A diterpene synthase from the sandfly Lutzomyia longipalpis produces the pheromone sobralene.

The phlebotomine sandfly, Lutzomyia longipalpis, a major vector of the Leishmania parasite, uses terpene pheromones to attract conspecifics for mating. Examination of the L. longipalpis genome revealed a putative terpene synthase (TPS), which-upon heterologous expression in, and purification from, Escherichia coli-yielded a functional enzyme. The TPS, termed LlTPS, converted geranyl diphosphate (GPP) into a mixture of monoterpenes with low efficiency, of which ß-ocimene was the major product. (E,E)-farnesyl diphosphate (FPP) principally produced small amounts of (E)-ß-farnesene, while (Z,E)- and (Z,Z)-FPP yielded a mixture of bisabolene isomers. None of these mono- and sesquiterpenes are known volatiles of L. longipalpis. Notably, however, when provided with (E,E,E)-geranylgeranyl diphosphate (GGPP), LlTPS gave sobralene as its major product. This diterpene pheromone is released by certain chemotypes of L. longipalpis, in particular those found in the Ceará state of Brazil. Minor diterpene components were also seen as products of the enzyme that matched those seen in a sandfly pheromone extract.

Pea aphid odorant-binding protein ApisOBP6 discriminates between aphid sex pheromone components, aphid alarm pheromone and a host plant volatile.

Olfactory perception of pheromones in insects involves odorant-binding proteins (OBPs), relatively small proteins (ca.110-240 amino acid residues) that can bind reversibly to behaviourally active olfactory ligands. In this study, we investigated the binding in silico and in vitro of the aphid sex pheromone components (1R,4aS,7S,7aR)-nepetalactol and (4aS,7S,7aR)-nepetalactone and the aphid alarm pheromone (E)-ß-farnesene by OBPs from the pea aphid, Acyrthosiphon pisum. Screening of protein models of ApisOBPs1-11 with the aphid sex pheromone components suggested that ApisOPB6 was a candidate. Fluorescence assays using ApisOBP6 suggested that ApisOBP6 was able to bind both sex pheromone components and discriminate from the aphid alarm pheromone and the generic plant compound (R/S)-linalool. Saturation transfer difference NMR experiments with ApisOBP6 yielded results consistent to those from the fluorescence experiments, with a clear interaction between ApisOBP6 and (4aS,7S,7aR)-nepetalactone. These results describe a novel interaction and potential function for ApisOBP6, point to pre-receptor odorant discrimination by OBPs, and provide a platform for investigating the function of other aphid olfactory proteins involved in aphid chemical ecology.

Osteological, multi-isotope and proteomic analysis of poorly-preserved human remains from a Dutch East India Company burial ground in South Africa.

Skeletal remains discovered in Simon's Town, South Africa, were hypothesised as being associated with a former Dutch East India Company (VOC) hospital. We report a novel combined osteological and biochemical approach to these poorly-preserved remains. A combined strontium (87Sr/86Sr), oxygen (δ18OVPDB) and carbon (δ13CVPDB) isotope analysis informed possible childhood origins and diet, while sex-specific amelogenin enamel peptides revealed biological sex. Osteological analyses presented evidence of residual rickets, a healed trauma, dental pathological conditions, and pipe notches. The combined isotope analyses yielded results for 43 individuals which suggested a diverse range of geological origins, including at least 16% of the population being non-local. The inclusion of δ13CVPDB had intriguing implications for three individuals who likely did not have origins in the Cape Town region nor in Europe. Peptide analysis on the dental enamel of 25 tested individuals confirmed they were all biologically male. We suggest that isolated enamel may provide crucial information about individuals' pathological conditions, geographical origins, diet, and biological sex. These data further demonstrated that a combined approach using multiple osteological and biochemical methods is advantageous for human remains which are poorly preserved and can contextualise a site with little direct evidence.

Characterisation of geranylgeranyl diphosphate synthase from the sandfly Lutzomyia longipalpis.

