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Conformational Changes in Ligand Binding Processes

dc.contributor.advisorGrubmüller, Helmut Prof. Dr.
dc.contributor.authorVoß, Béla
dc.titleConformational Changes in Ligand Binding Processesde
dc.contributor.refereeGrubmüller, Helmut Prof. Dr.
dc.description.abstractengLigand and protein-protein binding processes play a crucial role in many biological processes. In many cases ligand binding is associated with conformational changes within the receptor protein. While structure determination techniques resolve the end states of the binding process and kinetic measurements reveal the rates between them, the exact pathway between the two end states is hard to investigate with experimental apparatuses but in principle easier to access using atomistic computer simulations. In this work we focused on two questions related to the this pathway. First: What are the sub-steps in a binding process, what is their sequential order and what are the rates between them? Second: How does ligand binding lead to conformational changes and what are the functional consequences? To address the first question we investigated in the first part the binding of cAMP at the binding domain of the potassium channel MloK1. X-ray and NMR structures show a ligand-free open conformation and a ligand-bound closed conformation. Extending the simple two-state model to a four-state model with a ligand-free closed conformation and a ligand-bound open conformation provides the framework to distinguish between an induced fit model, where ligand binding occurs prior to the conformational change, and a conformational selection binding mechanism, where the conformational change occurs before the ligand binds. Using a combination of umbrella sampling simulations and large sets of unbiased simulations we determined a set of rates for transitions between the states of the 4-state model. The rates revealed that the ligand-free closed conformation is highly instable. Ligand binding at the open conformation however is observed in unbiased simulations as well as the conformational change from the ligand-bound open conformation to the ligand-bound closed conformation, which happens spontaneously on a timescale of tenth of microseconds. These findings showed that the binding is determined by an induced fit mechanism. To elucidate what determines the on-rate we postulated a binding funnel that increasingly confines the translational and rotational degrees of freedom. Quantifying the translational and rotational degrees of freedom in dependence of the ligand's RMSD to the bound state from a huge set of free binding simulations and combining it with the probability to actually reach the binding site from a certain distance results in a consistent on-rate estimate in the range of 15-35/(µs*mol/l) which is in good agreement with the experimentally determined on-rate. To address the second question, we investigated the allosteric binding of cargo proteins and RanGTP at the exportin CRM1. In the x-ray structure of the ternary complex CRM1 adopts a compact conformation, while the x-ray structure of free CRM1 reveals an extended conformation. On the basis of these structures, simulations of in silico mutants were carried out, revealing that the extended conformation of free CRM1 is stabilised by a C-terminal helix. For steric reasons, such a rearrangement is expected to be triggered by RanGTP binding as well. It was furthermore found that the change to the compact overall protein conformation leads to a shift in the cargo binding site to an open state compatible with cargo binding, thus increasing cargo affinity. These two findings are able to qualitatively explain the cooperative binding of cargo proteins and RanGTP at
dc.contributor.coRefereeEnderlein, Jörg Prof. Dr.
dc.subject.engLigand Bindingde
dc.subject.engMolecular Dynamics Simulationsde
dc.subject.engConformational Changesde
dc.subject.engComputer Simulationsde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

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