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. 2023 Jul 24;28(14):5603.
doi: 10.3390/molecules28145603.

New Crystal Form of Human Neuropilin-1 b1 Fragment with Six Electrostatic Mutations Complexed with KDKPPR Peptide Ligand

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New Crystal Form of Human Neuropilin-1 b1 Fragment with Six Electrostatic Mutations Complexed with KDKPPR Peptide Ligand

Ibrahima Goudiaby et al. Molecules. .

Abstract

Neuropilin 1 (NRP1), a cell-surface co-receptor of a number of growth factors and other signaling molecules, has long been the focus of attention due to its association with the development and the progression of several types of cancer. For example, the KDKPPR peptide has recently been combined with a photosensitizer and a contrast agent to bind NRP1 for the detection and treatment by photodynamic therapy of glioblastoma, an aggressive brain cancer. The main therapeutic target is a pocket of the fragment b1 of NRP1 (NRP1-b1), in which vascular endothelial growth factors (VEGFs) bind. In the crystal packing of native human NRP1-b1, the VEGF-binding site is obstructed by a crystallographic symmetry neighbor protein, which prevents the binding of ligands. Six charged amino acids located at the protein surface were mutated to allow the protein to form a new crystal packing. The structure of the mutated fragment b1 complexed with the KDKPPR peptide was determined by X-ray crystallography. The variant crystallized in a new crystal form with the VEGF-binding cleft exposed to the solvent and, as expected, filled by the C-terminal moiety of the peptide. The atomic interactions were analyzed using new approaches based on a multipolar electron density model. Among other things, these methods indicated the role played by Asp320 and Glu348 in the electrostatic steering of the ligand in its binding site. Molecular dynamics simulations were carried out to further analyze the peptide binding and motion of the wild-type and mutant proteins. The simulations revealed that specific loops interacting with the peptide exhibited mobility in both the unbound and bound forms.

Keywords: Hirshfeld interface; Neuropilin 1; X-ray crystallography; electrostatic influence; ligand; molecular dynamics simulation; variant.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(a) Ribbon view of the crystal structure of NRP1-b1 hexavariant. The six mutations and the disulfide bridge Cys275-Cys424 are shown as sticks and labelled. The loops L1–L5 that line the VEGF-binding pocket are highlighted in magenta and labelled. The PPR moiety of KDKPPR peptide is shown as sticks with refined 2mFo-DFc electron density contoured at 1.0 σ. (b) Repartition of the charged residues in NRP1-b1 hexavariant. The positive (blue) and negative (red) charges are shown as spheres on the protein structure. The mutated residues with change in charge are labeled and are shown in light blue and light yellow. The PR residues of KDKPPR peptide are shown on the top of the figure.
Figure 2
Figure 2
Highlights (in green) of the NRP1-b1 residues involved in contacts with a neighboring monomer in the crystal forms I to VI. Form VI corresponds to the NRP1-b1 hexavariant. Contacts are defined as residues with a proximity of less than 4 Å. Residues in bold characters forms intermolecular hydrogen bonds. The positions of the mutations have been highlighted with triangles above the two sets of residue numbering.
Figure 3
Figure 3
(a) Structure of the binding site of NRP1-b1 hexavariant in complex with the KDKPPR peptide. The pocket is mainly composed of five loops (L1 to L5), which are respectively colored blue, cyan, green, red and turquoise. The crystallographic model of the peptide includes only the PPR moiety, represented as sticks. The NRP1 residues in the close proximity of the peptide are also depicted as sticks. The four structural water molecules are highlighted as spheres, while hydrogen bonds are illustrated as dashed sticks. Various labels are provided to enhance clarity, indicating loops, peptide, residues and water molecules. (b) Nucleophilic Influence Zones associated with the oxygen atoms Asp320-Oδ1 (dark blue), Asp320-Oε2 (light blue), Tyr297-Oη (green) and Glu348-Oε2 (major conformer, orange) in the vicinity of the ligand-binding site of monomer A. The corresponding atomic nucleophilic sites are indicated by colored circles, and the PPR moiety of the KDKPPR peptide is highlighted in yellow.
Figure 4
Figure 4
Coordinate RMSD (Å) calculated on the backbone heavy atoms of NRP1-b1 with respect to the initial X-ray crystallographic structure. The curves measured on the triplicated trajectories are colored in black, green and gray, respectively.
Figure 5
Figure 5
Atomic root mean square fluctuations (RMSFs, Å) calculated along the molecular dynamics (MD) trajectories by superimposing the heavy backbone atoms of NRP1-b1 on the corresponding atoms in the initial crystal structure. The upper panel corresponds to the trajectories recorded on the WT protein, while the lower panel corresponds to the trajectories recorded on the NRP1-b1 hexavariant used to determine the crystal structure. The curves measured on the triplicated trajectories are colored black, green and grey, respectively. They are plotted as solid and dashed lines for NRP1-b1 in complex with the peptide and for the unbound form, respectively. The positions in the sequence of the mutated residues are marked with the letter ‘M’. The NRP1 loops interacting with the peptide are labelled as defined in the text.

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