Insights into Siglec-7 Binding to Gangliosides: NMR Protein Assignment and the Impact of Ligand Flexibility

Abstract

Gangliosides, sialylated glycosphingolipids abundant in the nervous system, play crucial roles in neurotransmission, interaction with regulatory proteins, cell-cell recognition, and signaling. Altered gangliosides expression has been correlated with pathological processes, including cancer, inflammatory disorders, and autoimmune diseases. Gangliosides are important endogenous ligands of Siglecs (Sialic acid-binding immunoglobulin-type lectins), I-type lectins mostly expressed by immune cells, that specifically recognize sialylated glycans. Siglec-7, an inhibitory immune receptor on human natural killer cells, represents a potential target for tumor immunotherapy. Notably, the expression of Siglec-7 ligands is high in various cancers, such as pancreatic cancer and melanoma and lead to tumor immune evasion. Siglec-7 binds the disialylated ganglioside GD3, a tumor-associated antigen overexpressed on cancer cells to suppress immune responses. Using a combination of structural biology techniques, including Nuclear Magnetic Resonance (NMR), biophysical, and computational methods, the binding of Siglec-7 to GD3 and Gb3 derivatives is investigated, revealing the importance of ligand conformation in modulating binding energetics and affinity. The greater flexibility of Gb3 derivatives appears to negatively impact binding entropy, leading to lower affinity compared to GD3. A thorough understanding of these interactions could contribute to elucidating molecular mechanisms of cancer immune evasion and facilitate the development of ganglioside-based diagnostic and therapeutic strategies for cancer.

Keywords: NMR; gangliosides; siglec‐7; structural biology.

Figure 1

Siglec‐7 expression, purification, and backbone assignment by NMR. A) SDS‐PAGE representation: Lysis step – (1) supernatant, (2) cell debris; Solubilization of Inclusion bodies I‐ (3) supernatant, (4) cell debris; Solubilization of Inclusion bodies II – (5) supernatant, (6) cell debris; Solubilization of Inclusion bodies III – (7) supernatant, (8) cell debris; Solubilization of Inclusion bodies IV – (9) supernatant, (10) cell debris; Solubilization of Inclusion bodies V – (11) supernatant, (12) cell debris; Solubilization of Inclusion bodies VI – (13) supernatant, (14) cell debris. B) SDS‐PAGE representation: HisTrap purification, 1st cycle – Flow‐through (15), Column wash (16), Elution (17); HisTrap purification, 2nd cycle – Flow‐through (18), Column wash (19), Elution (20), 1st Elution and 2nd Elution (21). C) SDS‐PAGE representation: Size‐exclusion chromatography – collected fraction of Siglec‐7 CRD. D) Size‐exclusion chromatogram representation of Siglec‐7 CRD as single peak. E) 2D ¹H‐¹⁵N HSQC NMR spectrum of the apo Siglec‐7 CRD in 20 mm KPi, 50 mm NaCl, pH 7.4 acquired on a spectrometer operating at 900 MHz at 298 K. NH amino acid resonances obtained by the protein assignment are reported in the spectrum.

Figure 2

Binding isotherms obtained by means of fluorescence spectroscopy for the complex formation between Siglec‐7 CRD and (A) ligand 1 (GD3), (B) ligand 2 (DSGb3α3), and (C) ligand 3 (DSGb3α6) at the temperatures of 10 °C (blue circles), 25 °C (green squares) and 35 °C (red diamonds). The solid lines represent the best fit of the experimental data according to a 1:1 binding model equation. All the experiments were performed in PBS buffer, pH 7.4.

Figure 3

Ligand‐ and Protein‐based NMR analysis of ligand GD3 in interaction with Siglec‐7. A) Epitope mapping of ligand GD3 highlights the protons most involved in interaction with Siglec‐7. %STD values are obtained by the ratio (I₀ − I_sat)/I₀, where (I₀ − I_sat) is the signal intensity in the STD‐NMR spectrum (red) and I₀ is the peak intensity of the off‐resonance spectrum (black). Despite some isochronous signals between N and K, the isolated signals from K, such as K8, as well as the diastereotopic K9 and K3 (axial and equatorial) protons were not recognized by the protein. However, an STD enhancement referring to the acetyl group (AcK) was observed, meaning a partial contribution of the internal sialic acid (K) occurred. Bioactive conformation of ligand GD3, the surface was colored according to the STD effects. B) Plots representing chemical shift perturbation (CSP) in red and decreases in signal intensity in blue. Surface representation of a model of Siglec‐7 with the residues experiencing the largest decrease in intensity in blue and CSP in red in the presence of 200 µm ligand GD3. Arg124 is colored green.

