Spectator no more, the role of the membrane in regulating ion channel function

CP is a Royal Society of Edinburgh (RSE) Personal Research Fellow, funded by the Scottish Government. Research funds for this study were also partly covered by a Tenovus Scotland grant (Tenovus Grant Application T15/41), awarded to CP. JHN is supported by the Chinese National Thousand Talents Program, Wellcome Trust Senior Investigator Award (WT100209MA) and Royal Society Wolfson Merit Award.A pressure gradient across a curved lipid bilayer leads to a lateral force within the bilayer. Following ground breaking work on eukaryotic ion channels, it is now known that many proteins sense this change in the lateral tension and alter their functions in response. It has been proposed that responding to pressure differentials may be one of the oldest signaling mechanisms in biology. The most well characterized mechanosensing ion channels are the bacterial ones which open when the pressure differential hits a threshold. Recent studies on one of these channels, MscS, have developed a simple molecular model for how they sense and adapt to pressure. Biochemical and structural studies on mechanosensitive channels from eukaryotes have disclosed pressure sensing mechanisms. In this review, we highlight these findings and discuss the potential for a general model for pressure sensing.PostprintPeer reviewe

Kinetic landscape of a peptide-bond-forming prolyl oligopeptidase

We thank Dr. Rafael Guimaraes da Silva for helpful discussions on enzyme kinetics. We also thank Professor David Lilley, Dr. Alasdair Freeman and Dr. Anne-Cecile Declais at the University of Dundee for training and usage of their QFM-4000 quenched-flow apparatus.Prolyl oligopeptidase B from Galerina marginata (GmPOPB) has recently been discovered as a peptidase capable of breaking and forming peptide bonds to yield a cyclic peptide. Despite the relevance of prolyl oligopeptidases in human biology and disease, a kinetic analysis pinpointing rate-limiting steps for a member of this enzyme family is not available. Macrocyclase enzymes are currently exploited to produce cyclic peptides with potential therapeutic applications. Cyclic peptides are promising drug-like molecules due to their stability and conformational rigidity. Here we describe an in-depth kinetic characterization of a prolyl oligopeptidase acting as a macrocyclase enzyme. By combining steady-state and pre-steady-state kinetics, we put forward a kinetic sequence in which a step after macrocyclization limits steady-state turnover. Additionally, product release is ordered, where cyclic peptide departs first followed by the peptide tail. Dissociation of the peptide tail is slow and significantly contributes to the turnover rate. Furthermore, trapping of the enzyme by the peptide tail becomes significant beyond initial-rate conditions. The presence of a burst of product formation and a large viscosity effect further support the rate-limiting nature of a physical step occurring after macrocyclization. This is the first detailed description of the kinetic sequence of a macrocyclase enzyme from this class. GmPOPB is amongst the fastest macrocyclases described to date, and this work is a necessary step towards designing broad specificity efficient macrocyclases.Publisher PDFPeer reviewe

Bacterial polysaccharide synthesis and export

The work is supported by the Chinese National Thousand Talents Program-, Wellcome Trust Senior Investigator Award (WT100209MA) and Royal Society Wolfson Merit Award.All domains of life make carbohydrate polymers and by anchoring them to lipid molecules they can decorate the outside of the cell. Polysaccharides are linked to proteins by glycosylation, a process found in both bacteria and in higher organisms. Bacteria do have other distinct uses for carbohydrate polymers; in gram-negative bacteria glycolipids form the outer leaflet of the outer membrane and in many pathogens (both gram-positive and gram-negative) sugar polymers are used to build a capsule or are secreted into the environment. There are parallels, but of course differences, in the biosynthesis of glycolipids between prokaryotes and eukaryotes, which occur at the membrane. The translocation of large sugar polymers across the outer membrane is unique to gram-negative bacteria. Recent progress in the molecular understanding of both the biosynthesis at the inner membrane and the translocation across the outer membrane are reviewed here.Publisher PDFPeer reviewe

Synthesis of Hybrid Cyclopeptides through Enzymatic Macrocyclization

Acknowledgements We thank Dr. G. Mann and Dr. A. Bent for supplying the enzymes and the useful discussions, and Dr. T. Lebl for the useful NMR discussions. This work was supported by the European Research Council (339367), UK Biotechnology and Biological Sciences Research Council (K015508/1), the Wellcome Trust [TripleTOF 5600 mass spectrometer (094476), the MALDI TOF-TOF Analyzer (079272AIA), 700 NMR], and the EPSRC UK National Mass Spectrometry Facility at Swansea University. J.H.N., M.A.J., and N.J.W. are named on patents that have been filed by St. Andrews and Aberdeen Universities to commercialize the enzymes (PatG) and (LynD) used in the study. No income derives from the patent.Peer reviewedPublisher PD

Mechanisms of cyanobactin biosynthesis

This work was supported by the European Research Council (339367), UK Biotechnology and Biological Sciences Research Council (K015508/1).Cyanobactins are a diverse collection of natural products that originate from short peptides made on a ribosome. The amino acids are modified in a series of transformations catalyzed by multiple enzymes. The patellamide pathway is the most well studied and characterized example. Here we review the structures and mechanisms of the enzymes that cleave peptide bonds, macrocyclise peptides, heterocyclise cysteine (as well as threonine and serine) residues, oxidize five-membered heterocycles and attach prenyl groups. Some enzymes operate by novel mechanisms which is of interest and in addition the enzymes uncouple recognition from catalysis. The normally tight relationship between these factors hinders biotechnology. The cyanobactin pathway may be particularly suitable for exploitation, with progress observed with in vivo and in vitro approaches.PostprintPeer reviewe

