Cardoso, M. V. C. | bioRxiv Science

bioRxiv2023

Uncovering the association mechanism between two intrinsically flexible proteins

The understanding of protein-protein interaction mechanisms is key to the atomistic description of cell signalling pathways and for the development of new drugs. In this context, the mechanism of intrinsically disordered proteins folding upon binding has attracted attention. The VirB9 C-terminal domain (VirB9Ct) and the VirB7 N-terminal motif (VirB7Nt) associate with VirB10 to form the outer membrane core complex of the Type IV Secretion System injectisome. Despite forming a stable and rigid complex, VirB7Nt behaves as a random coil while VirB9Ct is intrinsically dynamic in the free state. Here we combined NMR, stopped-flow fluorescence and computer simulations using structure-based models to characterize the VirB9Ct-VirB7Nt coupled folding and binding mechanism. Our data indicated that VirB9Ct binds to VirB7Nt by way of a conformational selection mechanism. However, at higher temperatures energy barriers between different VirB9Ct conformations are more easily surpassed. Under these conditions the formation of non-native initial encounter complexes may not be neglected, providing alternative pathways towards the native complex conformation. These observations highlight the intimate relationship between folding and binding, calling attention to the fact that the two molecular partners must search for the most favored intramolecular and intermolecular interactions on a rugged and funnelled conformational energy landscape, along which multiple intermediates may lead to the final native state.

biophysics↗

bioRxiv2019

CALX-CBD1 Ca2+-binding cooperativity studied by NMR spectroscopy and ITC with Bayesian statistics

The Na+/Ca2+ exchanger of Drosophila melanogaster, CALX, is the main Ca2+-extrusion mechanism in olfactory sensory neurons and photoreceptor cells. Na+/Ca2+ exchangers have two Ca2+ sensor domains, CBD1 and CBD2. In contrast to the mammalian homologues, CALX is inhibited by Ca2+-binding to CALX-CBD1, while CALX-CBD2 does not bind Ca2+ at physiological concentrations. CALX-CBD1 consists of a {beta}-sandwich and displays four Ca2+ binding sites at the tip of the domain. In this study, we used NMR spectroscopy and isothermal titration calorimetry (ITC) to investigate the cooperativity of Ca2+-binding to CALX-CBD1. We observed that this domain binds Ca2+ in the slow exchange regime at the NMR chemical shift time scale. Ca2+-binding restricts the dynamics in the Ca2+-binding region. Experiments of 15N CEST and 15N R2 dispersion allowed the determination of Ca2+ dissociation rates ({approx} 20 s-1). NMR titration curves of residues in the Ca2+-binding region were sigmoidal due to the contribution of chemical exchange to transverse magnetization relaxation rates, R2. Hence, a novel approach to analyze NMR titration curves was proposed. Ca2+-binding cooperativity was examined assuming two different stoichiometric binding models and using a Bayesian approach for data analysis. Fittings of NMR and ITC binding curves to the Hill model yielded nHill = 2.9 - 3.1, near maximum cooperativity (nHill = 4). By assuming a stepwise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca2+ is at least three orders of magnitude higher than that of binding a single Ca2+. Hence, four Ca2+ ions bind almost simultaneously to CALX-CBD1. Cooperative Ca2+-binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca2+-concentration in sensory neuronal cells.\n\nSIGNIFICANCECALX-CBD1 is the Ca2+-sensor domain of the Na+/Ca2+ exchanger of Drosophila melanogaster. It consists of a {beta}-sandwich, and contains four Ca2+ binding sites at the distal loops. In this study, we examined the cooperative binding of four Ca2+ ions to CALX-CBD1 using NMR spectroscopy and isothermal titration calorimetry (ITC) experiments. NMR and ITC data were analyzed using the framework of the binding polynomial formalism and Bayesian statistics. A novel approach to analyze NMR titration data in the slow exchange regime was proposed. These results support the view that CALX-CBD1 binds four Ca2+ with high cooperativity. The significant ligand binding cooperativity exhibited by this domain is determinant for the efficient allosteric regulation of this exchanger by intracellular Ca2+.

biophysics↗