Structure of the T6 Human Nickel Insulin Derivative at 1.35 Å Resolution

This paper presents results of the structural investigation on the human insulin hexamer derivative stabilised by nickel coordination. Single crystals of the Ni-insulin derivative were prepared by the hanging drop vapour diffusion crystallisation method using metal-free insulin and nickel(II) acetate tetrahydrate. The low-temperature crystal structure was determined by the single crystal X-ray crystallographic method with data extending to 1.35 Å spacing. The investigated insulin derivative exhibits the T6 form of insulin and crystallizes in the trigonal system in space group R3, with the unit cell parameters a = b = 81.41 Å and c = 33.75 Å. There are two nickel atoms per insulin hexamer and both are octahedrally coordinated by three Nε2 atoms of three symmetry-related HisB10/HisD10 and three oxygen atoms of three symmetry-related water molecules

Crystal structure of the putative peptide-binding protein AppA from Clostridium difficile

Peptides play an important signalling role in Bacillus subtilis, where their uptake by one of two ABC-type oligopeptide transporters, Opp and App, is required for efficient sporulation. Homologues of these transporters in Clostridium difficile have been characterized, but their role, and hence that of peptides, in regulating sporulation in this organism is less clear. Here, the oligopeptide-binding receptor proteins for these transporters, CdAppA and CdOppA, have been purified and partially characterized, and the crystal structure of CdAppA has been determined in an open unliganded form. Peptide binding to either protein could not be observed in Thermofluor assays with a set of ten peptides of varying lengths and compositions. Re-examination of the protein sequences together with structure comparisons prompts the proposal that CdAppA is not a versatile peptide-binding protein but instead may bind a restricted set of peptides. Meanwhile, CdOppA is likely to be the receptor protein for a nickel-uptake system

Crystal structures of the GH18 domain of the bifunctional peroxiredoxin-chitinase CotE from Clostridium difficile

CotE is a coat protein that is present in the spores of Clostridium difficile, an obligate anaerobic bacterium and a pathogen that is a leading cause of antibiotic-associated diarrhoea in hospital patients. Spores serve as the agents of disease transmission, and CotE has been implicated in their attachment to the gut epithelium and subsequent colonization of the host. CotE consists of an N-terminal peroxiredoxin domain and a C-terminal chitinase domain. Here, a C-terminal fragment of CotE comprising residues 349-712 has been crystallized and its structure has been determined to reveal a core eight-stranded β-barrel fold with a neighbouring subdomain containing a five-stranded β-sheet. A prominent groove running across the top of the barrel is lined by residues that are conserved in family 18 glycosyl hydrolases and which participate in catalysis. Electron density identified in the groove defines the pentapeptide Gly-Pro-Ala-Met-Lys derived from the N-terminus of the protein following proteolytic cleavage to remove an affinity-purification tag. These observations suggest the possibility of designing peptidomimetics to block C. difficile transmission

The molecular basis of thioalcohol production in human body odour

This work was supported by the BBSRC Grant BB/N006615/1.Body odour is a characteristic trait of Homo sapiens, however its role in human behaviour and evolution is poorly understood. Remarkably, body odour is linked to the presence of a few species of commensal microbes. Herein we discover a bacterial enzyme, limited to odour-forming staphylococci that are able to cleave odourless precursors of thioalcohols, the most pungent components of body odour. We demonstrated using phylogenetics, biochemistry and structural biology that this cysteine-thiol lyase (C-T lyase) is a PLP-dependent enzyme that moved horizontally into a unique monophyletic group of odour-forming staphylococci about 60 million years ago, and has subsequently tailored its enzymatic function to human-derived thioalcohol precursors. Significantly, transfer of this enzyme alone to non-odour producing staphylococci confers odour production, demonstrating that this C-T lyase is both necessary and sufficient for thioalcohol formation. The structure of the C-T lyase compared to that of other related enzymes reveals how the adaptation to thioalcohol precursors has evolved through changes in the binding site to create a constrained hydrophobic pocket that is selective for branched aliphatic thioalcohol ligands. The ancestral acquisition of this enzyme, and the subsequent evolution of the specificity for thioalcohol precursors implies that body odour production in humans is an ancient process.Publisher PDFPeer reviewe

