Energetic analysis of an antigen/antibody interface: Alanine scanning mutagenesis and double mutant cycles on the hyhel‐10/lysozyme interaction

Pons, Jaume; Rajpal, Arvind; Kirsch, Jack F.

doi:10.1110/ps.8.5.958

Cited by 101 publications

(131 citation statements)

References 41 publications

(30 reference statements)

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“…Thus, formation of HzKR127-preS1 complex is stabilized by the accumulation of many interactions throughout the binding interface, as found in the A6-IFN-␥ receptor complex (23) and the VEGFFab complex (24). This contrasts with the antibody D1.3-hen egg white lysozyme (HEL) complex (25) and the HyHEL-10-lysozyme complex (26), in which only a small subset of residues dominate the energetics of association (one and three hot spots of ⌬⌬G D of Ͼ4 kcal/mol, respectively). Second, major binding determinants are concentrated in light-chain CDRs (10 of 16 hot spots) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Broadly neutralizing anti-hepatitis B virus antibody reveals a complementarity determining region H3 lid-opening mechanism

Chi

Maeng²,

Kim

et al. 2007

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

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The humanized monoclonal antibody HzKR127 recognizes the preS1 domain of the human hepatitis B virus surface proteins with a broadly neutralizing activity in vivo. We present the crystal structures of HzKR127 Fab and its complex with a major epitope peptide. In the complex structure, the bound peptide forms a type IV ␤-turn followed by 310 helical turn, the looped-out conformation of which provides a structural basis for broad neutralization. Upon peptide binding, the antibody undergoes a dramatic complementarity determining region H3 lid opening. To understand the structural implication of the virus neutralization, we carried out comprehensive alanine-scanning mutagenesis of all complementarity determining region residues in HzKR127 Fab. The functional mapping of the antigen-combining site demonstrates the specific roles of major binding determinants in antigen binding, contributing to the rational design for maximal humanization and affinity maturation of the antibody.alanine-scanning mutagenesis ͉ crystal structure ͉ humanized antibody ͉ virus neutralization H uman hepatitis B virus (HBV) is a small enveloped DNA virus that causes acute and chronic hepatitis in humans (1-3). There are approximately 400 million carriers of HBV worldwide, posing a serious health threat. The current HBV vaccines have several limitations, including nonresponsiveness, escape mutants, and low immunogenicity (4). Recently, the potential of immunoprophylaxis for virus prevention has been increased by the discovery of monoclonal antibodies that have broad specificity toward various isolates of infectious viruses (5, 6).The HBV envelope consists of three surface glycoproteins called the large (L), middle (M), and small (S) proteins. All of these proteins are translated from a single ORF comprising preS1, preS2, and S domains (7). Translation of S, preS2ϩS, and preS1ϩpreS2ϩS domains results in S, M, and L proteins, respectively (Fig. 1). The L protein, preferentially localized in infectious HBV particles, plays an essential role in viral infection (7,8), and its preS1 domain is responsible for cell attachment in viral infection (9-12). In particular, the residue 21-47 region of preS1 has been suggested to contain a specific binding site for hepatocyte receptors and also has been shown to carry virusneutralizing epitopes (10-13).In the blood of HBV-infected persons, the noninfectious subviral particles with M and S proteins are present in excess amounts over the infectious virus particles containing L protein (7,8). Thus, neutralizing anti-preS1 antibodies are thought to have advantages over the neutralizing antibodies targeting S protein, which are currently undergoing clinical trials (4). Previously, we generated an anti-preS1 murine antibody KR127 that recognizes adr subtype preS1 (residues 37-45) (15). KR127 and the humanized version of KR127 antibody (HzKR127) bind to the preS1 domain of various clinical HBV isolates, including both adr and ayw subtypes, suggesting their broad neutralizing activity (15, 16). HzKR127 also has been sh...

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Section: Resultsmentioning

confidence: 99%

Broadly neutralizing anti-hepatitis B virus antibody reveals a complementarity determining region H3 lid-opening mechanism

Chi

Maeng²,

Kim

et al. 2007

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

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“…In many antibodies, Tyr residues located at the surface are critical for antigen binding (45)(46)(47)(48)(49)(50)(51)(52)(53), and Tyr residues often have an important role in other protein-protein interactions (45,46,54,55). In the following sections, we focus on the role of an aromatic ring to the polyether-antibody interaction, which was revealed by systematic mutation of L-Tyr-91.…”

Section: Role Of Each Residue In the Interaction Of Ctx3c-abc With Thmentioning

confidence: 99%

Critical Contribution of Aromatic Rings to Specific Recognition of Polyether Rings

