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.

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).

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.

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.

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.

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.

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 .

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.

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.

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.

Mass spectrometry insights into a tandem ubiquitin-binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin.

Unanchored polyubiquitin chains are emerging as important regulators of cellular physiology with diverse roles paralleling those of substrate-conjugated polyubiquitin. However tools able to discriminate unanchored polyubiquitin chains of different isopeptide linkages have not been reported. We describe the design of a linker-optimized ubiquitin-binding domain hybrid (t-UBD) containing two UBDs, a ZnF-UBP domain in tandem with a linkage-selective UBA domain, which exploits avidity effects to afford selective recognition of unanchored Lys48-linked polyubiquitin chains. Utilizing native MS to quantitatively probe binding affinities we confirm cooperative binding of the UBDs within the synthetic protein, and desired binding specificity for Lys48-linked ubiquitin dimers. Furthermore, MS/MS analyses indicate that the t-UBD, when applied as an affinity enrichment reagent, can be used to favor the purification of endogenous unanchored Lys48-linked polyubiquitin chains from mammalian cell extracts. Our study indicates that strategies for the rational design and engineering of polyubiquitin chain-selective binding in nonbiological polymers are possible, paving the way for the generation of reagents to probe unanchored polyubiquitin chains of different linkages and more broadly the 'ubiquitome'. All MS data have been deposited in the ProteomeXchange with identifier PXD004059 (http://proteomecentral.proteomexchange.org/dataset/PXD004059).

Ion mobility-mass spectrometry reveals conformational flexibility in the deubiquitinating enzyme USP5.

Many proteins exhibit conformation flexibility as part of their biological function, whether through the presence of a series of well-defined states or by the existence of intrinsic disorder. Ion mobility spectrometry, in combination with MS (IM-MS), offers a rapid and sensitive means of probing ensembles of protein structures through measurement of gas-phase collisional cross sections. We have applied IM-MS analysis to the multidomain deubiquitinating enzyme ubiquitin specific protease 5 (USP5), which is believed to exhibit significant conformational flexibility. Native ESI-MS measurement of the 94-kDa USP5 revealed two distinct charge-state distributions: [M + 17H](+) to [M + 21H](+) and [M + 24H](+) to [M + 29H](+). The collisional cross sections of these ions revealed clear groupings of 52 ± 4 nm(2) for the lower charges and 66 ± 6 nm(2) for the higher charges. Molecular dynamics simulation of a compact form of USP5, based on a crystal structure, produced structures of 53-54 nm(2) following 2 ns in the gas phase, while simulation of an extended form (based on small-angle X-ray scattering data) led to structures of 64 nm(2). These data demonstrate that IM-MS is a valuable tool in studying proteins with different discrete conformational states.

Ubiquitin-binding domains: mechanisms of ubiquitin recognition and use as tools to investigate ubiquitin-modified proteomes.

Ubiquitin-binding domains (UBDs) are modular units found within ubiquitin-binding proteins that mediate the non-covalent recognition of (poly)ubiquitin modifications. A variety of mechanisms are employed in vivo to achieve polyubiquitin linkage and chain length selectivity by UBDs, the structural basis of which have in some instances been determined. Here, we review current knowledge related to ubiquitin recognition mechanisms at the molecular level and explore how such information has been exploited in the design and application of UBDs in isolation or artificially arranged in tandem as tools to investigate ubiquitin-modified proteomes. Specifically, we focus on the use of UBDs to directly purify or detect (poly)ubiquitin-modified proteins and more broadly for the targeted manipulation of ubiquitin-mediated processes, highlighting insights into ubiquitin signalling that have been provided.

Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase.