Crystallization and preliminary crystallographic analysis of the bacterial capsule assembly-regulating tyrosine phosphatases Wzb of Escherichia coli and Cps4B of Streptococcus pneumoniae

The crystallization is reported of two bacterial tyrosine phosphatases which belong to different enzyme families despite their ability to catalyse identical reactions

MtsslWizard: In Silico Spin-Labeling and Generation of Distance Distributions in PyMOL

MtsslWizard is a computer program, which operates as a plugin for the PyMOL molecular graphics system. MtsslWizard estimates distances between spin labels on proteins quickly with user-configurable options through a simple graphical interface. In default mode, the program searches for ensembles of possible MTSSL conformations that do not clash with a static model of the protein. Once conformations are assigned, distance distributions between two or more ensembles are calculated, displayed, and can be exported to other software. The program’s use is evaluated in a number of challenging test cases and its strengths and weaknesses evaluated. The benefits of the program are its accuracy and simplicity

Understanding and manipulating non-templated peptide bond formation by macrocyclase enzymes

Peptide macrocycles are attractive molecules because they are drug-like, protease resistant, cell permeable, and possess a rigid structure. They have been shown to possess various biological activities and to be able to inhibit protein-protein interactions and other complex targets. Although several macrocyclases have been characterized to date, only two can catalyze the formation of cyclic peptides containing less than 9 amino acids in their core. PatGmac, from the biosynthesis of cyanobactins, is a versatile catalyst with very broad substrate specificity. It can utilize varied peptide sequences, incorporate unnatural amino acids, including substrates that are peptide “chimeras” containing triazoles, peg linkers and sugars (Figure 1A, bottom). Despite its remarkable substrate promiscuity, PatGmac is extremely slow, with turnover rates in the vicinity of once per day. In search for a more efficient macrocyclase we studied GmPOPB, a prolyl oligopeptidase from the mushroom Galerina marginata. GmPOPB (fast macrocyclase) participates in the biosynthesis of the toxic amanitins, catalyzing both peptide bond hydrolysis and peptide bond formation with equal efficiency (Figure 1A, top). We determined crystal structures of apoGmPOPB and GmPOPB mutants bound to a peptidase and a macrocyclase substrate unveiling a mechanism by which the enzyme controls which reaction will be catalyzed. We have also performed an extensive kinetic analysis of this enzyme in comparison to the slow PatGmac. Crucial differences exist between the fast and the slow macrocyclases. Substrate positioning plays an important role towards catalytic efficiency. For the fast macrocyclase GmPOPB there is product inhibition and the rate-limiting step for the reaction is product release. For the slow macrocyclase PatGmac product release is not rate determining for the majority of the substrates tested, and the rate-limiting step is coupled to chemistry. Guided by our kinetic studies, we have designed modified peptide substrates, which eliminate the requirement for a long peptide substrate from 25 amino acids to 13 amino acids for the fast macrocyclase. We are currently designing enzyme variants to improve the catalytic efficiency of the slow macrocyclase and to broaden the substrate scope of the fast macrocyclase. We hope our findings will result in a better, more efficient and substrate permissible macrocyclase that can be used for the biocatalytic generation of cyclic peptide libraries to be tested for biological function. Please click Additional Files below to see the full abstract

Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates

H.L. is funded by the George & Stella Lee Scholarship and Criticat EPSRC. This project was also funded by the European Research Council project 339367 NCB-TNT and by the BBSRC (K015508/1). JHN is 1000 talent scholar of the Chinese Academy of Sciences at the University of Sichuan.Peptide macrocycles are promising therapeutic molecules because they are protease resistant, structurally rigid, membrane permeable and capable of modulating protein-protein interactions. Here, we report the characterization of the dual function macrocyclase-peptidase enzyme involved in the biosynthesis of the highly toxic Amanitin toxin family of macrocycles. The enzyme first removes 10 residues from the N-terminus of a 35-residue substrate. Conformational trapping of the amino acid peptide forces the enzyme to release this intermediate rather than proceed to macrocyclization. The enzyme rebinds the 25 amino acid peptide in a different conformation and catalyzes macrocyclization of the N-terminal 8 residues. Structures of the enzyme bound to both substrates and biophysical analysis characterize the different binding modes rationalizing the mechanism. Using these insights simpler substrates with only five C-terminal residues were designed, allowing the enzyme to be more effectively exploited in biotechnology.Publisher PDFPeer reviewe

Mechanism of DNA loading by the DNA repair helicase XPD

Funding: Welcome Trust Programme Grant [WT091825MA to M.F.W., J.H.N.]; Wellcome Trust [099149/Z/12/Z]; Royal Society Wolfson Merit Award (to M.F.W., J.H.N.). Funding for open access charge: Wellcome Trust [WT091825MA].The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5′ to 3′ helicase with an essential iron–sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase from Thermoplasma acidophilum has been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD from Sulfolobus acidocaldiarius that lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD.Publisher PDFPeer reviewe