Peptide transport in Bacillus subtilis – structure and specificity in the extracellular solute binding proteins OppA and DppE

Peptide transporters play important nutritional and cell signalling roles in Bacillus subtilis, which are pronounced during stationary phase adaptations and development. Three high-affinity ATP-binding cassette (ABC) family transporters are involved in peptide uptake – the oligopeptide permease (Opp), another peptide permease (App) and a less well-characterized dipeptide permease (Dpp). Here we report crystal structures of the extracellular substrate binding proteins, OppA and DppE, which serve the Opp and Dpp systems, respectively. The structure of OppA was determined in complex with endogenous peptides, mod-elled as Ser-Asn-Ser-Ser, and with the sporulation-promoting peptide Ser-Arg-Asn-Val-Thr, which bind with Kd values of 0.4 and 2 µM, respectively, as measured by isothermal titration calorimetry. Differential scanning fluorescence experiments with a wider panel of ligands showed that OppA has highest affinity for tetra-and penta-peptides. The structure of DppE revealed the unexpected presence of a murein tripeptide (MTP) ligand, l-Ala-d-Glu-meso-DAP, in the peptide binding groove. The mode of MTP binding in DppE is different to that observed in the murein peptide binding protein, MppA, from Escherichia coli, suggesting independent evolution of these proteins from an OppA-like precursor. The presence of MTP in DppE points to a role for Dpp in the uptake and recycling of cell wall peptides, a conclusion that is supported by analysis of the genomic context of dpp, which revealed adjacent genes encoding enzymes involved in muropeptide catabolism in a gene organization that is widely conserved in Firmicutes

Overview of the CCP4 suite and current developments.

The CCP4 (Collaborative Computational Project, Number 4) software suite is a collection of programs and associated data and software libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims. The programs are from a wide variety of sources but are connected by a common infrastructure provided by standard file formats, data objects and graphical interfaces. Structure solution by macromolecular crystallography is becoming increasingly automated and the CCP4 suite includes several automation pipelines. After giving a brief description of the evolution of CCP4 over the last 30 years, an overview of the current suite is given. While detailed descriptions are given in the accompanying articles, here it is shown how the individual programs contribute to a complete software package

The CCP4 suite : integrative software for macromolecular crystallography

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world

Providing Programs for Protein Crystallography

The CCP4 (Collaborative Computational Project, number 4) aims to provide first a state-of-the-art suite consisting of a collection of programs plus associated data and subroutine libraries for the determination of macromolecular structure by X-ray crystallography. The programs are from a wide variety of sources but all use agreed standard data file formats. The suite is designed to be flexible, allowing users a number of methods of achieving their aims and so there may be more than one program to cover each function. The package has been ported to all the major platforms under both Unix and VMS and is freely distributed to academics by anonymous FTP from Daresbury Laboratory. It is widely used throughout the world. Secondly the Project has a responsibility to provide support, both in installing, documenting and maintaining the suite, and in educating budding crystallographers in methodological and computing techniques. key words: CCP4 / program suite / X-ray / macromolecular crystallog..

The crystal structures of the oligopeptide-binding protein OppA complexed with tripeptide and tetrapeptide ligands

AbstractBackground: The periplasmic oligopeptide-binding protein OppA has a remarkably broad substrate specificity, binding peptides of two to five amino-acid residues with high affinity, but little regard to sequence. It is therefore an ideal system for studying how different chemical groups can be accommodated in a protein interior. The ability of the protein to bind peptides of different lengths has been studied by co-crystallising it with different ligands.Results Crystals of OppA from Salmonella typhimurium complexed with the peptides Lys–Lys–Lys (KKK) and Lys–Lys–Lys–Ala (KKKA) have been grown in the presence of uranyl ions which form important crystal contacts. These structures have been refined to 1.4 å and 2.1 å, respectively. The ligands are completely enclosed, their side chains pointing into large hydrated cavities and making few strong interactions with the protein.Conclusion Tight peptide binding by OppA arises from strong hydrogen bonding and electrostatic interactions between the protein and the main chain of the ligand. Different basic side chains on the protein form salt bridges with the C terminus of peptide ligands of different lengths