Leishmaniasis is a debilitating and often fatal neglected tropical disease. Males from sub-populations of the Leishmania-harbouring sandfly, Lutzomyia longipalpis, produce the diterpene sex and aggregation pheromone, sobralene, for which geranylgeranyl diphosphate (GGPP) is the likely isoprenoid precursor. We have identified a GGPP synthase (lzGGPPS) from L. longipalpis, which was recombinantly expressed in bacteria and purified for functional and kinetic analysis. In vitro enzymatic assays using LC-MS showed that lzGGPPS is an active enzyme, capable of converting substrates dimethylallyl diphosphate (DMAPP), (E)-geranyl diphosphate (GPP), (E,E)-farnesyl diphosphate (FPP) with co-substrate isopentenyl diphosphate (IPP) into (E,E,E)-GGPP, while (Z,E)-FPP was also accepted with low efficacy. Comparison of metal cofactors for lzGGPPS highlighted Mg2+ as most efficient, giving increased GGPP output when compared against other divalent metal ions tested. In line with previously characterised GGPPS enzymes, GGPP acted as an inhibitor of lzGGPPS activity. The molecular weight in solution of lzGGPPS was determined to be ∼221 kDa by analytical SEC, suggesting a hexameric assembly, as seen in the human enzyme, and representing the first assessment of GGPPS quaternary structure in insects.

ERFVII action and modulation through oxygen-sensing in Arabidopsis thaliana.

Oxygen is a key signalling component of plant biology, and whilst an oxygen-sensing mechanism was previously described in Arabidopsis thaliana, key features of the associated PLANT CYSTEINE OXIDASE (PCO) N-degron pathway and Group VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factor substrates remain untested or unknown. We demonstrate that ERFVIIs show non-autonomous activation of root hypoxia tolerance and are essential for root development and survival under oxygen limiting conditions in soil. We determine the combined effects of ERFVIIs in controlling gene expression and define genetic and environmental components required for proteasome-dependent oxygen-regulated stability of ERFVIIs through the PCO N-degron pathway. Using a plant extract, unexpected amino-terminal cysteine sulphonic acid oxidation level of ERFVIIs was observed, suggesting a requirement for additional enzymatic activity within the pathway. Our results provide a holistic understanding of the properties, functions and readouts of this oxygen-sensing mechanism defined through its role in modulating ERFVII stability.

Mapping the Binding Interactions between Human Gasdermin D and Human Caspase-1 Using Carbene Footprinting.

Carbene footprinting is a recently developed mass spectrometry-based chemical labeling technique that probes protein interactions and conformation. Here, we use the methodology to investigate binding interactions between the protease human Caspase-1 (C285A) and full-length human Gasdermin D (hGSDMD), which are important in inflammatory cell death. GSDMD is cleaved by Caspase-1, releasing its N-terminal domain which oligomerizes in the membrane to form large pores, resulting in lytic cell death. Regions of reduced carbene labeling (masking), caused by protein binding, were observed for each partner in the presence of the other and were consistent with hCaspase-1 exosite and active-site interactions. Most notably, the results showed direct occupancy of hCaspase-1 (C285A) active-site by hGSDMD for the first time. Differential carbene labeling of full-length hGSDMD and the pore-forming N-terminal domain assembled in liposomes showed masking of the latter, consistent with oligomeric assembly and insertion into the lipid bilayer. Interactions between Caspase-1 and the specific inhibitor VRT-043198 were also studied by this approach. In wild-type hCaspase-1, VRT-043198 modifies the active-site Cys285 through the formation of a S,O-hemiacetal. Here, we showed by carbene labeling that this inhibitor can noncovalently occupy the active site of a C285A mutant. These findings add considerably to our knowledge of the hCaspase-1-hGSDMD system.

Ferric quinate (QPLEX) interacts with the major outer membrane protein (MOMP) of Campylobacter jejuni and enters through the porin channel into the periplasmic space.

Ferric chelates like ferric tyrosinate (TYPLEX) and the closely related ferric quinate (QPLEX) are structural mimics of bacterial siderophores. TYPLEX has been trialled as a feed additive in farming of commercial broilers, reducing Campylobacter loads by 2-3 log10 and leading to faster growth and better feed consumption. These ferric chelates offer a good alternative feed additive to antibiotics helping to reduce the indiscriminate use of preventative antibiotics in broiler farming to control Campylobacter infections. In this study, we show that QPLEX binds to the Major Outer Membrane Protein (MOMP) of C. jejuni NCTC11168. MOMP is an essential and abundant outer membrane porin on the surface of the bacteria, acting as an adhesin to help establish infection by mediating attachment of C. jejuni onto the gut epithelium of broilers and establish infection. Using carbene footprinting, we map the MOMP-QPLEX interaction and show by complementary in silico docking that QPLEX enters the porin channel through interactions at the extracellular face, translocates down the channel through a dipole transverse electric field towards the opposite end and is released into the periplasm at the intracellular face of MOMP. Our studies suggest a potential mechanism for the non-antibiotic anti-Campylobacter activity of these ferric chelates.

