DNA binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane pyrimidine dimer

Abstract

Monoclonal antibodies specific for the cyclobutane pyrimidine dimer (CPD) are widely used for detection and quantification of DNA photolesions. However, the mechanisms of antigen binding by anti-CPD antibodies are little understood. Here we report NMR analyses of antigen recognition by TDM-2, which is a mouse monoclonal antibody specific for the cis - syn -cyclobutane thymine dimer (T[ c, s ]T). (31)P NMR and surface plasmon resonance data indicated that the epitope recognized by TDM-2 comprises hexadeoxynucleotides centered on the CPD. Chemical shift perturbations observed for TDM-2 Fab upon binding to d(T[ c, s ]T) and d(TAT[ c, s ]TAT) were examined in order to identify the binding sites for these antigen analogs. It was revealed that d(T[ c, s ]T) binds to the central part of the antibody-combining site, while the CPD-flanking nucleotides bind to the positively charged area of the V(H)domain via electrostatic interactions. By applying a novel NMR method utilizing a pair of spin-labeled DNA analogs, the orientation of DNA with respect to the antigen-binding site was determined: CPD-containing oligonucleotides bind to TDM-2 in a crooked form, draping the 3'-side of the nucleotides onto the H1 and H3 segments, with the 5'-side on the H2 and L3 segments. These data provide valuable information for antibody engineering of TDM-2.

Figure 1

Structure of the cyclobutane pyrimidine dimer.

Figure 2

³¹P NMR spectra of d(TAT[c,s]TAT) (a and c) and d(GTAT[c,s]TATG) (b and d) in the absence (a and b) and presence (c and d) of 1.1 molar equivalents of TDM-2 Fab. All FIDs were multiplied by an exponential window function with a broadening factor of 1 Hz prior to Fourier transformation.

Figure 3

¹H-¹⁵N HSQC spectrum of the TDM-2 Fab labeled with [1-¹³C]Trp and [¹⁵N]Tyr. The boxed peak exhibiting a passive coupling with ¹J_CN is magnified in the inset. Protein concentration was 0.8 mM. The acquired data were zero filled once along the t₁ dimension, and multiplied by Gaussian window functions in the t₁ and t₂ dimensions.

Figure 4

Superpositions of ¹H-¹⁵N HSQC spectra of the TDM-2 Fab labeled with [¹⁵N]Tyr (a) in the absence (black) and presence (red) of 1.2 molar equivalents of antigen d(T[c,s]T), and (b) in the presence of 1.2 molar equivalents of d(T[c,s]T) (red) and d(TAT[c,s]TAT) (blue). Protein concentration was 0.5 mM.

Figure 5

The Fv model of TDM-2 built using the program AbM v.2.03 (Oxford Molecular). (a) Mapping of residues perturbed upon binding to d(T[c,s]T) on the Fv model of TDM-2. The residues whose chemical shift was perturbed and not perturbed upon binding of d(T[c,s]T) are colored red and green, respectively. The residues that gave no detectable peak in the absence of antigen but gave a detectable one in the presence of d(T[c,s]T) are colored yellow. (b) Mapping of the residues showing identical (green) and different (red) chemical shifts between the d(T[c,s]T)- and d(TAT[c,s]TAT)-bound forms. (c) Surface electrostatic potentials calculated and represented by use of the program Molmol 2.5.1. Blue, positive charge; red, negative charge.

Figure 6

Spin-labeled antigen analogs used in the present study. (a) 3′-TEMPO-d(AT[c,s]T) and (b) 5′-TEMPO-d(T[c,s]TA).

Figure 7

¹H-¹⁵N HSQC spectra of the TDM-2 Fab labeled with [¹⁵N]Phe and [¹⁵N]Tyr complexed with 3′-TEMPO-d(AT[c,s]T) (a) and 5′-TEMPO-d(T[c,s]TA) (b) in the absence (black) and in the presence (red) of ascorbic acid. Fab, 0.50 mM; 3′-TEMPO-d(AT[c,s]T) or 5′-TEMPO-d(T[c,s]TA), 0.55 mM; ascorbic acid, 0.66 mM.

Figure 8

Mapping of the residues close to the 3′- or 5′-end of d(T[c,s]T). The residues whose HSQC peak intensities were affected by 3′-TEMPO-d(AT[c,s]T) and 5′-TEMPO-d(T[c,s]TA) are colored yellow and red, respectively. Residues affected neither by 3′-TEMPO-d(AT[c,s]T) nor by 5′-TEMPO-d(T[c,s]TA) are colored green.