6LZ4

Crystal structure of PMab-1 Fv-clasp fragment with its antigen peptide

Summary for 6LZ4

• Entry DOI: 10.2210/pdb6lz4/pdb
• Descriptor: PMab-1 VH(S112C)-SARAH Chimera, PMab-1 VL-SARAH(S37C) Chimera, MAP peptide (3 entities in total)
• Functional Keywords: antibody fragment, fv-clasp, immune system
• Biological source: Rattus norvegicus; More
• Total number of polymer chains: 6
• Total formula weight: 78022.03

Authors

Arimori, T.,Takagi, J. (deposition date: 2020-02-18, release date: 2020-05-20, Last modification date: 2024-10-16)

Primary citation

Wakasa, A.,Kaneko, M.K.,Kato, Y.,Takagi, J.,Arimori, T.
Site-specific epitope insertion into recombinant proteins using the MAP tag system.
J.Biochem., 168:375-384, 2020

PubMed Abstract: The MAP tag system comprises a 14-residue peptide derived from mouse podoplanin and its high-affinity monoclonal antibody PMab-1. We determined the crystal structure of PMab-1 complexed with the MAP tag peptide and found that the recognition required only the N-terminal 8 residues of MAP tag sequence, enabling the shortening of the tag length without losing the affinity for PMab-1. Furthermore, the structure illustrated that the MAP tag adopts a U-shaped conformation when bound by PMab-1, suggesting that loop-inserted MAP tag would assume conformation compatible with the PMab-1 binding. We inserted the 8-residue MAP tag into multiple loop regions in various proteins including fibronectin type III domain and G-protein-coupled receptors and tested if they maintain PMab-1 reactivity. Despite the conformational restraints forced by the insertion position, all MAP-inserted mutants were expressed well in mammalian cells at levels comparable to the non-tagged proteins. Furthermore, the binding by PMab-1 was fully maintained even for the mutant where MAP tag was inserted at a structurally restricted β-hairpin, indicating that the MAP tag system has unique feature that allows placement in the middle of protein domain at desired locations. Our results indicate the versatile utility of the MAP tag system in 'site-specific epitope insertion' application.

PubMed: 32386302
DOI: 10.1093/jb/mvaa054

Experimental method

X-RAY DIFFRACTION (2.49 Å)