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Instruments / Technical Area	Publication Details	Published	Actions
I04-Macromolecular Crystallography	Calmodulin variant E140G associated with long QT syndrome impairs CaMKIIδ autophosphorylation and L-type calcium channel (Cav1.2) inactivation Ohm Prakash , Nitika Gupta , Amy Milburn , Liam Mccormick , Vishvangi Deugi , Pauline Fisch , Jacob Wyles , N. Lowri Thomas , Svetlana Antonyuk , Caroline Dart , Nordine Helassa Journal Of Biological Chemistry 120 DOI: 10.1016/j.jbc.2022.102777 Diamond Proposal Number(s): [21970] Open Access Show Abstract ▼ Abstract: Long QT syndrome (LQTS) is a human inherited heart condition that can cause life-threatening arrhythmia including sudden cardiac death. Mutations in the ubiquitous Ca2+-sensing protein calmodulin (CaM) are associated with LQTS, but the molecular mechanism by which these mutations lead to irregular heartbeats is not fully understood. Here, we use a multidisciplinary approach including protein biophysics, structural biology, confocal imaging and patch-clamp electrophysiology to determine the effect of the disease-associated CaM mutation E140G on CaM structure and function. We present novel data showing that mutant-regulated CaMKIIδ kinase activity is impaired with a significant reduction in enzyme autophosphorylation rate. We report the first high-resolution crystal structure of a LQTS-associated CaM variant in complex with the CaMKIIδ peptide, which shows significant structural differences, compared to the wild-type complex. Furthermore, we demonstrate that the E140G mutation significantly disrupted Cav1.2 Ca2+/CaM-dependent inactivation, while cardiac ryanodine receptor (RyR2) activity remained unaffected. In addition, we show that the LQTS-associated mutation alters CaM’s Ca2+ binding characteristics, secondary structure content and interaction with key partners involved in excitation-contraction coupling (CaMKIIδ, Cav1.2, RyR2). In conclusion, LQTS-associated CaM mutation E140G severely impacts the structure-function relationship of CaM and its regulation of CaMKIIδ and Cav1.2. This provides a crucial insight into the molecular factors contributing to CaM-mediated arrhythmias with a central role for CaMKIIδ.	Dec 2022
I03-Macromolecular Crystallography	CPVT-associated calmodulin variants N53I and A102V dysregulate calcium signalling via different mechanisms Ohm Prakash , Marie Held , Laura F. Mccormick , Nitika Gupta , Lu-Yun Lian , Svetlana Antonyuk , Lee P. Haynes , N. Lowri Thomas , Nordine Helassa Journal Of Cell Science DOI: 10.1242/jcs.258796 Diamond Proposal Number(s): [15991] Open Access Show Abstract ▼ Abstract: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited condition that can cause fatal cardiac arrhythmia. Human mutations in the Ca2+ sensor calmodulin (CaM) have been associated with CPVT susceptibility, suggesting that CaM dysfunction is a key driver of the disease. However, the detailed molecular mechanism remains unclear. Focusing on the interaction with the cardiac ryanodine receptor (RyR2), we determined the effect of CPVT-associated variants N53I and A102V on the structural characteristics of CaM and on Ca2+ fluxes in live cells. We provide novel data showing that binding of both Ca2+/CaM-N53I and Ca2+/CaM-A102V to RyR23583-3603 is decreased. Ca2+/CaM:RyR23583-3603 high-resolution crystal structures highlight subtle conformational changes for the N53I variant, with A102V being similar to wild-type. We show that co-expression of CaM-N53I or CaM-A102V with RyR2 in HEK293 cells significantly increased the duration of Ca2+ events, CaM-A102V exhibited a lower frequency of Ca2+ oscillations. In addition, we show that CaMKIIδ phosphorylation activity is increased for A102V, compared to CaM-WT. This paper provides novel insight into the molecular mechanisms of CPVT-associated CaM variants and will facilitate development of strategies for future therapies.	Dec 2021

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