30 research outputs found
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DNA-PKcs structure suggests an allosteric mechanism modulating DNA double-strand break repair
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a central component of nonhomologous end joining (NHEJ), repairing DNA double-strand breaks that would otherwise lead to apoptosis or cancer. We have solved its structure in complex with the C-terminal peptide of Ku80 at 4.3 angstrom resolution using x-ray crystallography. We show that the 4128–amino acid structure comprises three large structural units: the N-terminal unit, the Circular Cradle, and the Head. Conformational differences between the two molecules in the asymmetric unit are correlated with changes in accessibility of the kinase active site, which are consistent with an allosteric mechanism to bring about kinase activation. The location of KU80ct194 in the vicinity of the breast cancer 1 (BRCA1) binding site suggests competition with BRCA1, leading to pathway selection between NHEJ and homologous recombination.This work was funded by the Wellcome Trust (grant 093167/Z/10/Z)
Zn(II) mediates vancomycin polymerization and potentiates its antibiotic activity against resistant bacteria
Vancomycin is known to bind to Zn(II) and can induce a zinc starvation response in bacteria. Here we identify a novel polymerization of vancomycin dimers by structural analysis of vancomycin-Zn(II) crystals and fibre X-ray diffraction. Bioassays indicate that this structure is associated with an increased antibiotic activity against bacterial strains possessing high level vancomycin resistance mediated by the reprogramming of peptidoglycan biosynthesis to use precursors terminating in D-Ala-D-Lac in place of D-Ala-D-Ala. Polymerization occurs via interaction of Zn(II) with the N-terminal methylleucine group of vancomycin, and we show that the activity of other glycopeptide antibiotics with this feature can also be similarly augmented by Zn(II). Construction and analysis of a model strain predominantly using D-Ala-D-Lac precursors for peptidoglycan biosynthesis during normal growth supports the hypothesis that Zn(II) mediated vancomycin polymerization enhances the binding affinity towards these precursors.This work was supported by funding from the Royal Society, UK (516002.K5877/ROG) and the Medical Research Council, UK (G0700141). A.Z. was supported from the Said foundation, the Cambridge Trust and the Cambridge Philosophical Society
Crystal Structures of Yersinia enterocolitica Salicylate Synthase and its Complex with the Reaction Products Salicylate and Pyruvate.
Crystal structure of the beta-chain of human hepatocyte growth factor-like/macrophage stimulating protein.
Crystal strcuture of Escherichia coli ketopantoate reductase in a ternary complex with NADP+ and pantoate bound: substrate recognition, conformational change, and cooperativity
Crystal structures of NK1 heparin complexes reveal the basis for NK1 activity and enable engineering of potent agonists and the MET receptor
NK1 is a splice variant of the polypeptide growth factor HGF/SF, which consists of the N-terminal (N) and first kringle (K) domain and requires heparan sulfate or soluble heparin for activity. We describe two X-ray crystal structures of NK1–heparin complexes that define a heparin-binding site in the N domain, in which a major role is played by R73, with further contributions from main chain atoms of T61, K63 and G79 and the side chains of K60, T61, R76, K62 and K58. Mutagenesis experiments demonstrate that heparin binding to this site is essential for dimerization in solution and biological activity of NK1. Heparin also comes into contact with a patch of positively charged residues (K132, R134, K170 and R181) in the K domain. Mutation of these residues yields NK1 variants with increased biological activity. Thus, we uncover a complex role for heparan sulfate in which binding to the primary site in the N domain is essential for biological activity whereas binding to the K domain reduces activity. We exploit the interaction between heparin and the K domain site in order to engineer NK1 as a potent receptor agonist and suggest that dual (positive and negative) control may be a general mechanism of heparan sulfate-dependent regulation of growth factor activity
Nanostructured films from hierarchical self-assembly of amyloidogenic proteins.
In nature, sophisticated functional materials are created through hierarchical self-assembly of simple nanoscale motifs. In the laboratory, much progress has been made in the controlled assembly of molecules into one-, two- and three-dimensional artificial nanostructures, but bridging from the nanoscale to the macroscale to create useful macroscopic materials remains a challenge. Here we show a scalable self-assembly approach to making free-standing films from amyloid protein fibrils. The films were well ordered and highly rigid, with a Young's modulus of up to 5-7 GPa, which is comparable to the highest values for proteinaceous materials found in nature. We show that the self-organizing protein scaffolds can align otherwise unstructured components (such as fluorophores) within the macroscopic films. Multiscale self-assembly that relies on highly specific biomolecular interactions is an attractive path for realizing new multifunctional materials built from the bottom up