An ALS-associated variant of the autophagy receptor SQSTM1/p62 reprograms binding selectivity toward the autophagy-related hATG8 proteins.

Recognition of human autophagy-related 8 (hATG8) proteins by autophagy receptors represents a critical step within this cellular quality control system. Autophagy impairment is known to be a pathogenic mechanism in the motor neuron disorder amyotrophic lateral sclerosis (ALS). Overlapping but specific roles of hATG8 proteins belonging to the LC3 and GABARAP subfamilies are incompletely understood, and binding selectivity is typically overlooked. We previously showed that an ALS-associated variant of the SQSTM1/p62 (p62) autophagy receptor bearing an L341V mutation within its ATG8-interacting motif (AIM) impairs recognition of LC3B in vitro, yielding an autophagy-deficient phenotype. Improvements in understanding of hATG8 recognition by AIMs now distinguish LC3-interaction and GABARAP-interaction motifs and predict the effects of L341V substitution may extend beyond loss of function to biasing AIM binding preference. Through biophysical analyses, we confirm impaired binding of the L341V-AIM mutant to LC3A, LC3B, GABARAP, and GABARAPL1. In contrast, p62 AIM interactions with LC3C and GABARAPL2 are unaffected by this mutation. Isothermal titration calorimetry and NMR investigations provided insights into the entropy-driven GABARAPL2/p62 interaction and how the L341V mutation may be tolerated. Competition binding demonstrated reduced association of the L341V-AIM with one hATG8 manifests as a relative increase in association with alternate hATG8s, indicating effective reprogramming of hATG8 selectivity. These data highlight how a single AIM peptide might compete for binding with different hATG8s and suggest that the L341V-AIM mutation may be neomorphic, representative of a disease mechanism that likely extends into other human disorders.

Site-Selective Installation of Nϵ -Modified Sidechains into Peptide and Protein Scaffolds via Visible-Light-Mediated Desulfurative C-C Bond Formation.

Post-translational modifications (PTMs) enhance the repertoire of protein function and mediate or influence the activity of many cellular processes. The preparation of site-specifically and homogeneously modified proteins, to apply as tools to understand the biological role of PTMs, is a challenging task. Herein, we describe a visible-light-mediated desulfurative C(sp3 )-C(sp3 ) bond forming reaction that enables the site-selective installation of Nϵ -modified sidechains into peptides and proteins of interest. Rapid, operationally simple, and tolerant to ambient atmosphere, we demonstrate the installation of a range of lysine (Lys) PTMs into model peptide systems and showcase the potential of this technology by site-selectively installing an Nϵ Ac sidechain into recombinantly expressed ubiquitin (Ub).

Combined Chemical Modification and Collision Induced Unfolding Using Native Ion Mobility-Mass Spectrometry Provides Insights into Protein Gas-Phase Structure.

Native mass spectrometry is now an important tool in structural biology. Thus, the nature of higher protein structure in the vacuum of the mass spectrometer is an area of significant interest. One of the major goals in the study of gas-phase protein structure is to elucidate the stabilising role of interactions at the level of individual amino acid residues. A strategy combining protein chemical modification together with collision induced unfolding (CIU) was developed and employed to probe the structure of compact protein ions produced by native electrospray ionisation. Tractable chemical modification was used to alter the properties of amino acid residues, and ion mobility-mass spectrometry (IM-MS) utilised to monitor the extent of unfolding as a function of modification. From these data the importance of specific intramolecular interactions for the stability of compact gas-phase protein structure can be inferred. Using this approach, and aided by molecular dynamics simulations, an important stabilising interaction between K6 and H68 in the protein ubiquitin was identified, as was a contact between the N-terminus and E22 in a ubiquitin binding protein UBA2.

Mapping the interaction between eukaryotic initiation factor 4A (eIF4A) and the inhibitor hippuristanol using carbene footprinting and mass spectrometry.

