Structural Analysis of Anti-Hapten Antibodies to Identify Long-Range Structural Movements Induced by Hapten Binding

Abstract

Antibodies are well known for their high specificity that has enabled them to be of significant use in both therapeutic and diagnostic applications. Antibodies can recognize different antigens, including proteins, carbohydrates, peptides, nucleic acids, lipids, and small molecular weight haptens that are abundantly available as hormones, pharmaceuticals, and pesticides. Here we focus on a structural analysis of hapten-antibody couples and identify potential structural movements originating from the hapten binding by comparison with unbound antibody, utilizing 40 crystal structures from the Protein Data Bank. Our analysis reveals three binding surface trends; S1 where a pocket forms to accommodate the hapten, S2 where a pocket is removed when the hapten binds, and S3 where no pockets changes are found. S1 and S2 are expected for induced-fit binding, whereas S3 indicates that a pre-existing population of optimal binding antibody conformation exists. The structural analysis reveals four classifications of structural reorganization, some of which correlate to S2 but not to the other binding surface changes. These observations demonstrate the complexity of the antibody-antigen interaction, where structural changes can be restricted to the binding sites, or extend through the constant domains to propagate structural changes. This highlights the importance of structural analysis to ensure successful and compatible transformation of small antibody fragments at the early discovery stage into full antibodies during the subsequent development stages, where long-range structural changes are required for an Fc effector response.

Keywords: RMSD; RMSF; allosteric movement; antibody; antigen binding fragments (Fab); hapten.

FIGURE 1

Angle measurements. The IgG antibody can be divided into three fragments: two Fab regions and one Fc region. The enlarged Fab region illustrates the amino acid positions used to calculate the domain orientational changes. The selected amino acids are denoted as Cys: cysteine, Ser: serine, Gln: glutamine (in λ light chains), and Arg: arginine (in K light chains).

FIGURE 2

Binding sites surfaces. The binding surfaces can be grouped into three categories. In S1, there is a pocket binding site on the antibody-antigen complex and not on the antibody-free counterparts, as shown in (A). In S2, there is a pocket binding site on the antibody-free structure and not on the antibody-antigen complex, as can be observed in (B). In S3, there is no (or only slight) change in the binding sites as shown in (C). The heavy and light chains were colored as denoted in the figure, and the hapten antigen is shown as a red line drawing.

FIGURE 3

RMSF analysis. RMSF measurements were analyzed for anti-hapten Fabs. Classes B1, B2, B3, and B4 are represented by 2CGR_1CGS, 3CFD_3CFE, 2AJV_2AJU, and 1KEL_1KEM, respectively. The RMSF comparison was performed on the total chain (heavy and light) and independently on their specific domains (V_H, C_H1, V_L, and C_L). Heavy chain loops were highlighted in grey and light chain loops were highlighted in yellow. The x-axis denotes the amino acid positions of either the heavy or light chain, permitting them to be examined in the same figure.

FIGURE 4

Binding sites of antibody 7A1. Illustration of the crystal structures of antibody 7A1 as free form (2AJU) and six antigen bound states against Ecgonine methyl ester (2AJZ), Ecgonine methyl ester and benzoic acid (2AJY), Cocaine (2AJV), Benzoic acid (2AK1), 3-(hydroxy-phenyl-phosphinoyloxy)-8-methyl-8-aza-bicyclo[3.2.1]octane-2-carboxylic acid methyl ester (2AJX), and PEG330 (2AJS). The heavy and light chains of the antibody-free form of 2AJU were colored as red and yellow, respectively. Whilst green (heavy chains) and blue (light chains) was used to color the antigen bound structures. Chemical structure of each of the antigens was depicted at the bottom of each crystal structure.

FIGURE 5

RMSF analysis of antibody 7A1. RMSF measurements were analyzed for the six bound states of antibody 7A1. Blue dotted line was placed at RMSF 1 Å to allow easier comparison. The bound states were 2AJS, 2AJY, 2AJV, D 2AJZ, 2AJX, and 2AK1.