The giant muscle protein titin (connectin) is essential in the temporal and spatial control of the assembly of the highly ordered sarcomeres (contractile units) of striated muscle. Here we present the crystal structure of titin's only catalytic domain, an autoregulated serine kinase (titin kinase). The structure shows how the active site is inhibited by a tyrosine of the kinase domain. We describe a dual mechanism of activation of titin kinase that consists of phosphorylation of this tyrosine and binding of calcium/calmodulin to the regulatory tail. The serine kinase domain of titin is the first known non-arginine-aspartate kinase to be activated by phosphorylation. The phosphorylated tyrosine is not located in the activation segment, as in other kinases, but in the P + 1 loop, indicating that this tyrosine is a binding partner of the titin kinase substrate. Titin kinase phosphorylates the muscle protein telethonin in early differentiating myocytes, indicating that this kinase may act in myofibrillogenesis.

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Protein ab initio models predicted from sequence data alone can enable the elucidation of crystal structures by molecular replacement. However, the calculation of such ab initio models is typically computationally expensive. Here, a computational pipeline based on the clustering and truncation of cheaply obtained ab initio models for the preparation of structure ensembles is described. Clustering is used to select models and to quantitatively predict their local accuracy, allowing rational truncation of predicted inaccurate regions. The resulting ensembles, with or without rapidly added side chains, solved 43% of all test cases, with an 80% success rate for all-α proteins. A program implementing this approach, AMPLE, is included in the CCP4 suite of programs. It only requires the input of a FASTA sequence file and a diffraction data file. It carries out the modelling using locally installed Rosetta, creates search ensembles and automatically performs molecular replacement and model rebuilding.

The development of resistance to insecticides has become a classic exemplar of evolution occurring within human time scales. In this study we demonstrate how resistance to DDT in the major African malaria vector Anopheles gambiae is a result of both target-site resistance mechanisms that have introgressed between incipient species (the M- and S-molecular forms) and allelic variants in a DDT-detoxifying enzyme. Sequencing of the detoxification enzyme, Gste2, from DDT resistant and susceptible strains of An. gambiae, revealed a non-synonymous polymorphism (I114T), proximal to the DDT binding domain, which segregated with strain phenotype. Recombinant protein expression and DDT metabolism analysis revealed that the proteins from the susceptible strain lost activity at higher DDT concentrations, characteristic of substrate inhibition. The effect of I114T on GSTE2 protein structure was explored through X-ray crystallography. The amino acid exchange in the DDT-resistant strain introduced a hydroxyl group nearby the hydrophobic DDT-binding region. The exchange does not result in structural alterations but is predicted to facilitate local dynamics and enzyme activity. Expression of both wild-type and 114T alleles the allele in Drosophila conferred an increase in DDT tolerance. The 114T mutation was significantly associated with DDT resistance in wild caught M-form populations and acts in concert with target-site mutations in the voltage gated sodium channel (Vgsc-1575Y and Vgsc-1014F) to confer extreme levels of DDT resistance in wild caught An. gambiae.

The substrate-binding clefts and catalytic machinery of pectin and pectate lyases have diverged significantly. Specificity is dictated by both the nature of the protein-carbohydrate interaction and long-range electrostatic forces. Three potential catalytic residues have been identified in pectin lyase, two of these are common to pectate lyases. Pectin lyase A does not bind Ca2+ but an arginine residue is found in an equivalent position to the Ca2+ ion in pectate lyase, suggesting a similar role in catalysis. The activity of pectin lyase A is pH -dependent with an optimum activity at pH 5.5. The activity drops above pH 7.0 due to a conformational change at the binding cleft, triggered by the proximity of two buried aspartate residues.