3EOT

Crystal structure of LAC031, an engineered anti-VLA1 Fab

Summary for 3EOT

• Entry DOI: 10.2210/pdb3eot/pdb
• Related: 1MHP
• Descriptor: FAB FRAGMENT, HEAVY CHAIN, FAB FRAGMENT, LIGHT CHAIN (3 entities in total)
• Functional Keywords: antibody, fab, vla-1, domain swap, proten engineering, immune system
• Biological source: Mus musculus (mouse); More
• Total number of polymer chains: 2
• Total formula weight: 47738.90

Authors

Boriack-Sjodin, P.A.,Clark, L.A. (deposition date: 2008-09-29, release date: 2009-01-20, Last modification date: 2024-10-30)

Primary citation

Clark, L.A.,Boriack-Sjodin, P.A.,Day, E.,Eldredge, J.,Fitch, C.,Jarpe, M.,Miller, S.,Li, Y.,Simon, K.,van Vlijmen, H.W.
An antibody loop replacement design feasibility study and a loop-swapped dimer structure.
Protein Eng.Des.Sel., 22:93-101, 2009

PubMed Abstract: A design approach was taken to investigate the feasibility of replacing single complementarity determining region (CDR) antibody loops. This approach may complement simpler mutation-based strategies for rational antibody design by expanding conformation space. Enormous crystal structure diversity is available, making CDR loops logical targets for structure-based design. A detailed analysis for the L1 loop shows that each loop length takes a distinct conformation, thereby allowing control on a length scale beyond that accessible to simple mutations. The L1 loop in the anti-VLA1 antibody was replaced with the L2 loop residues longer in an attempt to add an additional hydrogen bond and fill space on the antibody-antigen interface. The designs expressed well, but failed to improve affinity. In an effort to learn more, one design was crystallized and data were collected at 1.9 A resolution. The designed L1 loop takes the qualitatively desired conformation; confirming that loop replacement by design is feasible. The crystal structure also shows that the outermost loop (residues Leu51-Ser68) is domain swapped with another monomer. Tryptophan fluorescence measurements were used to monitor unfolding as a function of temperature and indicate that the loop involved in domain swapping does not unfold below 60 degrees C. The domain-swapping is not directly responsible for the affinity loss, but is likely a side-effect of the structural instability which may contribute to affinity loss. A second round of design was successful in eliminating the dimerization through mutation of a residue (Leu51Ser) at the joint of the domain-swapped loop.

PubMed: 19074157
DOI: 10.1093/protein/gzn072

Experimental method

X-RAY DIFFRACTION (1.9 Å)