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Kinetic Regulation of Multi-Ligand Binding Proteins Salakhieva, D., Sadreev, I., Chen, M., Umezawa, Y., Evstifeev, A., Welsh, G., & Kotov, N. (2016). Kinetic regulation of multi-ligand binding proteins. BMC Systems Biology, 10(1), [32]. https://doi.org/10.1186/s12918-016-0277-0 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1186/s12918-016-0277-0 Link to publication record in Explore Bristol Research PDF-document University of Bristol - Explore Bristol Research General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/ebr-terms/ Salakhieva et al. BMC Systems Biology (2016) 10:32 DOI 10.1186/s12918-016-0277-0 RESEARCH ARTICLE Open Access Kinetic regulation of multi-ligand binding proteins Diana V. Salakhieva1†, Ildar I. Sadreev2†, Michael Z. Q. Chen3*, Yoshinori Umezawa4, Aleksandr I. Evstifeev5, Gavin I. Welsh6 and Nikolay V. Kotov5 Abstract Background: Second messengers, such as calcium, regulate the activity of multisite binding proteins in a concentration-dependent manner. For example, calcium binding has been shown to induce conformational transitions in the calcium-dependent protein calmodulin, under steady state conditions. However, intracellular concentrations of these second messengers are often subject to rapid change. The mechanisms underlying dynamic ligand-dependent regulation of multisite proteins require further elucidation. Results: In this study, a computational analysis of multisite protein kinetics in response to rapid changes in ligand concentrations is presented. Two major physiological scenarios are investigated: i) Ligand concentration is abundant and the ligand-multisite protein binding does not affect free ligand concentration, ii) Ligand concentration is of the same order of magnitude as the interacting multisite protein concentration and does not change. Therefore, buffering effects significantly influence the amounts of free ligands. For each of these scenarios the influence of the number of binding sites, the temporal effects on intermediate apo- and fully saturated conformations and the multisite regulatory effects on target proteins are investigated. Conclusions: The developed models allow for a novel and accurate interpretation of concentration and pressure jump-dependent kinetic experiments. The presented model makes predictions for the temporal distribution of multisite protein conformations in complex with variable numbers of ligands. Furthermore, it derives the characteristic time and the dynamics for the kinetic responses elicited by a ligand concentration change as a function of ligand concentration and the number of ligand binding sites. Effector proteins regulated by multisite ligand binding are shown to depend on ligand concentration in a highly nonlinear fashion. Keywords: Ligand-receptor binding, Transient kinetics, Calcium, Calmodulin Background ubiquitous protein, calmodulin as well as troponin A wide variety of intracellular events are initiated via andotherEF-handcontainingproteins[4,5].Tem- temporal change of ligand concentrations. One of the poral elevation of intracellular free Ca2+ is the key most important ligands in many cells is calcium (Ca2+). regulatory factor of the Ca2+-dependent protein activ- Calcium interacts with and regulates the activities of a ity [6–10]. The characteristics of the induced signal large number of calcium-binding proteins as well as are not fully understood. It remains to be determined numerous effectors. The number of functional calcium how a single ligand is able to govern numerous intra- binding sites within these proteins can range from one cellular properties. or two to ten or more [1–3]. The most common number Whilst the multisite ligand binding is not limited to of calcium binding sites is four: as observed in the most Ca2+ signaling, Ca2+ is probably the most versatile ion, regulating the largest number of cellular events. Several 2+ * Correspondence: [email protected] Ca -binding proteins can be considered as examples of Diana V. Salakhieva and Ildar I. Sadreev are first authors. multisite ligand protein interactions. Structural biology † Equal contributors investigations of calcium binding proteins in complexes 3Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong, China with target protein peptides have suggested that the 2+ Full list of author information is available at the end of the article specificity in Ca -CaM binding protein-dependent © 2016 Salakhieva et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Salakhieva et al. BMC Systems Biology (2016) 10:32 Page 2 of 19 target activation arises from the diversity of interaction Mathematical modelling of multisite protein kinetics in interfaces between the Ca2+-regulated protein and its response to rapid ligand changes presented in this paper target proteins [5, 11–21]. The most ubiquitous protein, provide new insights into the mechanism of conform- calmodulin (CaM), consists of two globular domains, ational kinetics of multisite proteins in complex with each domain containing a pair of helix-loop-helix Ca2 variable number of bound ligands for the two distinct +-binding motifs called EF-hands [1, 3, 5, 16, 17]. In earl- physiological situations described: ier studies the authors demonstrated that in addition to the diversity of CaM-target interfaces; the CaM selectiv- i) When the ligand concentration significantly exceeds ity emerges from its target specific Ca2+-affinity; the protein concentration, number of Ca2+ ions bound and the target specific coop- ii) When the total amount of ligand is conserved and erativity [22–24]. comparable with the protein concentration. Another major factor that contributes to the selectiv- ity of seemingly simultaneous regulation of several In the first case, the buffering effects are negligible multisite Ca2+ binding proteins and Ca2+-mediated whereas in the latter, the ligand-protein interactions processesisthetemporalalterationsofCa2+ [25–27]. have a significant impact on the amount of available The remarkable variety of Ca2+ signals in cells, ranging ligand and the binding kinetics. In this work, the equa- from infrequent spikes to sustained oscillations and tions for the dependence of the characteristic time plateaus, requires an understanding of how fast intracellu- constants and the temporal distribution of individual lar calcium changes regulate the kinetics of multiple conformations as a function of the ligand concentra- multisite Ca2+ binding proteins. Therefore mathematical tion, the number of binding sites and the binding modeling of Ca2+ jump induced responses could prove to affinities have been derived. The impact of the number be invaluable in the interpretation of transient kinetic of binding sites, temporal effects on conformations, experiments. and regulation by multisite proteins of their effector Cooperative binding is a special case of molecular in- proteins have been investigated by employing the devel- teractions where ligand binding to one site of a mol- oped models. The analysis of the ligand-multisite pro- eculedependsontheligandbindingtotheothersites. tein mediated regulation of effector proteins suggests The first quantitative determination of the dynamic that significant degree of selectivity in regulation can be properties of cooperative binding was proposed by [28]. achieved by a single ligand by employing mechanisms In this work the authors emphasized the significance of described in this study. the cooperativity by studying the fast dynamics of Ca2+ binding to calretenin (CR), which has one independent and four cooperative binding sites. The investigation of Results cooperative effects of Ca2+ binding to CR was per- A new model for multisite protein ligand binding kinetics formed both experimentally and using mathematical The majority of studies of the activation of multisite pro- modeling. The authors employed the simplified version teins consider only the ligand concentration-dependent of the Adair-Klotz model [29, 30] to describe the dy- profiles. One of the interesting questions about these namics of the interactions involved in Ca2+ binding to multisite proteins is how temporal alterations of ligand CR. This approach was then extended to the binding of concentration contribute to their function. The shape of Ca2+ to CaM [31]. The models proposed in these stud- distribution of multisite proteins in complex with variable ies [28, 31] demonstrated excellent fitting results to the numbers of bound ligand is known or can be experimen- experimental data, in comparison with the previously tally elucidated in many cases [4, 5, 16, 22–24, 36–38]. published models. However, the described approach is However, the role of temporal transitions caused by fast rather limited, as it describes fitting instead of provid- alteration of ligand concentration
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