(667a) Investigation of Hetero-Multivalent Binding Dynamics between Cholera Toxin and Glycolipids on Membranes Via Kinetic Monte Carlo Model and Nanocube-Based Measurements

Cholera toxin (CTx) is an AB5 toxin that consists of a virulent A-subunit and pentameric cholera toxin B-subunits (CTB) [1]. After secreted by Vibrio cholerae bacteria, CTx toxins will lead to main symptoms of cholera infection such as severe dehydration and electrolyte imbalance, which can threaten a patient’s life if untreated [2]. In order for CTx toxins to initiate their virulent actions, CTB proteins need to first bind with their receptors expressed on a host cell membrane and enters the cytoplasm of the host cell. It is important to elucidate the binding mechanisms, in order to understand cholera pathogenesis and develop new therapeutics [3]. In the literature, it has been believed that GM1, a type of glycolipids, is the primary receptor for CTB due to its high binding affinity [1,3]. However, this line of thought has been questioned by a few studies [4-6]. First, the GM1 surface density is extremely low in human intestinal epithelial cells (less than 0.003 mol% of glycosphingolipids) [6]. Second, the presence of other receptors, such as GM2, significantly increases the CTB binding while GM2 alone cannot induce noticeable CTB binding [4-5]. These studies have casted doubts on the physiological role of GM1 in CTB binding processes.

Previously, we examined the multivalent process of a tetravalent lectin, LecA, which is secreted from Pseudomonas aeruginosa bacteria [7-9]. From these studies, we have concluded that glycolipid receptors with lower binding affinities can make significant contributions to the overall LecA binding as long as the sufficient number of glycolipid receptors is present on a heterogeneous cell membrane [7-9]. Such phenomena are mainly mediated by a mechanism called “reduction in dimensionality” (RD). [10] The reduced dimension of receptor diffusion on a cell membrane surface activates LecA binding to weaker receptors, leading to the increase of protein attachment [7].

Motivated by the LecA studies, this work examines how the RD mechanism influences the CTB-glycolipid binding system on a heterogeneous cell membrane containing both GM1 and GM2 receptors. LecA has only two binding sites that can interact with receptors on the same cell membrane. In contrast, all five binding pockets of a pentavalent CTB can simultaneously bind to multiple receptors on a cell membrane. The increase of valency can raise the complexity of this nonlinear binding phenomenon. To address this issue, we have developed an on-lattice kinetic Monte Carlo simulation framework by incorporating detailed step-wise binding mechanisms between CTB proteins and a cell membrane with GM1 and GM2 glycolipids [8]. Here, the kMC framework is chosen as it is a natural tool to simulate stochasticity that exists in the underlying dynamics due to a low number of molecules involved in the process [8]. Also, the on-lattice kMC model also takes into account spatial effects of the heterogeneity glycolipid distribution on the overall binding dynamics [8]. As a result, the kMC modeling framework can simulate hetero-multivalent binding process and the temporal evolution of binding configurations in detail. Since kinetic parameters for the developed kMC model have not been quantitatively calibrated before, we use a nanocube-based biosensor, which can monitor the binding dynamics in a high-throughput manner, to observe the CTB-GM1-GM2 binding dynamics [4,7,11]. The obtained measurements are used for tuning the parameter values so that the resultant kMC model prediction is accurate. Lastly, the calibrated kMC model is used to perform a series of perturbation studies to examine how a physical condition such as densities of glycolipid receptors will influence the overall binding dynamics. Specifically, we varied binding affinities and densities of glycolipid receptors as well as their diffusion rates on the membrane to examine their importance in the binding dynamics and outcomes. Through these modeling and experimentation, we found the RD mechanisms play an important role in the CTB binding system as the LecA system, and the importance of various physical conditions with respect to the RD mechanism in the CTB systems was assessed.

References

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