Figure 4

3D model of Siglec‐7CRD ‐ligand GD3 complex. A) 3D complex with the protein surface colored according to the chemical shift perturbation (in red) and intensity decreased (in blue) detected by protein‐based NMR titration. B) 3D views of the Siglec‐7−GD3 complex: the amino acids involved in the interactions as revealed by protein‐based NMR experiments were represented as sticks; the ligand surface was colored according to the STD edit code. C) 2D plot of the interactions occurring at the protein‐ligand interface. D) RMSD along MD simulation. E) Comparison between experimental (blue) and calculated %STD for protons of ligand 1 by RedMat analysis. A NOE R‐factor of 0.2 was calculated.

Figure 5

Representation of the Siglec‐7−GD3 complex with the BC, CC’ and GG’ loops highlighted in cyan, green, and orange, respectively. Different views highlighting the H‐bonds monitored by MD simulation were shown (in the zoom, the amino acids were colored according to the loops.

Figure 6

Ligand‐ and Protein‐based NMR analysis of ligand DSGb3α3 in interaction with Siglec‐7. A) Epitope mapping of ligand DSGb3α3 highlights the protons more involved in interaction with Siglec‐7. %STD values are obtained by the ratio (I₀ − I_sat)/I₀, where (I₀ − I_sat) is the signal intensity in the STD‐NMR spectrum (red) and I₀ is the peak intensity of the off‐resonance spectrum (black). Bioactive conformation of ligand DSGb3α3, the surface was colored according to the STD effects. B) Plots representing chemical shift perturbation (CSP) in red and decreases in signal intensity in blue. Surface representation of a model of Siglec‐7 with the residues experiencing the largest decrease in intensity in blue and CSP in red in the presence of 200 µm of ligand DSGb3α3. Arg124 is colored green.

Figure 7

3D model of Siglec‐7CRD ‐ligand DSGb3α3 complex. A) 3D complex with the protein surface colored according to the chemical shift perturbation (in red) and intensity decreases (in blue) detected by protein‐based NMR titration. B) 3D views of the Siglec‐7− DSGb3α3 complex: the amino acids involved in the interactions as revealed by protein‐based NMR experiments were represented as sticks; the ligand surface was colored according to the STD edit code. C) 2D plot of the interactions establishing at the protein‐ligand interface. D) RMSD along MD simulation. E) Comparison between experimental (blue) and calculated %STD for protons of ligand DSGb3α3 by RedMat analysis. A NOE R‐factor of 0.3 was calculated.

Figure 8

Representation of the Siglec‐7− DSGb3α3 complex with the BC, CC’ and GG’ loops highlighted in cyan, green, and orange, respectively. Different views highlighting the H‐bonds monitored by MD simulation were shown (in the zoom, the amino acids were colored according to the loops legend).

Figure 9

Ligand‐ and Protein‐based NMR analysis of ligand DSGb3α6 in interaction with Siglec‐7. A) Epitope mapping of ligand 3 highlights the protons more involved in interaction with Siglec‐7. %STD values are obtained by the ratio (I₀ − I_sat)/I₀, where (I₀ − I_sat) is the signal intensity in the STD‐NMR spectrum (red) and I₀ is the peak intensity of the off‐resonance spectrum (black). Bioactive conformation of ligand DSGb3α6, the surface was colored according to the STD effects. B) Plots representing chemical shift perturbation (CSP) in red and decreases in signal intensity in blue. Surface representation of a model of Siglec‐7 with the residues experiencing the largest decrease in intensity in blue and CSP in red in the presence of 200 µm ligand DSGb3α6. Arg124 is colored green.

Figure 10

A) Superimposition of 3D models of Siglec‐7CRD bound to ligand DSGb3α6 in tg and gt. B) 3D views of the Siglec‐7− DSGb3α6 complex: the amino acids involved in the interactions as revealed by protein‐based NMR experiments were represented as sticks; the ligand surface was colored according to the STD edit code. 2D diagram of the interactions established at the protein‐ligand interface, with DSGb3α6 in tg (A) and gt (B) conformations.

Figure 11

Representation of the Siglec‐7− DSGb3α6 complex in both gt (pink) and tg (cyan) conformations with the BC, CC’, and GG’ loops highlighted in cyan, green and orange, respectively. A) Different views of the Siglec‐7− DSGb3α6 complex in the gt conformation highlighting the H‐bonds monitored by MD simulation were shown (in the zoom, the amino acids were colored according to the loops legend). B) Different views of the Siglec‐7− DSGb3α6 complex in the tg conformation highlighting the H‐bonds monitored by MD simulation were shown (in the zoom, the amino acids were colored according to the loops legend).