4w68

Cytoplasmically Produced Homodimeric Single Domain Antibody (sdAb) C22A/C99V variant against Staphylococcal enterotoxin B (SEB)

spinqualitylabelsall models 4w68, resolution 2.00Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 4w68 is a 2 chain structure with sequence from Lama glama. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2Å Ligands: SO4 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Publication Abstract from PubMed Camelid single domain antibodies (sdAb) are known for their thermal stability and reversible refolding. We have characterized an unusually stable sdAb recognizing Staphylococcal enterotoxin B with one of the highest reported melting temperatures (Tm = 85 degrees C). Unexpectedly, ~10-20% of the protein formed a dimer in solution. Three other cases where <20% of the sdAb dimerized have been reported; however, this is the first report of both the monomeric and dimeric X-ray crystal structures. Concentration of the monomer did not lead to the formation of new dimer suggesting a stable conformationally distinct species in a fraction of the cytoplasmically expressed protein. Comparison of periplasmic and cytoplasmic expression showed that the dimer was associated with cytoplasmic expression. The disulfide bond was partially reduced in the WT protein purified from the cytoplasm and the protein irreversibly unfolded. Periplasmic expression produced monomeric protein with a fully formed disulfide bond and mostly reversible refolding. Crystallization of a disulfide-bond free variant, C22A/C99A, purified from the periplasm yielded a structure of a monomeric form, while crystallization of C22A/C99A from the cytoplasm produced an asymmetric dimer. In the dimer, a significant conformational asymmetry was found in the loop residues of the edge beta-strands (S50-Y60) containing the highly variable complementarity determining region, CDR2. Two dimeric assemblies were predicted from the crystal packing. Mutation of a residue at one of the interfaces, Y98A, disrupted the dimer in solution. The pleomorphic homodimer may yield insight into the stability of misfolded states and the importance of the conserved disulfide bond in preventing their formation. (c) Proteins 2014;. (c) 2014 Wiley Periodicals, Inc. Structural and mutational analysis of a monomeric and dimeric form of a single domain antibody with implications for protein misfolding.,George J, Compton JR, Leary DH, Olson MA, Legler PM Proteins. 2014 Aug 18. doi: 10.1002/prot.24671. PMID:25136772 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Antibody 3D structures 3D structures of non-human antibody

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