Recognition Mechanisms between a Nanobody and Disordered Epitopes of the Human Prion Protein: An Integrative Molecular Dynamics Study

Abstract

Immunotherapy using antibodies to target the aggregation of flexible proteins holds promise for therapeutic interventions in neurodegenerative diseases caused by protein misfolding. Prions or PrP^Sc, the causal agents of transmissible spongiform encephalopathies (TSE), represent a model target for immunotherapies as TSE are prototypical protein misfolding diseases. The X-ray crystal structure of the wild-type (WT) human prion protein (HuPrP) bound to a camelid antibody fragment, denoted as Nanobody 484 (Nb484), has been previously solved. Nb484 was found to inhibit prion aggregation in vitro through a unique mechanism of structural stabilization of two disordered epitopes, that is, the palindromic motif (residues 113-120) and the β2-α2 loop region (residues 164-185). The study of the structural basis for antibody recognition of flexible proteins requires appropriate sampling techniques for the identification of conformational states occurring in disordered epitopes. To elucidate the Nb484-HuPrP recognition mechanisms, here we applied molecular dynamics (MD) simulations complemented with available NMR and X-ray crystallography data collected on the WT HuPrP to describe the conformational spaces occurring on HuPrP prior to Nb484 binding. We observe the experimentally determined binding competent conformations within the ensembles of pre-existing conformational states in solution before binding. We also described the Nb484 recognition mechanisms in two HuPrP carrying a polymorphism (E219K) and a TSE-causing mutation (V210I). Our hybrid approaches allow the identification of dynamic conformational landscapes existing on HuPrP and highly characterized by molecular disorder to identify physiologically relevant and druggable transitions.

Figure 1

Cartoon representation of the NMR structure of WT HuPrP (residues 117–226, superposition of the 20 lowest-energy NMR structures, PDB ID 2lsb) in panel A and of the X-ray crystal structure of WT HuPrP (residues 117–226) bound to Nb484 (PDB ID 4kml) in panel B. The secondary structure motifs are highlighted and include the β1, α1, β2, α2, and α3 in free HuPrP and β0, β1, α1, β2, α2, and α3 in HuPrP bound to Nb484. The Nanobody binds to HuPrP through two complementarity-determining regions (CDR), named CDR2 and CDR3. Interacting surfaces on HuPrP side include the palindromic motif (residues 113–120) and the β2−α2 loop region (residues 164–185). In C, superposition of Nb484 X-ray crystal structures in the bound (PDB ID 4kml, coloured in red) and unbound states (PDB ID 6heq, coloured in magenta).

Figure 2

Overall computational flowchart and root mean square fluctuations (RMSFs) of apo and holo HuPrP structures. (A) Workflow to study the conformational energy landscapes of HuPrP by means of MD starting from available experimental NMR and X-ray diffraction (XRD) protein crystallography data. As NMR structures, we used the following PDB IDs: 2LSB (WT), 2LFT (E219K), and 2LEJ (V210I); as XRD structure, we used the PDB ID 4N9O (WT HuPrP-Nb484). (B) Analysis of RMSF in the MD structures. The regions that show higher flexibility are highlighted with a different colour in the MD 3D structures of WT and mutant HuPrP (see in the inset). The first and the last residues have a RMSF value higher than 0.3 nm since they are in a terminal position and are omitted here from the analysis. In black, green and red the RMSF of the WT, V210I, and E219K HuPrP MD structures, respectively. (C) Analysis of the 3 μs-RMSF trajectories of the WT, E219K, and V210I HuPrP-Nb484 complexes. The HuPrP regions involved in the Nb484 binding are highlighted in yellow color (residues 123 to 125 and from 164 to 185). In the inset, a representative MD ensemble of the WT HuPrP-Nb484. On the top, secondary structures of HuPrP indicate the positions of E219K and V210I mutations.

