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

Molecular basis of sugar recognition by collectin-K1 and the effects of mutations associated with 3MC syndrome

Background Collectin-K1 (CL-K1, or CL-11) is a multifunctional Ca2+-dependent lectin with roles in innate immunity, apoptosis and embryogenesis. It binds to carbohydrates on pathogens to activate the lectin pathway of complement and together with its associated serine protease MASP-3 serves as a guidance cue for neural crest development. High serum levels are associated with disseminated intravascular coagulation, where spontaneous clotting can lead to multiple organ failure. Autosomal mutations in the CL-K1 or MASP-3 genes cause a developmental disorder called 3MC (Carnevale, Mingarelli, Malpuech and Michels) syndrome, characterised by facial, genital, renal and limb abnormalities. One of these mutations (Gly204Ser in the CL-K1 gene) is associated with undetectable levels of protein in the serum of affected individuals. Results In this study, we show that CL-K1 primarily targets a subset of high-mannose oligosaccharides present on both self- and non-self structures, and provide the structural basis for its ligand specificity. We also demonstrate that three disease-associated mutations prevent secretion of CL-K1 from mammalian cells, accounting for the protein deficiency observed in patients. Interestingly, none of the mutations prevent folding nor oligomerization of recombinant fragments containing the mutations in vitro. Instead, they prevent Ca2+ binding by the carbohydrate-recognition domains of CL-K1. We propose that failure to bind Ca2+ during biosynthesis leads to structural defects that prevent secretion of CL-K1, thus providing a molecular explanation of the genetic disorder. Conclusions We have established the sugar specificity of CL-K1 and demonstrated that it targets high-mannose oligosaccharides on self- and non-self structures via an extended binding site which recognises the terminal two mannose residues of the carbohydrate ligand. We have also shown that mutations associated with a rare developmental disorder called 3MC syndrome prevent the secretion of CL-K1, probably as a result of structural defects caused by disruption of Ca2+ binding during biosynthesis

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

Effect of hypertrophic and dilated cardiomyopathies associated mutations in troponin I on cardiac thin filament dynamics

Troponin I mutations have been linked to genetic hypertrophic and dilated cardiomyopathies. We aimed to understand, at the molecular level, how six HCM mutations (R21C, Q130R, R145G, G203S, and K206Q) and one DCM mutation (A2V) in troponin I affect its structure and function. Circular dichroism, co-sedimentation with actin and ATPase assays demonstrated that these mutations had little or no effect on the folding or the thermal stability of the troponin complex. Isothermal calorimetry, fluorescence spectroscopy, and transient kinetics were used to assess the effect of these mutations on the function of troponin I. We found that: 1) all TnI mutations increased the affinity of the troponin complex for actin in the presence of Ca²+ and increased the Ca²+ affinity of troponin within thin filaments. This suggests an uncoupling between Ca²+ binding and actin binding. 2) The size of the cooperative unit n was not affected by troponin I mutations. 3) A2V, R21C, Q130R, A157V, G203S, and K206Q mutations did not affect the proportion of thin filaments in the blocked state (at low Ca²+). In contrast R145G mutation dramatically reduced the amount of thin filaments switched to the blocked state. This effect was also observed using electron microscopy and helical reconstruction. 4) A2V, R21C, Q130R, R145G, G203S, and K206Q did not affect the observed rate constant of Ca²+ dissociation from troponin and thin filaments. In contrast troponin I A157V showed a decrease in the Ca²+ dissociation rate constant. 5) Finally, we found that calcium alone is sufficient to fully activate the cardiac thin filament while skeletal muscle thin filaments complete activation required both Ca²+ and myosin heads. Overall these results provide insight into the mechanism by which troponin I mutations affect contractility in hypertrophic and dilated cardiomyopathy. These findings could have important clinical consequences.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