Biology subjects

Robinson, C. V.

Publications and source records attributed to Robinson, C. V..

45 records · Page 3Linked to original sources

bioRxiv2020

Correlating glycoforms of DC-SIGN with stability using a combination of enzymatic digestion and ion mobility MS

The immune scavenger protein DC-SIGN interacts with glycosylated proteins and has a putative role in facilitating viral infection. How these recognition events take place with different viruses is not clear and the effects of glycosylation on the folding and stability of DC-SIGN have not been reported. Here, we develop and apply a mass spectrometry-based approach to both uncover and characterise the effects of O-glycans on the stability of DC-SIGN. We first quantify the Core 1 & 2 O-glycan structures on the carbohydrate recognition and extracellular domains of the protein via sequential exoglycosidase sequencing. We then use ion mobility mass spectrometry to show how specific O-glycans, and/or single monosaccharide substitutions, alter both the overall collision cross section and the gas-phase stability of the glycoprotein isoforms of DC-SIGN. We find that rather than the mass or length of glycoprotein modifications, the stability of DC-SIGN is better correlated with the number of glycosylation sites. Collectively, our results exemplify a combined multi-dimensional MS approach, proficient in evaluating protein stability in response to both glycoprotein macro- and micro-heterogeneity and adding structural detail to the infection enhancer DC-SIGN.

biophysics↗

bioRxiv2020

Propylene glycol-based antifreeze as an effective preservative for DNA metabarcoding of benthic arthropods

Maintaining the integrity of DNA in bulk environmental samples from source to laboratory is crucial for capturing the true range of taxa present within an ecosystem. Preservation consideration of DNA is particularly important if samples are being collected in remote areas and by non-specialist citizen scientists in nationwide programs. Traditionally, absolute ethanol is used as the preferred preservative for environmental samples collected for downstream DNA metabarcoding analyses. However, transport, shipping and DNA extraction of samples preserved in ethanol is a lengthy procedure due to safety restrictions and the requirement of full ethanol evaporation prior to extraction. We examined the efficacy of an easily accessible, non-toxic propylene glycol-based antifreeze as an alternative to absolute ethanol for preserving macroinvertebrate DNA from bulk-benthos DNA samples. We tested the differences in both cytochrome oxidase I (COI) exact sequence variants (ESVs) and COI taxonomic orders detected in both ethanol and antifreeze samples using two processing methods (no evaporation of preservative versus full evaporation). In addition, we assessed the detection of families and genera within the Arthropoda phylum for preservative type, site and processing method. Our results suggest that antifreeze is a suitable alternative to ethanol, a greater global ESV richness reported for antifreeze samples. Additionally, a higher proportion of arthropod reads in ESVs were detected in antifreeze (average 69%) compared with ethanol (average 53%). Finally, antifreeze samples produced similar results for the different processing methods, whereas ethanol samples failed to produce similar results without prior evaporation. Although ethanol is currently widely used for DNA preservation, our results demonstrate that by using antifreeze, it is possible to achieve similar taxonomic coverage and community assemblages of bulk-benthos DNA samples for macroinvertebrates, with the added simplicity and shorter laboratory processing time achieved using an easily available, unregulated preservative.

genomics↗

bioRxiv2019

Pyocin S5 import into Pseudomonas aeruginosa reveals a generic mode of bacteriocin transport

Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen P. aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analysis and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked three helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli towards PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria.

biochemistry↗

bioRxiv2019

Spontaneous oligomerization of BAK/BAX is suppressed by hetero-dimerization with MCL-1

BCL-2 proteins control the intrinsic pathway of programmed cell death. Composed of anti- and pro-apoptotic members, their network of interactions forms a molecular switch that controls mitochondrial outer-membrane permeability. Apoptotic stimulation leads to BAK/BAX oligomerization and pore formation, yet the molecular details of this pivotal step remain poorly understood, and controversy persists regarding the activation mechanism. Here we use native mass spectrometry and kinetics to show that the homo-oligomerization of BAK and BAX is spontaneous in hydrophobic environments. This process is abrogated by hetero-dimerization of both BAK and BAX with the anti-apoptotic BCL-2 protein MCL-1. Pro-apoptotic BH3-only proteins disrupt these hetero-dimers by binding competitively to MCL-1, releasing BAK/BAX for homo-oligomerization. Thus, we infer that their oligomeric states are thermodynamically favored at the membrane. Our approach provides the framework for future quantitative biophysical characterizations of the BCL-2 network, and advances our molecular understanding of apoptosis.