Protein-ligand interactions are central to protein activity and cell functionality. Improved knowledge of these relationships greatly benefits our understanding of key biological processes and aids in rational drug design towards the treatment of clinically relevant diseases. Carbene footprinting is a recently developed mass spectrometry-based chemical labelling technique that provides valuable information relating to protein-ligand interactions, such as the mapping of binding sites and associated conformational change. Here, we show the application of carbene footprinting to the interaction between eIF4A helicase and a natural product inhibitor, hippuristanol, found in the coral Isis hippuris. Upon addition of hippuristanol we identified reduced carbene labelling (masking) in regions of eIF4A previously implicated in ligand binding. Additionally, we detected hippuristanol-associated increased carbene labelling (unmasking) around the flexible hinge region of eIF4A, indicating ligand-induced conformational change. This work represents further development of the carbene footprinting technique and demonstrates its potential in characterising medicinally relevant protein-ligand interactions.

Iridoid Sex Pheromone Biosynthesis in Aphids Mimics Iridoid-Producing Plants.

Biosynthesis of (1R,4aS,7S,7aR)-nepetalactol (1) and (4aS,7S,7aR)-nepetalactone (2) in plants involves iridoid synthase (ISY), an atypical reductive cyclase that catalyses the reduction of 8-oxogeranial into the reactive enol of (S)-8-oxocitronellal, and cyclization of this enol intermediate, either non-enzymatically or by a nepetalactol-related short chain dehydrogenase enzyme (NEPS) that yields the nepetalactols. In this study, we investigated the biosynthesis in vivo of 1 and 2 in the pea aphid, Acyrthosiphon pisum, using a library of isotopically-labelled monoterpenoids as molecular probes. Topical application of deuterium-labelled probes synthesized from geraniol and nerol resulted in production of 2 H4 -lactol 1 and 2 H4 -lactone 2. However, deuterium incorporation was not evident using labelled probes synthesized from (S)-citronellol. These results suggest that iridoid biosynthesis in animals, specifically aphids, may follow a broadly similar route to that characterised for plants.

Analysis of insulin glulisine at the molecular level by X-ray crystallography and biophysical techniques.

This study concerns glulisine, a rapid-acting insulin analogue that plays a fundamental role in diabetes management. We have applied a combination of methods namely X-ray crystallography, and biophysical characterisation to provide a detailed insight into the structure and function of glulisine. X-ray data provided structural information to a resolution of 1.26 Å. Crystals belonged to the H3 space group with hexagonal (centred trigonal) cell dimensions a = b = 82.44 and c = 33.65 Å with two molecules in the asymmetric unit. A unique position of D21Glu, not present in other fast-acting analogues, pointing inwards rather than to the outside surface was observed. This reduces interactions with neighbouring molecules thereby increasing preference of the dimer form. Sedimentation velocity/equilibrium studies revealed a trinary system of dimers and hexamers/dihexamers in dynamic equilibrium. This new information may lead to better understanding of the pharmacokinetic and pharmacodynamic behaviour of glulisine which might aid in improving formulation regarding its fast-acting role and reducing side effects of this drug.

Structural basis for chain release from the enacyloxin polyketide synthase.

Modular polyketide synthases and non-ribosomal peptide synthetases are molecular assembly lines that consist of several multienzyme subunits that undergo dynamic self-assembly to form a functional megacomplex. N- and C-terminal docking domains are usually responsible for mediating the interactions between subunits. Here we show that communication between two non-ribosomal peptide synthetase subunits responsible for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii, is mediated by an intrinsically disordered short linear motif and a ß-hairpin docking domain. The structures, interactions and dynamics of these subunits were characterized using several complementary biophysical techniques to provide extensive insights into binding and catalysis. Bioinformatics analyses reveal that short linear motif/ß-hairpin docking domain pairs mediate subunit interactions in numerous non-ribosomal peptide and hybrid polyketide-non-ribosomal peptide synthetases, including those responsible for assembling several important drugs. Short linear motifs and ß-hairpin docking domains from heterologous systems are shown to interact productively, highlighting the potential of such interfaces as tools for biosynthetic engineering.

PepFoot: A Software Package for Semiautomated Processing of Protein Footprinting Data.

Covalent footprinting of proteins using reactive intermediates such as radicals and carbenes is emerging as a valuable tool for mapping surface accessibility, and hence binding sites of proteins. The approach generates a significant amount of mass spectrometry (MS) data, which can be time-consuming to process manually. PepFoot, a software package that allows semiautomated processing of MS data from footprinting experiments, is described. By using the open source .mz5 file format, it is able to accept data from all the major instrument manufacturers. Following manual user interrogation of one data file within a user-friendly GUI, the software then automates determination of the degree of fractional modification ( fm) with the footprinting agent across a batch of experimental data. This greatly increases efficiency and throughput compared to manual analysis of each file, and provides initial scrutiny and confidence compared to fully automated analysis. Histogram plots of fm for each peptide from the footprinted protein may be displayed within PepFoot and mapped onto an imported protein structure to reveal differential labeling patterns and hence binding sites. The software has been tested on data from carbene and hydroxyl radical labeling experiments to demonstrate its broad utility. PepFoot is released under the LGPL version 3 license, and is available for Windows, MacOS, and Linux systems at github.com/jbellamycarter/pepfoot .

