Deciphering the crystal structure of a novel nanobody against the NEIL1 DNA glycosylase

Abstract

Nanobodies (VHHs) are single-domain antibodies with three antigenic CDR regions and are used in diverse scientific applications. Here, an ∼14 kDa nanobody (A5) specific for the endonuclease VIII (Nei)-like 1 or NEIL1 DNA glycosylase involved in the first step of the base-excision repair pathway was crystallized and its structure was determined to 2.1 Å resolution. The crystals posed challenges due to potential twinning and anisotropic diffraction. Despite inconclusive twinning indicators, reprocessing in an orthorhombic setting and molecular replacement in space group P2₁2₁2 enabled the successful modeling of 96% of residues in the asymmetric unit, with final R_work and R_free values of 0.199 and 0.229, respectively.

Keywords: NEIL1 DNA glycosylase; X-ray crystallography; nanobodies; orthorhombic space group; pseudo-merohedral twinning.

Figure 1

Increased crystal quality with decreasing construct disorder. (a) A5-10×His-FLAG PONDR analysis (left) predicted the largest region of disorder at the C-terminus. A5-10×His-FLAG produced small, clustered crystals that were unable to produce a diffraction pattern (right). (b) PONDR predicted A5-6×His to have a shorter disordered region at the C-terminus than the 10×His-FLAG construct above. The A5-6×His protein produced slightly larger, plate-like crystal clusters that produced low-resolution diffraction (∼7 Å). (c) The cleavable A5-TEV-6×His protein was predicted to have minimal disorder within the C-terminal region. The cleaved A5-TEV-6×His produced individual, three-dimensional, rod-shaped crystals that diffracted to 2.08 Å resolution.

Figure 2

The diffraction pattern of A5 reveals the possibility of twinning. Initial close observation of the spot intensities indicated the appearance of smearing caused by partially overlapping reflections that could be a result of multiple crystals, crystal fragmentation or possible twinning within the data. The enlarged view of the diffraction pattern (insets) displays a single spot (top), minimally overlapping spots (bottom left) and split spots (bottom right). The green box adjacent to each spot is the corresponding 3D peak profile, with the pixel intensity represented as counts. The figure was directly taken from Proteum III and compiled using Biorender.

Figure 3

The overall three-dimensional structure of A5. (a) The overall three-dimensional structure of A5 colored as follows: β-strands, purple arrows; π-helices, light blue; CDR1, green; CDR2, blue; CDR3, orange. The β-strands (β1–β11) and π-helices (π1–π2) are labeled in sequential order. The disulfide bond formed between Cys24 and Cys99 is highlighted in yellow and is displayed in the inset surrounded by a composite omit map contoured at 1σ cut to an area around the atoms. (b) The A5 sequence with the major secondary structures displayed as purple arrows (β-strands) and coils (π-helices). (c) Topology map of A5. (d) Organization of the four molecules (chains A–D) within the asymmetric unit.

Figure 4

Stabilizing interactions between chain A and neighboring molecules. (a, b) Hydrogen-bond interaction network at the interface of chains A, B and C in the asymmetric unit. (c, d, e) Hydrogen-bond interaction network highlighting interactions between chain A and symmetry mates of chain C.