Structural basis of G-quadruplex recognition by a camelid antibody fragment

Abstract

Apart from the iconic Watson-Crick duplex, DNA can fold into different noncanonical structures, of which the most studied are G-quadruplexes (G4s). Despite mounting structural and biophysical evidence, their existence in cells was controversial until their detection using G4-specific antibodies. However, it remains unknown how antibodies recognize G4s at the molecular level and why G4-specific antibodies have low selectivity and are unable to distinguish different G4 sequences. Here, we present the crystal structure of a nanobody bound to the archetypical G4 structure, the thrombin-binding aptamer (TBA). The nanobody exhibits strong selectivity against different G4 sequences and utilizes an unusual scaffold-based paratope, with very limited involvement of complementarity-determining region. The nanobody effectively mimics the binding interface of thrombin, a natural binding partner of TBA, by using isosteric interactions at key positions. The presented structure sheds light on the molecular basis of how antibodies, essential G4-detection tools, recognize noncanonical G4 structures.

Graphical Abstract

Figure 1.

Biophysical characterization of the Nb55–TBA interaction. (A) A G-quartet structural motif is stabilized by Hoogsteen-type hydrogen bonds (dotted lines) and a central monovalent cation. (B) Thermal melting of TBA and TBA–Nb55 complex, monitored by circular dichroism (CD) spectroscopy, shows an increase in TBA melting temperature in the presence of Nb55. (C) Isothermal titration calorimetry (ITC) thermogram (top) and the integrated heat (bottom) for the titration of Nb55 into TBA at 25°C. A representative thermogram of N= 3 independent experiments is shown. The fit of a single-site binding model is represented by a solid line. (D) Monodispersity and molecular weight analysis of Nb55–TBA complex monitored by size-exclusion chromatography coupled with multi-angle light scattering (SEC–MALS). Chromatograms of individual components are shown for comparison. (E) CD spectra of free and Nb55-bound TBA show stabilization of TBA upon Nb55 binding. (F) Binding selectivity of Nb55 to different DNA structures. The bar chart shows the logarithm of binding affinity measured by ITC for different types of DNA structures and quadruplexes with data shown in Supplementary Table S3.

Figure 2.

Asymmetric unit of the Nb55–TBA crystals. (A) The asymmetric unit consists of four molecules (two Nb55 and two TBA) that interact through three unique interfaces, shown with black dashed lines. TBA loops are shown in red. A close-up view highlights the positions of mutated residues that abrogate: interface 1 through the Gln81Asp mutation, interface 2 through the Arg45Asp or the Trp105Glu mutations, and interface 3 through the Thr56Asp mutation. (B) The corresponding ITC titrations for Nb55 mutated at each interface are shown in color, while that for wild-type Nb55 is shown in black for comparison. Only mutations at interface 2 (Arg45Asp or Trp105Glu) abrogate the Nb55–TBA interaction indicating that in solution Nb55–TBA complex forms though interface 2 (green check mark). Red crosses denote that interfaces 1 and 3 are not formed in solution.

Figure 3.

Molecular basis of TBA quadruplex recognition by Nb55. (A) The structure of Nb55-TBA complex. TBA loops are shown in red. (B) Topology representation of the nanobody and TBA, with dashed lines showing interactions color coded by nanobody’s structural elements. (C) The only CDR3 loop interaction is mediated by the main chain hydrogen bond to thymine T3 of TT loop 1. Arg45 interacts with both TT loops and G-quartet. (D) The C–C′ loop extends along the major groove, reaching the TGT loop. (E) The two Trp–Tyr pairs of the aromatic core interact with T12 and T4 on both TT loops. (F) Structural superposition of free (orange) and TBA-bound (blue) Nb55. Upon interaction, the two β-hairpins open up to accommodate TBA. The shift of the FG β-hairpin likely disrupts CDR1, which becomes disordered in the TBA-bound structure.

Figure 4.

Nb55 mimics the key interactions of the thrombin–TBA complex. (A) Structural superposition of the thrombin–TBA and Nb55–TBA complexes. Exosite I loop is shown in dark violet. For simplicity, only conformation of the TBA bound to thrombin is shown. (B) Isosteric interactions with TT loop 1 and the second G-quartet, formed by Arg45 (Nb55) or Arg77 (thrombin). (C) The aromatic rings of Tyr37 (Nb55) and Tyr76 (thrombin) adopt the same relative position in both complexes. (D) Isosteric interactions with thymine T4 by Tyr94 (Nb55) or Asn79 (thrombin). (E) The thrombin electrostatic potential surface shows that the thrombin–TBA interface is stabilized by charge complementarity, whereas the Nb55–TBA interface exhibits a predominantly hydrophobic character.

Figure 5.

Nanobodies SG4 and Nb55 recognize overlapping epitopes. (A) 1D ¹H NMR spectra of TBA at different SG4 equivalents, with annotated imino signals visible due to hydrogen bonding. (B) ITC thermogram (top) and the integrated heat (bottom) for the titration of SG4 into TBA at 25°C. A representative thermogram of N= 3 independent experiments is shown. The fit of a single-site binding model is represented by a solid line. (C) Change in NMR signal intensity at 0.05 and 0.1 molar equivalents of SG4 relative to TBA. (D) Chemical shift changes of imino protons at 0.1 molar equivalent of SG4. (E) The two TT loops function as interaction hotspots for different binders: nanobodies Nb55 (yellow) and SG4 (pink), as well as thrombin (mint).