PDBsum entry 3eot

Immune system

PDB id: 3eot

Contents

Protein chains

212 a.a. *

214 a.a. *

Waters ×204

* Residue conservation analysis

PDB id:

3eot

Name:

Immune system

Title:

Crystal structure of lac031, an engineered anti-vla1 fab

Structure:

Fab fragment, heavy chain. Chain: h. Engineered: yes. Mutation: yes. Fab fragment, light chain. Chain: l. Engineered: yes

Source:

Mus musculus. Mouse. Organism_taxid: 10090. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_taxid: 562

Resolution:

1.90Å R-factor: 0.234 R-free: 0.270

Authors:

P.A.Boriack-Sjodin,L.A.Clark

Key ref:

L.A.Clark et al. (2009). An antibody loop replacement design feasibility study and a loop-swapped dimer structure. Protein Eng Des Sel, 22, 93. PubMed id: 19074157

Date:

29-Sep-08 Release date: 20-Jan-09

Protein chain

No UniProt id for this chain

Struc: 212 a.a.

Protein chain

No UniProt id for this chain

Struc: 214 a.a.

Protein Eng Des Sel 22:93 (2009)

PubMed id: 19074157

An antibody loop replacement design feasibility study and a loop-swapped dimer structure.

L.A.Clark, P.A.Boriack-Sjodin, E.Day, J.Eldredge, C.Fitch, M.Jarpe, S.Miller, Y.Li, K.Simon, H.W.van Vlijmen.

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.