Tyrosinase-Mediated Bioconjugation. A Versatile Approach to Chimeric Macromolecules.

We present a method for tyrosine-selective and reversible bioconjugation; tyrosines are enzymatically converted into catechols and in situ "clicked" onto boronic acids. Importantly, our process selectively produces catechols and avoids quinones, thereby improving the control over the chemical identity of the products. We have conjugated boronic acid-containing hyaluronic acid (HyA) to peptides bearing tyrosines in variable number and position; the use of tagging peptides for the provision of well exposed tyrosine residues-in our case the hemagglutinin-derived HA-tag-makes our approach applicable to virtually any protein; we have demonstrated this concept by conjugating HA-tagged ovalbumin to HyA, thereby also showing the feasibility of producing chimeric proteoglycans. A caveat of this appproach is that, although the formation of boronic esters does not affect the biological recognition of substrates (ovalbumin and HyA), the introduction of catechols may alter some of their biological properties: for example, only after tyrosinase treatment ovalbumin directly induced dendritic cell maturation, either alone or as a HyA conjugate.

Mechanism of intersubunit ketosynthase-dehydratase interaction in polyketide synthases.

Modular polyketide synthases (PKSs) produce numerous structurally complex natural products that have diverse applications in medicine and agriculture. PKSs typically consist of several multienzyme subunits that utilize structurally defined docking domains (DDs) at their N and C termini to ensure correct assembly into functional multiprotein complexes. Here we report a fundamentally different mechanism for subunit assembly in trans-acyltransferase (trans-AT) modular PKSs at the junction between ketosynthase (KS) and dehydratase (DH) domains. This mechanism involves direct interaction of a largely unstructured docking domain (DD) at the C terminus of the KS with the surface of the downstream DH. Acyl transfer assays and mechanism-based crosslinking established that the DD is required for the KS to communicate with the acyl carrier protein appended to the DH. Two distinct regions for binding of the DD to the DH were identified using NMR spectroscopy, carbene footprinting, and mutagenesis, providing a foundation for future elucidation of the molecular basis for interaction specificity.

Carbene Footprinting Reveals Binding Interfaces of a Multimeric Membrane-Spanning Protein.

Mapping the interaction sites between membrane-spanning proteins is a key challenge in structural biology. In this study a carbene-footprinting approach was developed and applied to identify the interfacial sites of a trimeric, integral membrane protein, OmpF, solubilised in micelles. The diazirine-based footprinting probe is effectively sequestered by, and incorporated into, the micelles, thus leading to efficient labelling of the membrane-spanning regions of the protein upon irradiation at 349 nm. Areas associated with protein-protein interactions between the trimer subunits remained unlabelled, thus revealing their location.

A front-face 'SNi synthase' engineered from a retaining 'double-SN2' hydrolase.

SNi-like mechanisms, which involve front-face leaving group departure and nucleophile approach, have been observed experimentally and computationally in chemical and enzymatic substitution at α-glycosyl electrophiles. Since SNi-like, SN1 and SN2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus ß-glycosidase, which originally catalyzed double SN2 substitution, changed its mode to SNi-like. Destruction of the first SN2 nucleophile through E387Y mutation created a ß-stereoselective catalyst for glycoside synthesis from activated substrates, despite lacking a nucleophile. The pH profile, kinetic and mutational analyses, mechanism-based inactivators, X-ray structure and subsequent metadynamics simulations together suggest recruitment of substrates by π-sugar interaction and reveal a quantum mechanics-molecular mechanics (QM/MM) free-energy landscape for the substitution reaction that is similar to those of natural, SNi-like glycosyltransferases. This observation of a front-face mechanism in a ß-glycosyltransfer enzyme highlights that SNi-like pathways may be engineered in catalysts with suitable environments and suggests that 'ß-SNi' mechanisms may be feasible for natural glycosyltransfer enzymes.

Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions.

Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.