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

Mutations in repeating structural motifs of tropomyosin cause gain of function in skeletal muscle myopathy patients

The congenital myopathies include a wide spectrum of clinically, histologically and genetically variable neuromuscular disorders many of which are caused by mutations in genes for sarcomeric proteins. Some congenital myopathy patients have a hypercontractile phenotype. Recent functional studies demonstrated that ACTA1 K326N and TPM2 ΔK7 mutations were associated with hypercontractility that could be explained by increased myofibrillar Ca(2+) sensitivity. A recent structure of the complex of actin and tropomyosin in the relaxed state showed that both these mutations are located in the actin–tropomyosin interface. Tropomyosin is an elongated molecule with a 7-fold repeated motif of around 40 amino acids corresponding to the 7 actin monomers it interacts with. Actin binds to tropomyosin electrostatically at two points, through Asp25 and through a cluster of amino acids that includes Lys326, mutated in the gain-of-function mutation. Asp25 interacts with tropomyosin K6, next to K7 that was mutated in the other gain-of-function mutation. We identified four tropomyosin motifs interacting with Asp25 (K6-K7, K48-K49, R90-R91 and R167-K168) and three E-E/D-K/R motifs interacting with Lys326 (E139, E181 and E218), and we predicted that the known skeletal myopathy mutations ΔK7, ΔK49, R91G, ΔE139, K168E and E181K would cause a gain of function. Tests by an in vitro motility assay confirmed that these mutations increased Ca(2+) sensitivity, while mutations not in these motifs (R167H, R244G) decreased Ca(2+) sensitivity. The work reported here explains the molecular mechanism for 6 out of 49 known disease-causing mutations in the TPM2 and TPM3 genes, derived from structural data of the actin–tropomyosin interface

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

Insights into the Importance of Hydrogen Bonding in the γ-Phosphate Binding Pocket of Myosin: Structural and Functional Studies of Serine 236†,‡

The active site of myosin contains a group of highly conserved amino acid residues whose roles in nucleotide hydrolysis and energy transduction might appear to be obvious from the initial structural and kinetic analyses but become less clear on deeper investigation. One such residue is Ser236 (Dictyostelium discoideum myosin II numbering) which was proposed to be involved in a hydrogen transfer network during γ-phosphate hydrolysis of ATP, which would imply a critical function in ATP hydrolysis and motility. The S236A mutant protein shows a comparatively small decrease in hydrolytic activity and motility, and thus this residue does not appear to be essential. To understand better the contribution of Ser236 to the function of myosin, structural and kinetic studies have been performed on the S236A mutant protein. The structures of the D. discoideum motor domain (S1dC) S236A mutant protein in complex with magnesium pyrophosphate, MgAMPPNP, and MgADP·vanadate have been determined. In contrast to the previous structure of wild-type S1dC, the S236A·MgAMPPNP complex crystallized in the closed state. Furthermore, transient-state kinetics showed a 4-fold reduction of the nucleotide release step, suggesting that the mutation stabilizes a closed active site. The structures show that a water molecule approximately adopts the location of the missing hydroxyl of Ser236 in the magnesium pyrophosphate and MgAMPPNP structures. This study suggests that the S236A mutant myosin proceeds via a different structural mechanism than wild-type myosin, where the alternate mechanism is able to maintain near normal transient-state kinetic values

Evolution structurale jurassique de la partie nord du Moyen Atlas (Maroc)

CNRS TD Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc

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