Application of CoLD-CoP to Detecting Competitively and Cooperatively Binding Ligands

Abstract

NMR utilization in fragment-based drug discovery requires techniques to detect weakly binding fragments and to subsequently identify cooperatively binding fragments. Such cooperatively binding fragments can then be optimized or linked in order to develop viable drug candidates. Similarly, ligands or substrates that bind macromolecules (including enzymes) in competition with the endogenous ligand or substrate are valuable probes of macromolecular chemistry and function. The lengthy and costly process of identifying competitive or cooperative binding can be streamlined by coupling computational biochemistry and spectroscopy tools. The Clustering of Ligand Diffusion Coefficient Pairs (CoLD-CoP) method, previously developed by Snyder and co-workers, detects weakly binding ligands by analyzing pairs of diffusion spectra, obtained in the absence and the presence of a protein. We extended the CoLD-CoP method to analyze spectra pairs (each in the presence of a protein) with or without a critical ligand, to detect both competitive and cooperative binding.

Keywords: cooperative/competitive ligand binding; diffusion spectroscopy (DOSY); fragment-based drug discovery (FBDD); nuclear magnetic resonance (NMR).

Scheme 1

Detection of cooperative and competitive binding using CoLD-CoP.

Scheme 2

Use of CoLD-CoP to detect competitive and cooperative binding. Interactions identified by CoLD-CoP map to the yellow-highlighted portion of the plots. (A) Traditional CoLD-CoP (Scheme 1, Step #3.B.i): Diffusion coefficients measured in the presence and absence of protein (ligand+protein vs. ligand-only). Ligand–protein binding is identified by slower diffusion in the presence of the protein. (B) Competitive binding (Scheme 1, Step #3.B.ii): diffusion coefficients measured in the absence and presence of a known ligand (ligand+protein vs. ligand+protein+known ligand). Competitive binding is identified by slower diffusion in the absence of the known ligand. (C) Cooperative binding (Scheme 1, Step #3.B.iii): diffusion coefficients measured in the presence and absence of a known ligand (ligand+protein+known ligand vs. ligand+protein). Cooperative binding is identified by slower diffusion in the presence of the known ligand.

Figure 1

DOSY spectra for mixtures (as labeled in Table 1) (A) TL, (B) TLP, (C) TLPK, (D) LL, (E) LLP, and (F) LLPK, obtained using the tyrosinase (A–C) and lysozyme systems (D–F). Vertical arrows of color-coordinated (to match the color scheme used in Figure 2 and Figure 3) compounds show peak correspondences between panels. Horizontal line styles correspond to diffusion coefficients in related mixtures. Solid lines indicate the average (by eye) diffusion coefficient for a given compound in the ligand+only (A,D), ligand+protein (B,E), and ligand+protein+known ligand (C,F) mixtures. The dashed lines indicate the comparable diffusion coefficients in the ligand+only (B,E) and ligand+protein (C,F) mixtures. The blue solid line in panel (A) and blue dashed line in panel (B) indicate the diffusion coefficient of salicylic acid in the absence of protein. The red solid line in panel (B) and red dashed line in panel (C) indicate the diffusion coefficient of salicylic acid in the presence of tyrosinase (but without 4-HCCA present). The muted red solid line in panel (C) indicates the diffusion coefficient of salicylic acid in the presence of both tyrosinase and 4-HCCA. Note that the muted red line is below the dashed red line in panel (C), indicating faster diffusion in the presence of 4-HCCA than in its absence, and hence that salicylic acid and 4-HCCA bind tyrosinase competitively. By way of comparison, the diffusion coefficients of tris and tartaric acid, indicated by the cyan annotations, do not change much between panels (B,C), as indicated by the proximity of the dashed cyan line and the muted cyan line in panel (C). The proximity of the yellow dashed line and yellow-orange solid line in panel (E) indicates the extremely weak binding of imidazole in the absence of GlcNAc. The larger gap between the solid and dashed lines in panel (F) indicates the cooperative binding of GlcNAc and imidazole to lysozyme. The near coincidence of the dashed and solid green lines in panel (E) indicates that tris binds lysozyme so weakly that its binding cannot be detected even by CoLD-CoP. However, the gap between the solid green line and the dashed mint green line in panel (F) indicates that GlcNAc and tris cooperatively bind lysozyme.

