. The second type of RIPs are heterodimers with an approximate molecular weight of66kDa, comprising two chains linked by a disulfide bridge. Chain A is an N-glycosidase with an approximate molecular weight of32kDa, and Chain B exhibits the lectin properties with an approximate molecular weight of34kDa. Ricin belongs to the second type of RIPs, which depurinates the sarcin-ricin loop region of ribosome28S rRNA.6C2is a mouse-human chimeric monoclonal antibody, possessing potent neutralizing ability against ricin, but the neutralization mechanism by which6C2protects against ricin remains unknown.Here, we solved the structure of6C2Fab complexed with the A-chain of ricin (RTA) at2.8A resolution. This is the first structure that deciphers the detailed interaction of RTA with a Fab fragment of monoclonal antibody. The structure indicates that residues Gln98, Glu99, Glu102and Thr105on the epitope of RTA are recognized by the pocket formed by the CDR loops, which is confirmed by the ELISA experiment. The results based on the inhibition of protein synthesis in a cell-free system reveal that disruption of the interface between6C2Fab and RTA would induce the lost of the neutralization ability of6C2against Ricin. Finally, according to the pull down and SPR results, we propose the neutralization mechanism of6C2against ricin. Altogether, our findings provides structural basis for the rational design of antibody drugs with higher affinity and better specificity. KeyWords:RIPs, N-glycosidase, RTA, RTB, Antibody, Antibody-antigen complex, Fab... |
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