The clamp-loader complex plays a crucial role in DNA replication by loading the ß-clamp onto primed DNA to be used by the replicative polymerase. Relatively little is known about the stoichiometry, structure and assembly pathway of this complex, and how it interacts with the replicative helicase, in Gram-positive organisms. Analysis of full and partial complexes by mass spectrometry revealed that a hetero-pentameric τ3-δ-δ' Bacillus subtilis clamp-loader assembles via multiple pathways, which differ from those exhibited by the Gram-negative model Escherichia coli. Based on this information, a homology model of the B. subtilis τ3-δ-δ' complex was constructed, which revealed the spatial positioning of the full C-terminal τ domain. The structure of the δ subunit was determined by X-ray crystallography and shown to differ from that of E. coli in the nature of the amino acids comprising the τ and δ' binding regions. Most notably, the τ-δ interaction appears to be hydrophilic in nature compared with the hydrophobic interaction in E. coli. Finally, the interaction between τ3 and the replicative helicase DnaB was driven by ATP/Mg(2+) conformational changes in DnaB, and evidence is provided that hydrolysis of one ATP molecule by the DnaB hexamer is sufficient to stabilize its interaction with τ3.

Acyl-chain elongation drives ketosynthase substrate selectivity in trans-acyltransferase polyketide synthases.

Type I modular polyketide synthases (PKSs), which are responsible for the biosynthesis of many biologically active agents, possess a ketosynthase (KS) domain within each module to catalyze chain elongation. Acylation of the KS active site Cys residue is followed by transfer to malonyl-ACP to yield an extended ß-ketoacyl chain (ACP = acyl carrier protein). To date, the precise contribution of KS selectivity in controlling product fidelity has been unclear. Six KS domains from trans-acyltransferase (trans-AT) PKSs were subjected to a mass spectrometry based elongation assay, and higher substrate selectivity was identified for the elongating step than in preceding acylation. A close correspondence between the observed KS selectivity and that predicted by phylogenetic analysis was seen. These findings provide insights into the mechanism of KS selectivity in this important group of PKSs, can serve as guidance for engineering, and show that targeted mutagenesis can be used to expand the repertoire of acceptable substrates.

Native ESI-MS and Collision-Induced Unfolding (CIU) of the Complex between Bacterial Elongation Factor-Tu and the Antibiotic Enacyloxin IIa.

Collision-induced unfolding (CIU) of protein ions, monitored by ion mobility-mass spectrometry, can be used to assess the stability of their compact gas-phase fold and hence provide structural information. The bacterial elongation factor EF-Tu, a key protein for mRNA translation in prokaryotes and hence a promising antibiotic target, has been studied by CIU. The major [M + 12H]12+ ion of EF-Tu unfolded in collision with Ar atoms between 40 and 50 V, corresponding to an Elab energy of 480-500 eV. Binding of the cofactor analogue GDPNP and the antibiotic enacyloxin IIa stabilized the compact fold of EF-Tu, although dissociation of the latter from the complex diminished its stabilizing effect at higher collision energies. Molecular dynamics simulations of the [M + 12H]12+ EF-Tu ion showed similar qualitative behavior to the experimental results.

Expanding the diversity of chemical protein modification allows post-translational mimicry.

One of the most important current scientific paradoxes is the economy with which nature uses genes. In all higher animals studied, we have found many fewer genes than we would have previously expected. The functional outputs of the eventual products of genes seem to be far more complex than the more restricted blueprint. In higher organisms, the functions of many proteins are modulated by post-translational modifications (PTMs). These alterations of amino-acid side chains lead to higher structural and functional protein diversity and are, therefore, a leading contender for an explanation for this seeming incongruity. Natural protein production methods typically produce PTM mixtures within which function is difficult to dissect or control. Until now it has not been possible to access pure mimics of complex PTMs. Here we report a chemical tagging approach that enables the attachment of multiple modifications to bacterially expressed (bare) protein scaffolds: this approach allows reconstitution of functionally effective mimics of higher organism PTMs. By attaching appropriate modifications at suitable distances in the widely-used LacZ reporter enzyme scaffold, we created protein probes that included sensitive systems for detection of mammalian brain inflammation and disease. Through target synthesis of the desired modification, chemistry provides a structural precision and an ability to retool with a chosen PTM in a manner not available to other approaches. In this way, combining chemical control of PTM with readily available protein scaffolds provides a systematic platform for creating probes of protein-PTM interactions. We therefore anticipate that this ability to build model systems will allow some of this gene product complexity to be dissected, with the aim of eventually being able to completely duplicate the patterns of a particular protein's PTMs from an in vivo assay into an in vitro system.

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.

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.