biophysics↗

bioRxiv2019

Dynamic architecture of the Escherichia coli structural maintenance of chromosomes (SMC) complex, MukBEF

Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organise chromosomes, thereby facilitating chromosome segregation. They utilise cycles of ATP binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contacts. We have analysed changes in the architecture of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis. This builds upon previous work showing that MukF kleisin directs formation of a MukBEF tripartite ring as a consequence of functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively (Zawadzka et al., 2018). Using both model truncated substrates and complexes containing full length MukB, we now demonstrate formation of MukBEF dimers of dimers, dependent on MukF dimerization, MukB head-engagement and MukE, which plays an essential role in organizing MukBEF complexes.

biochemistry↗

bioRxiv2018

State-Dependent Lipid Interactions with the A2a Receptor Revealed by MD Simulations Using In Vivo-Mimetic Membranes

G protein-coupled receptors (GPCRs) are the largest family of integral membrane proteins and a major class of drug targets. Membranes are known to have modulatory effects on GPCRs via specific lipid interactions. However, the mechanisms of such modulations in cell membranes and how they influence GPCR functions remain unclear. Here we report coarse-grained MD simulations on the Adenosine A2a receptor embedded in an in vivo mimetic membrane model comprised of 10 different lipid species. Three conformational states of the receptor, i.e. the inactive state, the active state, and the active state with a mini-GS protein bound were simulated to study the impact of protein-lipid interactions on the receptor activation. The simulations revealed three specific lipids (GM3, cholesterol and PIP2) that form stable and preferential interactions with the receptor, differentiating these from bulk lipids such as PS, PE and PC. In total, nine specific lipid-binding sites were revealed. The strength of lipid interaction with these sites depends on the conformational state of the receptor, suggesting that these lipids may regulate the conformational dynamics of the receptor. In particular, we revealed a dual role of PIP2 in promoting A2aR activation, which involves stabilization of both the characteristic outward tilt of helix TM6 within receptor and also the association of A2aR and mini-Gs when the activated complex forms. Structural comparisons suggested that PIP2 may facilitate G activation. Our results reveal likely allosteric effects of bound lipids in regulating the functional behaviour of GPCRs, providing a springboard for design of allosteric modulators of these biomedically important receptors.

biophysics↗

bioRxiv2018

Structures of DPAGT1 explain glycosylation disease mechanisms and advance TB antibiotic design

Protein glycosylation is a widespread post-translational modification. The first committed step to the lipid-linked glycan used for this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and congenital disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use as antibiotics. However, little is known about the mechanism or the effects of disease-associated mutations in this essential enzyme. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (and thus cause disease) and allow design of non-toxic lipid-altered tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo thereby providing a promising new class of antimicrobial drug.\n\nHighlightsO_LIStructures of DPAGT1 with UDP-GlcNAc and tunicamycin reveal mechanisms of catalysis\nC_LIO_LIDPAGT1 mutants in patients with glycosylation disorders modulate DPAGT1 activity\nC_LIO_LIStructures, kinetics and biosynthesis reveal role of lipid in tunicamycin\nC_LIO_LILipid-altered, tunicamycin analogues give non-toxic antibiotics against TB\nC_LI

biochemistry↗

bioRxiv2018

Structure of the Core of the Type Three Secretion System ExportApparatus

Export of proteins through type three secretion systems is critical for bacterial motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP/FliQ/FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. We here present the structure of this complex at 4.2 [A] by cryo-electron microscopy. None of the subunits adopt canonical integral membrane protein topologies and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems localize the export gate as a core component of the periplasmic portion of the machinery, and cross-linking experiments confirm this observation. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.\n\nOne Sentence SummaryThe core of the T3SS export gate forms a supra-membrane helical assembly

microbiology↗

bioRxiv2017

Architecture of TAF11/TAF13/TBP complex suggests novel regulatory state in General Transcription Factor TFIID function

General transcription factor TFIID is a key component of RNA polymerase II transcription initiation. