6AL1

The NZ-1 Fab complexed with the PDZ tandem fragment of A. aeolicus S2P homolog with the PA12 tag inserted between the residues 181 and 184

Summary for 6AL1

• Entry DOI: 10.2210/pdb6al1/pdb
• Descriptor: PDZ tandem fragment with PA tag insertion, Heavy chain of antigen binding fragment, Fab of NZ-1, Light chain of antigen binding fragment, Fab of NZ-1 (3 entities in total)
• Functional Keywords: protease, signaling protein
• Biological source: Aquifex aeolicus VF5; More
• Total number of polymer chains: 3
• Total formula weight: 67706.61

Authors

Tamura, R.,Oi, R.,Kaneko, M.K.,Kato, Y.,Nogi, T. (deposition date: 2018-09-05, release date: 2019-02-13, Last modification date: 2024-10-16)

Primary citation

Tamura, R.,Oi, R.,Akashi, S.,Kaneko, M.K.,Kato, Y.,Nogi, T.
Application of the NZ-1 Fab as a crystallization chaperone for PA tag-inserted target proteins.
Protein Sci., 28:823-836, 2019

PubMed Abstract: An antibody fragment that recognizes the tertiary structure of a target protein with high affinity can be utilized as a crystallization chaperone. Difficulties in establishing conformation-specific antibodies, however, limit the applicability of antibody fragment-assisted crystallization. Here, we attempted to establish an alternative method to promote the crystallization of target proteins using an already established anti-tag antibody. The monoclonal antibody NZ-1 recognizes the PA tag with an extremely high affinity. It was also established that the PA tag is accommodated in the antigen-binding pocket in a bent conformation, compatible with an insertion into loop regions on the target. We, therefore, explored the application of NZ-1 Fab as a crystallization chaperone that complexes with a target protein displaying a PA tag. Specifically, we inserted the PA tag into the β-hairpins of the PDZ tandem fragment of a bacterial Site-2 protease. We crystallized the PA-inserted PDZ tandem mutants with the NZ-1 Fab and solved the co-crystal structure to analyze their interaction modes. Although the initial insertion designs produced only moderate-resolution structures, eliminating the solvent-accessible space between the NZ-1 Fab and target PDZ tandem improved the diffraction qualities remarkably. Our results demonstrate that the NZ-1-PA system efficiently promotes crystallization of the target protein. The present work also suggests that β-hairpins are suitable sites for the PA insertion because the PA tag contains a Pro-Gly sequence with a propensity for a β-turn conformation.

PubMed: 30666745
DOI: 10.1002/pro.3580

Experimental method

X-RAY DIFFRACTION (3.2 Å)