Hetero-Multivalency of Pseudomonas aeruginosa Lectin LecA Binding to Model Membranes

Abstract

A single glycan-lectin interaction is often weak and semi-specific. Multiple binding domains in a single lectin can bind with multiple glycan molecules simultaneously, making it difficult for the classic "lock-and-key" model to explain these interactions. We demonstrated that hetero-multivalency, a homo-oligomeric protein simultaneously binding to at least two types of ligands, influences LecA (a Pseudomonas aeruginosa adhesin)-glycolipid recognition. We also observed enhanced binding between P. aeruginosa and mixed glycolipid liposomes. Interestingly, strong ligands could activate weaker binding ligands leading to higher LecA binding capacity. This hetero-multivalency is probably mediated via a simple mechanism, Reduction of Dimensionality (RD). To understand the influence of RD, we also modeled LecA's two-step binding process with membranes using a kinetic Monte Carlo simulation. The simulation identified the frequency of low-affinity ligand encounters with bound LecA and the bound LecA's retention of the low-affinity ligand as essential parameters for triggering hetero-multivalent binding, agreeing with experimental observations. The hetero-multivalency can alter lectin binding properties, including avidities, capacities, and kinetics, and therefore, it likely occurs in various multivalent binding systems. Using hetero-multivalency concept, we also offered a new strategy to design high-affinity drug carriers for targeted drug delivery.

Figure 1

Schematic for the Reduction of Dimensionality (RD) model. (a) A schematic representation of RD influencing LecA interactions with the cellular membrane. LecA first diffuses from solution to a membrane surface and attaches to the high-affinity ligand, Gb3. Then, free membrane ligands move two dimensionally, enabling subsequent binding. The reduced dimensionality of diffusion enhances the effective concentrations of membrane ligands; thus, a weak ligand, such as LacCer, can contribute to LecA binding. (b) Graphical representation of LecA complexed with galactose as observed in the crystal structure (PDB code 1OKO). Four binding sites are indicated by arrows. Protein and carbohydrate are displayed in a cartoon representation with coloring done by subunit using JSmol. (c) Cartoon representations of glycolipids used on this study.

Figure 2

Saturation binding curves of LecA binding to common galactose terminated glycolipids and Gb3/LacCer mixtures that show positive cooperativity. The saturation binding curves’ dash lines represent the curve fits to Hill’s equation, fitted parameters are listed in SI Table 4. Data points are reported as mean ± S.D (n = 8). To better show the data points at low concentrations, the same binding curves on a semi-log scale are shown in the supplementary information. (a and b) Saturation binding curves of LecA binding to bilayers of common galactose terminated glycolipids. (c) Saturation binding curves of LecA binding to bilayers containing Gb3/LacCer mixtures. (d) ϕ values for 1 mol% of Gb3 mixed with different densities of LacCer. Dash line representing the fit of ϕ to the sigmoidal function is a guide to the eye.

Figure 3

Modeling LecA binding kinetics using kMC simulation. LecA binding to a membrane surface containing 1 mol% of high-affinity ligands and various low-affinity ligand densities, (a) 0 mol%, (b) 0.5 mol%, (c) 3 mol%, and (d) 9 mol%. The affinity of the low-affinity ligand is 300-fold lower than the high-affinity ligand. (${\mathit{K}}_{\mathit{d},\mathit{low}}=300{\mathit{K}}_{\mathit{d},\mathit{high}}$ where ${\mathit{K}}_{\mathit{d}}={\mathit{k}}_{-1}/{\mathit{k}}_1$) Each curve represents the number of bound LecA in different binding configurations. The dashed line shows the maximum number of bound LecA at 2000 s without the high-affinity ligand at the same membrane density of low-affinity ligand. All data represented as average ± S.D from 50 kMC simulations. (e) A binding mechanism observed in the kMC simulation when the low-affinity ligand density is higher than the high-affinity ligand. (1) A LecA molecule moves from the solution phase to the membrane surface, and attaches to a high-affinity ligand. Then, a low-affinity ligand encounters the bound LecA completing the hetero-multivalent binding. (2) The high-affinity ligand dissociates from the bound LecA. (3) LecA binding to one low-affinity ligand is relatively unstable. At sufficient density, a low-affinity ligand can reach the free binding site before the LecA dissociates from the surface. (4) LecA binding to two low-affinity ligands is relatively stable. (5) The high-affinity ligand can facilitate the binding between LecA and low-affinity ligands by continuing the same process. (The figure shows only two binding sites that are participating in reactions happening on the surface. The other two binding sites facing in the opposite direction are not shown).

Figure 4

Colloid aggregation kinetics. (a) A schematic drawing of silica particle aggregation induced by LecA-glycolipid binding. (b) A snapshot of particle aggregation mediated by LecA tethering. (c) A snapshot of particle dispersion without LecA. (d) Particle aggregation at different conditions. The decay rate of the singlet ratio (θ) is associated with the binding avidity between LecA and membrane ligands.

Figure 5

Cooperativity between strong, moderate, and weak ligands. (a) Binding curves of LecA to the mixture of Gb3/GalßCer. (b) 1 mol% Gb3 mixed with GalNAc terminated glycolipids at 3 μM LecA. (c) Binding curves of LecA to the mixture of the strong (Gb3)/moderate (GM1 or AGM1) ligands. (d) Binding curves of LecA to the mixture of the moderate (GM1 or AGM1) and LacCer. (e) Binding curves of LecA to the mixture of the moderate (GM1 or AGM1) and GalβCer. All data points are reported as mean ± S.D (n = 8). The dashed lines represent Hill equation fits to the data.

Figure 6

Liposome binding to P. aeruginosa. Retention of fluorescent liposomes on P. aeruginosa ((a) PAO1 and (b) Xen41) was quantified by normalized fluorescence intensity per colony forming unit (CFU). The liposome concentration given is mass concentration. Control (yellow) is 99.5 mol% POPC/0.5 mol% TR-DHPE. LacCer (green) is 10 mol% LacCer/89.5 mol% POPC/0.5 mol% TR-DHPE. Gb3 (orange) is 10 mol% Gb3/89.5 mol% POPC/0.5 mol% TR-DHPE. Gb3/LacCer (blue) is 5 mol% LacCer/5 mol% Gb3/89.5 mol% POPC/0.5 mol% TR-DHPE. The error bars are standard deviation (n = 3). The stars indicate t-test unequal variance p-values of p < 0.1 (*), p < 0.05 (**), and p < 0.01 (***). (SI Table 5).