Supervisors info:
Καθηγητής Κωνσταντίνος Βοργιάς (επιβλέπων)
Τομέας Βιοχημείας και Μοριακής Βιολογίας,
Τμήμα Βιολογίας, Πανεπιστήμιο Αθηνών
Δρ. Γεώργιος Νούνεσης
Ερευνητής Α’ ΕΚΕΦΕ «Δημόκριτος»
Επίκουρη Καθηγήτρια Βασιλική Οικονομίδου
Τομέας Βιολογίας Κυττάρου και Βιοφυσικής,
Τμήμα Βιολογίας, Πανεπιστήμιο Αθηνών
Summary:
For complexes with biological activity, structural information is an important step towards a better understanding of both their functional behavior and mechanisms of regulation of their corresponding biological pathways. Nowdays, access to state-of-the-art bioinformatic tools, has greatly expanded the study of biomolecular interactions, even without any prior knowledge of their exact conformation. Today, through molecular simulations, realistic molecules and complexes of biochemical interest can be produced for a wide range of physicochemical conditions.
The scope of this master thesis is the structural study of the interaction of calmodulin (CaM) with specific regions of the type 2 ryanodine receptor [RyR2] (Peptide B and Peptide F), which according to the literature are the main binding sites of CaM in RyR2. The molecular mechanism of interaction and regulation of CaM/Ry2 is not fully understood, mainly due to the absence of structural information about RyR2. Data so far have shown that RyR2 acts as a molecular switch on the Ca2+ release channel of the Sarcoplasmic Reticulum (SR), having an important role in the excitation-contraction coupling mechanism of the cardiac muscle and hence the heartbeat.
In addition, recent research data identified 17 mutations in genes encoding CaM (CALM1, CALM2 and CALM3), mainly in young patients with cardiac dysfunctions including severe arrhythmias and heart failure. The molecular mechanism giving rise to such phenotypes remains unknown and therefore there is a need to clarify the effect of CaM mutants on Ca2+ homeostasis mechanisms of heart cells. To date, there are three propable scenarios for the effect of CaM mutants: 1) either a drastic change in CaM affinity for RyR2, 2) a drastic change for the Ca2+ ions binding affinity in CaM, or 3) a significant change in the thermodynamic stability of the CaM.
To fully explore all possible scenarios, docking simulations were performed in key areas of the CaM/RyR2 interaction interface and evaluation of the resulting complexes was made. Furthermore, de novo synthesis of RyR2 peptides B and F was performed with proper bioinformatic tools followed by analysis of their physicochemical characteristics. Finally, structural models of calmodulin mutations were constructed via specialized molecular dynamics programs, in order to study their effect on both structure and activity of the molecule.
All findings were compared to recent experimental results and existing theoretical frameworks on the conformation of the CaM/RyR2 complex to validate the conclusions of this study. As a result, a new structural model is poposed for the regulation of human RyR2 as well as associating specific biophysicall changes of CaM with pathological conditions.
Keywords:
Calmodulin (CaM), Ryanodine Receptor Type 2 (RyR2), Calcium ions (Ca2+), Arrhythmias, Docking, Mutagenesis
