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 semi-automated 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

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

Decoding Protein Gas‐Phase Stability with Alanine Scanning and Collision‐Induced Unfolding Ion Mobility Mass Spectrometry

Native mass spectrometry is a widely used tool in structural biology, providing information on protein structure and interactions through preservation of complexes in the gas phase. Herein, the importance of intramolecular non‐covalent interactions in the gas phase has been studied by alanine scanning and collision‐induced unfolding (CIU) ion mobility‐mass spectrometry. Mutation of specific polar and ionic residues on the surface of an acyl carrier protein (ACP) were found to destabilise the compact gas‐phase structure with mutants E31A, D32A, D41A and D65A being particularly destabilised. Molecular dynamics simulations of the ACP 7+ and 8+ ions showed extended intramolecular interactions, resulting from sidechain collapse of polar surface residues, which were confined to the gas phase and consistent with the CIU data. These findings provide evidence for the importance of specific ionic residues, and their interactions, in the maintenance of compact protein gas‐phase structure

Structural Insights into Dissimilatory Sulfite Reductases: Structure of Desulforubidin from Desulfomicrobium Norvegicum

Dissimilatory sulfite reductases (dSiRs) are crucial enzymes in bacterial sulfur-based energy metabolism, which are likely to have been present in some of the earliest life forms on Earth. Several classes of dSiRs have been proposed on the basis of different biochemical and spectroscopic properties, but it is not clear whether this corresponds to actual physiological or structural differences. Here, we describe the first structure of a dSiR from the desulforubidin class isolated from Desulfomicrobium norvegicum. The desulforubidin (Drub) structure is assembled as α2β2γ2, in which two DsrC proteins are bound to the core [DsrA]2[DsrB]2 unit, as reported for the desulfoviridin (Dvir) structure from Desulfovibrio vulgaris. Unlike Dvir, four sirohemes and eight [4Fe–4S] clusters are present in Drub. However, the structure indicates that only two of the Drub coupled siroheme-[4Fe–4S] cofactors are catalytically active. Mass spectrometry studies of purified Drub and Dvir show that both proteins present different oligomeric complex forms that bind two, one, or no DsrC proteins, providing an explanation for conflicting spectroscopic and biochemical results in the literature, and further indicating that DsrC is not a subunit of dSiR, but rather a protein with which it interacts

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)-β-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

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

Terpenes are widely used in nature for chemical communication, but our understanding of how these structurally diverse natural products are produced by insects is only now beginning to emerge. Males of the sandfly, Lutzomyia longipalpis, use terpene pheromones to lure females and other males to mating sites. This insect attracts considerable attention due to its role as a vector for the Leishmania parasite, which causes the neglected tropical disease leishmaniasis. In this study, a diterpene synthase that produces the pheromone component sobralene is identified, heterologously expressed and functionally characterized. This represents identification of a terpene synthase (TPS) from Lutzomyia and shows that insects are capable of biosynthesizing diterpenes. It offers the potential for sustainable production of this compound through biocatalysis

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

Design of nucleotide-mimetic and non-nucleotide inhibitors of the translation initiation factor eIF4E: Synthesis, structural and functional characterisation.

Eukaryotic translation initiation factor 4E (eIF4E) is considered as the corner stone in the cap-dependent translation initiation machinery. Its role is to recruit mRNA to the ribosome through recognition of the 5'-terminal mRNA cap structure (m7GpppN, where G is guanosine, N is any nucleotide). eIF4E is implicated in cell transformation, tumourigenesis, and angiogenesis by facilitating translation of oncogenic mRNAs; it is thus regarded as an attractive anticancer drug target. We have used two approaches to design cap-binding inhibitors of eIF4E by modifying the N7-substituent of m7GMP and replacing the phosphate group with isosteres such as squaramides, sulfonamides, and tetrazoles, as well as by structure-based virtual screening aimed at identifying non-nucleotide cap-binding antagonists. Phosphomimetic nucleotide derivatives and highly ranking virtual hits were evaluated in a series of in vitro and cell-based assays to identify the first non-nucleotide eIF4E cap-binding inhibitor with activities in cell-based assays, N-[(5,6-dihydro-6-oxo-1,3-dioxolo[4,5-g]quinolin-7-yl)methyl]-N'-(2-methyl-propyl)-N-(phenyl-methyl)thiourea (14), including down-regulation of oncogenic proteins and suppression of RNA incorporation into polysomes. Although we did not observe cellular activity with any of our modified m7GMP phosphate isostere compounds, we obtained X-ray crystallography structures of three such compounds in complex with eIF4E, 5'-deoxy-5'-(1,2-dioxo-3-hydroxycyclobut-3-en-4-yl)amino-N7-methyl-guanosine (4a), N7-3-chlorobenzyl-5'-deoxy-5'-(1,2-dioxo-3-hydroxy-cyclobut-3-en-4-yl)amino-guanosine (4f), and N7-benzyl-5'-deoxy-5'-(trifluoromethyl-sulfamoyl)guanosine (7a). Collectively, the data we present on structure-based design of eIF4E cap-binding inhibitors should facilitate the optimisation of such compounds as potential anticancer agents

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

Design of nucleotide-mimetic and non-nucleotide inhibitors of the translation initiation factor eIF4E: Synthesis, structural and functional characterisation.

Eukaryotic translation initiation factor 4E (eIF4E) is considered as the corner stone in the cap-dependent translation initiation machinery. Its role is to recruit mRNA to the ribosome through recognition of the 5'-terminal mRNA cap structure (m7GpppN, where G is guanosine, N is any nucleotide). eIF4E is implicated in cell transformation, tumourigenesis, and angiogenesis by facilitating translation of oncogenic mRNAs; it is thus regarded as an attractive anticancer drug target. We have used two approaches to design cap-binding inhibitors of eIF4E by modifying the N7-substituent of m7GMP and replacing the phosphate group with isosteres such as squaramides, sulfonamides, and tetrazoles, as well as by structure-based virtual screening aimed at identifying non-nucleotide cap-binding antagonists. Phosphomimetic nucleotide derivatives and highly ranking virtual hits were evaluated in a series of in vitro and cell-based assays to identify the first non-nucleotide eIF4E cap-binding inhibitor with activities in cell-based assays, N-[(5,6-dihydro-6-oxo-1,3-dioxolo[4,5-g]quinolin-7-yl)methyl]-N'-(2-methyl-propyl)-N-(phenyl-methyl)thiourea (14), including down-regulation of oncogenic proteins and suppression of RNA incorporation into polysomes. Although we did not observe cellular activity with any of our modified m7GMP phosphate isostere compounds, we obtained X-ray crystallography structures of three such compounds in complex with eIF4E, 5'-deoxy-5'-(1,2-dioxo-3-hydroxycyclobut-3-en-4-yl)amino-N7-methyl-guanosine (4a), N7-3-chlorobenzyl-5'-deoxy-5'-(1,2-dioxo-3-hydroxy-cyclobut-3-en-4-yl)amino-guanosine (4f), and N7-benzyl-5'-deoxy-5'-(trifluoromethyl-sulfamoyl)guanosine (7a). Collectively, the data we present on structure-based design of eIF4E cap-binding inhibitors should facilitate the optimisation of such compounds as potential anticancer agents