Characterisation of the effects of mutation of the caldesmon sequence 691glu-trp-leu-thr-lys-thr696 to pro-gly-his-tyr-asn-asn on caldesmon-calmodulin interaction

AbstractWe have investigated the functional properties of a mutant (Cg1) derived from the C-terminal 99 amino acids of chicken caldesmon, 658–756 (658C) where the sequence 691glu-trp-leu-thr-lys-thr696 is changed to pro-gly-his-tyr-asn-asn. Cg1 bound Ca2+-calmodulin with (1/7)th of the affinity as compared to 658C or whole caldesmon. NMR titrations indicate that the contacts of Ca2+-calmodulin with the Trp-722 region of the peptide are retained but that those at the mutated site are lost. Most importantly Ca2+-calmodulin is not able to reverse the Cg1-induced inhibition. We conclude that the interaction of calmodulin with this caldesmon sequence is crucial for the reversal of caldesmon inhibition of actin-tropomyosin activation of myosin ATPase. The results are interpreted in terms of multi-site attachment of actin and Ca2+-calmodulin to overlapping sequences in caldesmon domain 4b

The crystal structure of Pneumolysin at 2.0 Å resolution reveals the molecular packing of the pre-pore complex

Pneumolysin is a cholesterol-dependent cytolysin (CDC) and virulence factor of Streptococcus pneumoniae. It kills cells by forming pores assembled from oligomeric rings in cholesterol-containing membranes. Cryo-EM has revealed the structures of the membrane-surface bound pre-pore and inserted-pore oligomers, however the molecular contacts that mediate these oligomers are unknown because high-resolution information is not available. Here we have determined the crystal structure of full-length pneumolysin at 1.98 Å resolution. In the structure, crystal contacts demonstrate the likely interactions that enable polymerisation on the cell membrane and the molecular packing of the pre-pore complex. The hemolytic activity is abrogated in mutants that disrupt these intermolecular contacts, highlighting their importance during pore formation. An additional crystal structure of the membrane-binding domain alone suggests that changes in the conformation of a tryptophan rich-loop at the base of the toxin promote monomer-monomer interactions upon membrane binding by creating new contacts. Notably, residues at the interface are conserved in other members of the CDC family, suggesting a common mechanism for pore and pre-pore assembly

A kinetic insight into troponin T mutations related to dilated and hypertrophic cardiomyopathies

Dilated and Hypertrophic Cadiomyopathy can be caused by mutations of genes encoding sarcomeric proteins. Mutations in cTnT are of particular interest since they are generally associated with mild or no ventricular hypertrophy but a high incidence of sudden death. Previous investigations have focused on steady state parameters such as maximal activation and inhibition of actomyosin ATPase and force and Ca2+ sensitivity. We have aimed to use transient kinetics to investigate the effects of 7 cTnT mutations on the dynamics of thin filament switching. We have studied two DCM mutations (R141W, ∆K210) and five HCM (∆E160, S179F, K273E, ∆14, ∆28+7) mutations present in two functional domains of TnT (T1 and T2). Overall circular dichroism studies showed that the structure of these mutant proteins is not grossly affected although minor changes in the α–helical content were found for cTnT mutants K273E, ∆14, ∆28+7 and ∆E160. Co-sedimentation with actin suggested that most of cTnT mutations do not interfere with the association between cTn and thin filament except for the truncated mutations. Cooperativity along thin filament was changed for all deletion mutations (∆K210, ∆E160, ∆14 and ∆28+7) but unchanged by the point mutations. In this study we also demonstrated that the equilibrium constant between the blocked and closed states (KB) for DCM mutations were unchanged but increased dramatically for HCM mutations suggesting loss of blocked state specifically for those in the T2 region. We assessed Ca2+ binding of the regulatory site of cardiac TnC using IAANS attached to C35 and C84 of cTnC. Ca2+ binding affinity (pCa50 =6.65) of reconstituted Tn complex was unaffected by all mutations with the exception of ∆28+7 which caused a decrease (pCa50 0.34). In contrast when incorporated into thin filament, all HCM mutations and DCM ∆K210 showed increased Ca2+ affinity. The observed rate constant of Ca2+ dissociation was unchanged for all mutations except for ∆28+7. In conclusion, we have observed multiple structural and functional consequences from different TnT mutations that occur in different regions of the molecule. Overall the data suggests that it is the functional changes caused by mutations that are critical in developing the disease and not the specific location of the mutation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays

Abstract The large unmet demand for new heart failure therapeutics is widely acknowledged. Over the last decades the contractile myofilaments themselves have emerged as an attractive target for the development of new therapeutics for both systolic and diastolic heart failure. However, the clinical use of myofilament-directed drugs has been limited, and further progress has been hampered by incomplete understanding of myofilament function on the molecular level and screening technologies for small molecules that accurately reproduce this function in vitro. In this study we have designed, validated and characterized new high throughput screening platforms for small molecule effectors targeting the interactions between the troponin C and troponin I subunits of the cardiac troponin complex. Fluorescence polarization-based assays were used to screen commercially available compound libraries, and hits were validated using secondary screens and orthogonal assays. Hit compound-troponin interactions were characterized using isothermal titration calorimetry and NMR spectroscopy. We identified NS5806 as novel calcium sensitizer that stabilizes active troponin. In good agreement, NS5806 greatly increased the calcium sensitivity and maximal isometric force of demembranated human donor myocardium. Our results suggest that sarcomeric protein-directed screening platforms are suitable for the development of compounds that modulate cardiac myofilament function

The Calponin Regulatory Region Is Intrinsically Unstructured: Novel Insight into Actin-Calponin and Calmodulin-Calponin Interfaces Using NMR Spectroscopy

Calponin is an actin- and calmodulin-binding protein believed to regulate the function of actin. Low-resolution studies based on proteolysis established that the recombinant calponin fragment 131–228 contained actin and calmodulin recognition sites but failed to precisely identify the actin-binding determinants. In this study, we used NMR spectroscopy to investigate the structure of this functionally important region of calponin and map its interaction with actin and calmodulin at amino-acid resolution. Our data indicates that the free calponin peptide is largely unstructured in solution, although four short amino-acid stretches corresponding to residues 140–146, 159–165, 189–195, and 199–205 display the propensity to form α-helices. The presence of four sequential transient helices probably provides the conformational malleability needed for the promiscuous nature of this region of calponin. We identified all amino acids involved in actin binding and demonstrated for the first time, to our knowledge, that the N-terminal flanking region of Lys137-Tyr144 is an integral part of the actin-binding site. We have also delineated the second actin-binding site to amino acids Thr180-Asp190. Ca2+-calmodulin binding extends beyond the previously identified minimal sequence of 153–163 and includes most amino acids within the stretch 143–165. In addition, we found that calmodulin induces chemical shift perturbations of amino acids 188–190 demonstrating for the first time, to our knowledge, an effect of Ca2+-calmodulin on this region. The spatial relationship of the actin and calmodulin contacts as well as the transient α-helical structures within the regulatory region of calponin provides a structural framework for understanding the Ca2+-dependent regulation of the actin-calponin interaction by calmodulin