Figure 3

Local structural variation at the β2−α2 loop region. (A) Superposition of WT, E219K, and V210I NMR (A) and WT HuPrP-Nb484 (B) structures in the region encompassing the β2−α2 loop (residues 164–185). Conformation transitions of Tyr169 residue (black, red, and green for WT, E219K, and V210I, respectively) pointing outside (toward the solvent) in the NMR bundles (“open” conformation) or inside (“close” conformation) as observed in the WT HuPrP-Nb484 XRD structure. Ramachandran phi (upper panel) and psi (lower panel) dihedral angle trajectory distributions in MD apo (C) and holo (D) structures (black, green, and red for WT, V210I, and E219K, respectively) and in the Nb484-bound WT HuPrP-Nb484 XRD structure (blue bar).

Figure 4

Ramachandran plots for selected residues of WT, E219K, and V210I HuPrP. The numbers of key residues involved in conformational changes (G124, M166, D167, and N171) are indicated on the right, while the HuPrP construct (WT, E219K, and V210I) is indicated on the top. In black dots, phi/psi pairs from MD snapshots (apo MD) are reported; in red dots, the ones extracted from the NMR structures in the deposited bundle (apo NMR); in green dots, the ones from simulated WT, E219K, and V210I HuPrP bound to Nb484 (holo MD); and in blue squares with green background, the ones extracted from the crystallographic structure of WT HuPrP bound to Nb484 (holo XRD). In the M166 and D167 panels, corresponding to the V210I mutant, the arrows highlight the arrangements of dihedral angles of these residues that occupy totally different positions in the Ramachandran space compared to the XRD structure. To better appreciate these arrangements, we show in Figure S6 the same Ramachandran plots without the green dots corresponding to holo MD.

Figure 5

Mapping the β2−α2 loop conformational ensembles of WT, E219K, and V210I. (A) RMSD of the backbone of apo WT HuPrP (black), apo E219K (red), and apo V210I (green) computed on the backbone atoms with respect to corresponding NMR bundles. (B) RMSD of the backbone of apo WT HuPrP (black), apo E219K (red), and apo V210I (green) computed on the backbone atoms with respect to the XRD structure of the WT in complex with Nb484. The data with and without (w/o) the inclusion of the β2−α2 loop (residues 163–173) are reported as continuous and dashed lines, respectively.

Figure 6

Principal component analyses (PCA). Projection of the motions of the proteins in phase space along the PC1 and PC2 is drawn for WT, E219K, and V210I in panels A, B, and C, respectively. Heatmap visualization of the distribution probability of the motion projections. The centroid populations (expressed in arbitrary values calibrated with a maximum height equal to 35 counts) are indicated with a letter code. In bold, the centroids showing the lower RMSD compared to the HuPrP-Nb484 structure (Table S3).

Figure 7

Mechanisms of WT HuPrP-Nb484 molecular recognition in the β2−α2 loop epitope region. (A) Ramachandran plots for selected residues within the β2−α2 loop. In black and green dots, the phi/psi pairs from MD snapshots from apo MD and holo MD, respectively. The dotted square indicates the residues involved in Nb484 interaction and subjected to adaptable changes during the conformational selection steps. (B) Schematic representation of the β2−α2 loop region, where black and white circles represent the residues involved in unchanged or adaptable states, respectively, during the binding with key residues (indicated in green) of Nb484. (C) Close overview of a MD snapshot from holo HuPrP with highlighted the residues involved in dynamic interactions (in gray and magenta color the HuPrP and Nb484, respectively).

Figure 8

Proposed models for Nb484 recognition mechanisms in HuPrP carrying disease-related point mutations. On the left, the HuPrP β2−α2 loop and the palindromic motif (circled in yellow) are key sites for Nb484 interaction and highly flexible segments. The recognition of these antigens by Nb484 mainly follows the conformational selection model for the three HuPrP systems here investigated. In the CJD-linked V210I mutant, we also described a contribution of the induced fit mechanism for some residues within the loop.