Figure 2

CoLD-CoP comparisons: circles and asterisks are colored (automatically by the MATLAB toolbox implementing CoLD-CoP) based on the cluster to which CoLD-CoP assigns them. Ideally, each cluster represents one and only one molecule, but molecules of similar size whose diffusion coefficient does not change between the solutions being compared may not be distinguishable based on paired diffusion coefficients. Open circles indicate diffusion coefficients that do not significantly change between the compared solutions while (filled) asterisks indicate a significant result. (A) CoLD-CoP comparison of solutions TL and TLP. In this comparison, the chemical shifts in the cluster denoted by blue asterisks are assignable to salicylate; that they are asterisks (rather than open circles) indicates that it binds tyrosinase; (B) comparison of solutions TLP and TLPK to detect competitive binding as described in Scheme 1; (C) Same as (B), but zoomed in to show competitive binding more clearly: asterisks indicate clusters with significantly higher diffusion coefficients in the presence of 4-HCCA, indicating competitive binding. Each significant cluster (yellow and red asterisks) is associated with chemical shifts assigned to salicylate. Note that salicylate, tartrate, and tris all have similar molecular masses and hence similar diffusion coefficients. This approach cannot distinguish between tris and tartrate (empty cyan circles), but does distinguish (albeit dividing it into two clusters) salicylate as competing with 4-HCCA for tyrosinase binding.

Figure 3

(A) The purpose of this comparison, performed as described in Scheme 1, Step 3.B.ii, is to detect any ligands binding competitively with GlcNAc via CoLD-CoP analysis of solution LLP (Lysozyme Ligands: Caffeine, Citrate, Imidazole, Tartrate, Tris, Tryptamine) against LLPK (Lysozyme +Ligands+Known Ligand: GlcNAc). CoLD-CoP does identify any ligand as competing with GlcNAc for lysozyme binding. (B) A similar comparison performed as described in Scheme 1, Step 3.B.iii. (C) Zoomed-in portion of panel (B). Points are colored by cluster as identified by CoLD-CoP, which identifies two diffusion coefficient pairs (closed circles) as diffusing significantly slower in the presence of (i.e., cooperatively binding with) GlcNAc: the closed orange circle corresponds (by chemical shift) to imidazole, and the closed green circle corresponds to tris.

Figure 4

Model of the lysozyme/GlcNAc/tris ternary complex, illustrating the accommodation of both GlcNAc and tris in lysozyme’s active site. The binding surface of lysozyme is colored by charge (positive, blue; negative, red). Hydrogen bonds between GlcNAc (blue) and tris (green) are depicted as dotted red lines.

Figure 5

Interactions in the (A) lysozyme/GlcNAc complex, (B) lysozyme/tris complex, and (C,D) lysozyme/GlcNAc/tris complex highlighting hydrogen bonds with (C) GlcNAc and (D) tris. Lysozyme residues hydrogen-bonded to a ligand are shown in green. The presence of tris does not affect the hydrogen-bonding between GlcNAc and lysozyme. Two hydrogen bonds between tris and GlcNAc compensate for the loss of hydrogen bonds to Asn59 and Gln57 in the ternary complex and suggest a mechanism for cooperative lysozyme binding by tris and GlcNAc.

Scheme 3

Model for cooperative GlcNAc and imidazole binding. (A) Lysozyme monomer (active), (B) Lysozyme dimer (inactive), (C) Lysozyme monomer + imidazole (competitively inhibited), (D) Lysozyme monomer + GlcNAc (competitively inhibited), (E) Lysozyme monomer + 2 imidazole, (F) Lysozyme monomer + GlcNAc + imidazole. GlcNAc and the imidazole at the dimer interface are shown where they dock to lysozyme. GlcNAc and imidazole structures are highlighted by purple hexagons and yellow pentagons, respectively, for clarity.