Thermodynamic Consequences of Grafting Enhanced Affinity toward the Mutated Antigen onto an Antibody

Nishimiya, Yoshiyuki; Tsumoto, Kouhei; Shiroishi, Mitsunori; Koyanagi, Izumi

doi:10.1074/jbc.275.17.12813

Cited by 20 publications

(14 citation statements)

References 51 publications

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“…However, the covered area of VH-VL interface of SFSF⅐TEL upon complexation is larger than SFSF⅐HEL, and SFSF has more than 5-fold greater affinity for TEL than for HEL (28). This suggests that an increase in complementary association of VH with VL does not always lead to the enhancement of the affinity of antibodies.…”

Section: Discussionmentioning

confidence: 91%

“…mini-libraries containing random mutations at four identical sites in the CDR-H2 region). Phage display was used to enhance its specificity toward turkey egg white lysozyme (28). Several mutants were selected.…”

Section: Resultsmentioning

confidence: 99%

“…The expression vector for the HyHEL-10 variable region fragment (Fv) (wild-type and an SFSF mutant in which sites 53, 54, 56, and 58 are Ser, Phe, Ser, and Phe, respectively) was described previously (28). All other reagents were of biochemical research grade.…”

Section: Methodsmentioning

confidence: 99%

“…The enhance its specificity toward turkey lysozyme (a naturally mutated antigen) (28). Isothermal titration calorimetric studies have revealed the following (28).…”

mentioning

confidence: 99%

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Structural Consequences of Target Epitope-directed Functional Alteration of an Antibody

Koyanagi¹,

Nishimiya²,

Kondo³

et al. 2003

Journal of Biological Chemistry

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Decreased affinity of an antibody for a mutated epitope in an antigen can be enhanced and reversed by mutations in certain antibody residues. Here we describe the crystal structures of (a) the complex between a naturally mutated proteinaceous antigen and an antibody that was mutated and selected in vitro, and (b) the complex between the normal antigen and the mutated antibody. The mutated and selected antibody recognizes essentially the same epitope as in the wild-type antibody, indicating successful target site-directed functional alteration of the antibody. In comparing the structure of the mutated antigen-mutant antibody complex with the previously established structure of the wild-type antigen-wild-type antibody complex, we found that the enhanced affinity of the mutated antibody for the mutant antigen originated not from improvements in local complementarity around the mutated sites but from subtle and critical structural changes in nonmutated sites, including an increase in variable domain interactions. Our findings indicate that only a few mutations in the antigen-binding region of an antibody can lead to some structural changes in its paratopes, emphasizing the critical roles of the plasticity of loops in the complementarity-determining region and also the importance of the plasticity of the interaction between the variable regions of immunoglobulin heavy and light chains in determining the specificity of an antibody.

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Section: Discussionmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The enhance its specificity toward turkey lysozyme (a naturally mutated antigen) (28). Isothermal titration calorimetric studies have revealed the following (28).…”

mentioning

confidence: 99%

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Structural Consequences of Target Epitope-directed Functional Alteration of an Antibody

Koyanagi¹,

Nishimiya²,

Kondo³

et al. 2003

Journal of Biological Chemistry

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“…We focused on the interaction between hen egg white lysozyme (HEL) and the variable domain fragment (Fv) of the anti-HEL monoclonal antibody HyHEL-10, which is one of the most studied proteinaceous antigen-antibody interactions in terms of structural and functional features (42)(43)(44)(45)(46)(47)(48)(49)(50). The bacterial expression system for the HyHEL-10 Fv fragment has been established (51)(52)(53), and the Fv-HEL interactions have been investigated by using the wild-type and/or mutant Fv fragments (42-44, 46, 47, 54), including the x-ray crystal structure of its complex with HEL (45,47,48,55).…”

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confidence: 99%

Contribution of Asparagine Residues to the Stabilization of a Proteinaceous Antigen-Antibody Complex, HyHEL-10-Hen Egg White Lysozyme

Yokota

Tsumoto

Shiroishi

et al. 2010

Journal of Biological Chemistry

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Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of antigen-antibody complexes, we examined the interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of L-Asn-31, L-Asn-32, and L-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the antigen-antibody interaction can be attributed to their limited flexibility and accessibility at the complex interface.The specific recognition of ligands by proteins is a fundamental biological phenomenon (1), and the interaction between antigen and antibody in the immune system is a typical example. Antibodies acquire their affinity and specificity for various target antigens by changing the amino acid residue composition of their six hyper-variable regions, known as complementarity-determining regions (CDRs) 3 (2). Despite the small number of amino acid residues composing the CDRs, antibodies can precisely bind a large number of target antigens (3). Some of these residues, serine and asparagine, for example, are located at specific positions in the CDRs in the germ line protein (supplemental Fig. S1). Many structural studies have shown that these residues are essential for the affinity and specificity of the antigen-antibody interaction (4 -9, 11).Recent high resolution analyses of three-dimensional structures of protein-ligand complexes, including the antigen-antibody complexes, and analyses of the kinetic and thermodynamic parameters underlying these interactions (12-19) have indicated that protein-ligand interactions require a good geometric fit according to the lock-and-key (20) and induced fit models (21) as well as a high degree of complementarity of hydrophobic and polar parts of each binding site (17,22,2...

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Protein Dynamics and the Evolution of Novel Protein Function

Zimmermann¹,

Thielges²,

Yu³

et al. 2008

Protein Engineering Handbook

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Thermodynamic Consequences of Grafting Enhanced Affinity toward the Mutated Antigen onto an Antibody

Cited by 20 publications

References 51 publications

Structural Consequences of Target Epitope-directed Functional Alteration of an Antibody

Structural Consequences of Target Epitope-directed Functional Alteration of an Antibody

Contribution of Asparagine Residues to the Stabilization of a Proteinaceous Antigen-Antibody Complex, HyHEL-10-Hen Egg White Lysozyme

Protein Dynamics and the Evolution of Novel Protein Function

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