Tsumoto

Yokota

Tanaka

et al. 2008

Journal of Biological Chemistry

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To address how proteins recognize polyether toxin compounds, we focused on the interaction between the ABC ring compound of ciguatoxin 3C and its specific antibody, 1C49. Surface plasmon resonance analyses indicated that Escherichia coliexpressed variable domain fragments (Fv) of 1C49 had the high affinity constants and slow dissociation constants typical of antigen-antibody interactions. Linear van't Hoff analyses suggested that the interaction is enthalpy-driven. We resolved the crystal structure of 1C49 Fv bound to ABC ring compound of ciguatoxin 3C at a resolution of 1.7 Å . The binding pocket of the antibody had many aromatic rings and bound the antigen by shape complementarity typical of hapten-antibody interactions. Three hydrogen bonds and many van der Waals interactions were present. We mutated several residues of the antibody to Ala, and we used surface plasmon resonance to analyze the interactions between the mutated antibodies and the antigen. This analysis identified Tyr-91 and Trp-96 in the light chain as hot spots for the interaction, and other residues made incremental contributions by conferring enthalpic advantages and reducing the dissociation rate constant. Systematic mutation of Tyr-91 indicated that CH-and -interactions between the aromatic ring at this site and the antigen made substantial contributions to the association, and van der Waals interactions inhibited dissociation, suggesting that aromaticity and bulkiness are critical for the specific recognition of polyether compounds by proteins.Ciguatera is a form of food poisoning caused by the ingestion of reef fish that have accumulated trace amounts of ciguatoxins of dinoflagellate origin via the food chain. More than 50,000 people suffer from ciguatera annually, making it one of the most common sources of food poisoning (1-4). The disease is characterized by gastrointestinal, neurological, and cardiovascular disturbances that often persist for months or years, and in severe cases, paralysis, coma, and death may occur (1). Ciguatoxins exert their effects by binding to voltage-sensitive sodium ion channels, causing persistent activation of the channels (1). Ciguatoxin and its congener, CTX3C, are structurally classified as ladder-like polyethers ( Fig. 1) (5, 6). A major obstacle to avoiding the disease is that ciguateric fish look, taste, and smell the same as uncontaminated fish. In addition, neither cooking nor freezing detoxifies the heat-stable ciguatoxins. Despite the seriousness of ciguatera, there is currently no rapid and reliable method for detecting these toxins at fisheries. The traditional method is a mouse bioassay of lipid extracts (7). Several additional methods for detecting ciguatoxins have recently been developed, including assays based on cytotoxicity (8), radioligand binding (8, 9), high performance chromatography (10), mass spectrometry (11-13), and an antibody-based immunoassay (14 -16). Among these, the antibody-based immunoassay is attractive, because it is accurate, sensitive, easily performed, and portable. Dur...

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“…⌬⌬G int = ⌬⌬G A→ AЈ + ⌬⌬G B→ BЈ − ⌬⌬G AB→ AЈBЈ ( 1) This method, while theoretically powerful, requires either very precise experimental data or that the interrogated ⌬⌬G int values be large (Pons et al 1999).…”

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confidence: 99%

A general method for the quantitative analysis of functional chimeras: Applications from site‐directed mutagenesis and macromolecular association

Luong

Kirsch

2001

Protein Science

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Two new parameters, I and C, are introduced for the quantitative evaluation of functional chimeras: I (impact) and C (context dependence) are the free energy difference and sum, respectively, of the effects on a given property measured in forward and retro chimeras. The forward chimera is made by substitution of a part "a" from ensemble A into the analogous position of homologous ensemble B (S B → A ). The C value is a measure of the interaction of the interrogated position with its surroundings, whereas I is an expression of the quantitative importance of the probed position. Both I and C vary with the evaluated property, for example, kinetics, binding, thermostability, and so forth. The retro chimera is the reverse substitution of the analogous part "b" from B into A, S A → B . The I and C values derived from original data for forward and retro mutations in aspartate and tyrosine aminotransferase, from literature data for quasi domain exchange in oncomodulin and for the interaction of Tat with bovine and human TAR are evaluated. The most salient derived conclusions are, first, that Thr 109 (AATase) or Ser 109 (TATase) is an important discriminator for dicarboxylic acid selectivity by these two enzymes (I < −2.9 kcal/mol). The T109S mutation in AATase produces a nearly equal and opposite effect to S109T in TATase (C < 0.4 kcal/mol). Second, an I value of 5.5 kcal/mol describes the effects of mirror mutations D94S (site 1) and S55D (site 2) in the Ca 2+ binding sites of oncomodulin on Ca 2+ affinity. The second mirror set, G98D (site 1) and D59G (site 2), yields a smaller impact (I ‫ס‬ −3.4 kcal/mol) on Ca 2+ binding; however, the effect is significantly more nearly context independent (C ‫ס‬ −0.6 versus C ‫ס‬ −2.7 kcal/mol). Third, the stem and loop regions of HIV and BIV TAR are predominantly responsible for the species specific interaction with BIV Tat 65-81 (I ‫ס‬ −1.5 to −1.6 kcal/mol), whereas I ‫ס‬ 0.1 kcal/mol for bulge TAR chimeras. The C values are from −0.3 to −1.2 kcal/mol. The analysis described should have important applications to protein design.

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Energetic analysis of an antigen/antibody interface: Alanine scanning mutagenesis and double mutant cycles on the hyhel‐10/lysozyme interaction

Cited by 101 publications

References 41 publications

Broadly neutralizing anti-hepatitis B virus antibody reveals a complementarity determining region H3 lid-opening mechanism

Broadly neutralizing anti-hepatitis B virus antibody reveals a complementarity determining region H3 lid-opening mechanism

Critical Contribution of Aromatic Rings to Specific Recognition of Polyether Rings

A general method for the quantitative analysis of functional chimeras: Applications from site‐directed mutagenesis and macromolecular association

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