Discriminating between negative cooperativity and ligand binding to independent sites using pre-equilibrium properties of binding curves

Abstract

Negative cooperativity is a phenomenon in which the binding of a first ligand or substrate molecule decreases the rate of subsequent binding. This definition is not exclusive to ligand-receptor binding, it holds whenever two or more molecules undergo two successive binding events. Negative cooperativity turns the binding curve more graded and cannot be distinguished from two independent and different binding events based on equilibrium measurements only. The need of kinetic data for this purpose was already reported. Here, we study the binding response as a function of the amount of ligand, at different times, from very early times since ligand is added and until equilibrium is reached. Over those binding curves measured at different times, we compute the dynamic range: the fold change required in input to elicit a change from 10 to 90% of maximum output, finding that it evolves in time differently and controlled by different parameters in the two situations that are identical in equilibrium. Deciphering which is the microscopic model that leads to a given binding curve adds understanding on the molecular mechanisms at play, and thus, is a valuable tool. The methods developed in this article were tested both with simulated and experimental data, showing to be robust to noise and experimental constraints.

The archaeal glutamate transporter homologue GltPh shows heterogeneous substrate binding

“…Interestingly, a recent saturation transfer difference NMR study reported an unusually low Hill coefficient of 0.69 for aspartate binding to liposome-reconstituted Glt Ph ( Hall et al, 2020 ). A Hill coefficient below one may reflect negative cooperativity or result from multiple binding states with distinct affinities ( Cattoni et al, 2015 ; Sevlever et al, 2020 ; Wang and Pan, 1996 ). Distinguishing these possibilities requires a kinetic approach ( Cattoni et al, 2009 ).…”

Heterogeneous substrate binding in a glutamate transporter homologue

“…Interestingly, a recent saturation transfer difference NMR study reported an unusually low Hill coefficient of 0.69 for aspartate binding to liposome-reconstituted GltPh (Hall et al, 2020). A Hill coefficient below 1 may reflect negative cooperativity or result from multiple binding states with distinct affinities (Cattoni, Chara, Kaufman, & Gonzalez Flecha, 2015;Sevlever, Di Bella, & Ventura, 2020; Z. X. Wang & Pan, 1996).…”

The Use of the Statistical Entropy in Some New Approaches for the Description of Biosystems

“…Although low-molecular-weight ligands do not take part in the processes of protein folding [ 32 , 33 , 34 ] and DNA melting, the latter are substantially cooperative. Thus, cooperativity significantly affects both intracellular and intercellular regulation of life processes [ 25 , 35 , 36 , 37 , 38 ].…”

“…There is also a known problem that the results of calculating the curves in a model with included anticooperative interactions between ligands coincide with the results of calculations in the presence of heterogeneous binding, but not cooperative. To solve this problem, the researcher needs to set up binding kinetics experiments [ 38 ].…”