Switching Antibody Specificity through Minimal Mutation

Piatesi, Andrea; Aldag, Caroline; Hilvert, Donald

doi:10.1016/j.jmb.2008.01.069

Cited by 5 publications

(15 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although the Arg H100 Trp mutation does not significantly change the affinity for the 1E9 TSA, it enhances the affinity for steroids (12). Residue H47, however, primarily acts as a specificity switch: Replacement of Leu H47 by the bulky Trp H47 leads to a complete reorganization of the 1E9 binding site and three orders of magnitude weaker TSA binding (12), because it sterically forces the Trp H50 indole to rotate Ϸ145°around its C␤-C␥ bond [ Fig. 4A and B and supporting information (SI) Figs.…”

Section: Resultsmentioning

confidence: 87%

“…However, this progesterone binding mode does not translate to any significantly lower binding affinity. The K d values of the progesterone complexes of 1E9dm and DB3 are both low nanomolar (12). The aforementioned ligand binding differences are caused by the discriminative space requirements of the corresponding H100b residues.…”

Section: Resultsmentioning

confidence: 97%

“…As in DB3, the Trp H100 side chain can either act as a surrogate ligand for the unliganded binding pocket, or in the open conformation, as a hydrophobic platform that can adapt to distinct ligands to provide van der Waals interactions. Although the Arg H100 Trp mutation does not significantly change the affinity for the 1E9 TSA, it enhances the affinity for steroids (12). Residue H47, however, primarily acts as a specificity switch: Replacement of Leu H47 by the bulky Trp H47 leads to a complete reorganization of the 1E9 binding site and three orders of magnitude weaker TSA binding (12), because it sterically forces the Trp H50 indole to rotate Ϸ145°around its C␤-C␥ bond [ Fig.…”

Section: Resultsmentioning

confidence: 99%

“…DB3 thus provides hydrophobic interactions with the steroid skeleton and hydrogen bonding with either steroid keto or hydroxyl groups, whereas 1E9dm uses a more extended arsenal of hydrophobic interactions that are associated with deeper penetration of the ligands into the protein. Remarkably, despite the different binding mechanisms, DB3 and 1E9dm exhibit similar high affinities for these structurally distinct ligands (12) ¶ , indicating that the increased number of hydrophobic interactions between 1E9dm and the steroids energetically compensates for the lack of hydrogen bonds (16).…”

Section: Resultsmentioning

confidence: 99%

“…Piatesi et al (12) recently showed that the specificity and function of 1E9 can be gradually switched to that of DB3 by mutation of only five residues (Phe L89 Ser/Leu H47 Trp/Thr H97 Tyr/Arg H100 Trp/Met H100b Phe) in the combining site. As expected, because of its increased resemblance of DB3, 1E9 loses its ability to catalyze the Diels-Alder ¶ Despite their structural differences, the functional mimicry of 1E9dm and DB3 extends to low affinity ligands like testosterone.…”

Section: Discussionmentioning

confidence: 99%

See 4 more Smart Citations

Closely related antibody receptors exploit fundamentally different strategies for steroid recognition

Verdino¹,

Aldag

Hilvert

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Molecular recognition by the adaptive immune system relies on specific high-affinity antibody receptors that are generated from a restricted set of starting sequences through homologous recombination and somatic mutation. The steroid binding antibody DB3 and the catalytic Diels-Alderase antibody 1E9 derive from the same germ line sequences but exhibit very distinct specificities and functions. However, mutation of only two of the 36 sequence differences in the variable domains, Leu H47 Trp and Arg H100 Trp, converts 1E9 into a high-affinity steroid receptor with a ligand recognition profile similar to DB3. To understand how these changes switch binding specificity and function, we determined the crystal structures of the 1E9 Leu H47 Trp/Arg H100 Trp double mutant (1E9dm) as an unliganded Fab at 2.05 Å resolution and in complex with two configurationally distinct steroids at 2.40 and 2.85 Å. Surprisingly, despite the functional mimicry of DB3, 1E9dm employs a distinct steroid binding mechanism. Extensive structural rearrangements occur in the combining site, where residue H47 acts as a specificity switch and H100 adapts to different ligands. Unlike DB3, 1E9dm does not use alternative binding pockets or different sets of hydrogen-bonding interactions to bind configurationally distinct steroids. Rather, the different steroids are inserted more deeply into the 1E9dm combining site, creating more hydrophobic contacts that energetically compensate for the lack of hydrogen bonds. These findings demonstrate how subtle mutations within an existing molecular scaffold can dramatically modulate the function of immune receptors by inducing unanticipated, but compensating, mechanisms of ligand interaction.antibody-antigen complex ͉ modulation of receptor specificity ͉ molecular recognition ͉ protein engineering ͉ x-ray crystallography

show abstract

Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

Closely related antibody receptors exploit fundamentally different strategies for steroid recognition

Verdino¹,

Aldag

Hilvert

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

Natural Diels–Alderases: Elusive and Irresistable

et al. 2015

View full text Add to dashboard Cite

Eight examples of biosynthetic pathways wherein a natural enzyme has been identified and claimed to function as a catalyst for the [4+2] cycloaddition reaction, namely, Diels-Alderases, are briefly reviewed. These are discussed in the context of the mechanistic challenges associated with the technical difficulty of proving that the net formal [4+2] cycloaddition under study, indeed proceeds through a synchronous, mechanism and that the putative biosynthetic enzyme deploys the pericyclic transition state required for a Diels-Alder cycloaddition reaction.

show abstract

Importance of Polar Solvation for Cross-Reactivity of Antibody and Its Variants with Steroids

2011

View full text Add to dashboard Cite

Understanding the factors determining the binding of ligands to receptors in detail is essential for rational drug design. Here, the free energies of binding of the steroids progesterone (PRG) and 5β-androstane-3,17-dione (5AD) to the Diels-Alderase antibody 1E9, as well as the Leu(H47)Trp/Arg(H100)Trp 1E9 double mutant (1E9dm) and the corresponding single mutants, have been estimated and decomposed using the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. Also the difference in binding free energies between the PRG-1E9dm complex and the complex of PRG with the antiprogesterone antibody DB3 have been evaluated and decomposed. The steroids bind less strongly to 1E9 than to DB3, but the mutations tend to improve the steroid affinity, in quantitative agreement with experimental data. Although the complexes formed by PRG or 5AD with 1E9dm and by PRG with DB3 have similar affinity, the binding mechanisms are different. Reduced van der Waals interactions as observed for 5AD-1E9dm versus PRG-1E9dm or for PRG-1E9dm versus PRG-DB3 are energetically compensated by an increased solvation of polar groups, partly contrasting previous conclusions based on structural inspection. Our study illustrates that deducing binding mechanisms from structural models alone can be misleading. Therefore, taking into account solvation effects as in MM-PBSA calculations is essential to elucidate molecular recognition.

show abstract

Switching Antibody Specificity through Minimal Mutation

Cited by 5 publications

References 22 publications

Closely related antibody receptors exploit fundamentally different strategies for steroid recognition

Closely related antibody receptors exploit fundamentally different strategies for steroid recognition

Natural Diels–Alderases: Elusive and Irresistable

Importance of Polar Solvation for Cross-Reactivity of Antibody and Its Variants with Steroids

Contact Info

Product

Resources

About