Abstract
Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody-antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 215-222 |
| Number of pages | 8 |
| Journal | Journal of Structural Biology |
| Volume | 185 |
| Issue number | 2 |
| DOIs | |
| State | Published - Feb 2014 |
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Keywords
- Antibody
- Binding affinity
- Computer-aided design
- Crosslinking
- L-DOPA
- Non-canonical amino acid
- Rosetta
- Structure-based design
ASJC Scopus subject areas
- Structural Biology
Cite this
Structure-based non-canonical amino acid design to covalently crosslink an antibody-antigen complex. / Xu, Jianqing; Tack, Drew; Hughes, Randall A.; Ellington, Andrew D.; Gray, Jeffrey J.
In: Journal of Structural Biology, Vol. 185, No. 2, 02.2014, p. 215-222.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Structure-based non-canonical amino acid design to covalently crosslink an antibody-antigen complex
AU - Xu, Jianqing
AU - Tack, Drew
AU - Hughes, Randall A.
AU - Ellington, Andrew D.
AU - Gray, Jeffrey J
PY - 2014/2
Y1 - 2014/2
N2 - Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody-antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes.
AB - Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody-antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes.
KW - Antibody
KW - Binding affinity
KW - Computer-aided design
KW - Crosslinking
KW - L-DOPA
KW - Non-canonical amino acid
KW - Rosetta
KW - Structure-based design
UR - http://www.scopus.com/inward/record.url?scp=84894965507&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84894965507&partnerID=8YFLogxK
U2 - 10.1016/j.jsb.2013.05.003
DO - 10.1016/j.jsb.2013.05.003
M3 - Article
C2 - 23680795
AN - SCOPUS:84894965507
VL - 185
SP - 215
EP - 222
JO - Journal of Structural Biology
JF - Journal of Structural Biology
SN - 1047-8477
IS - 2
ER -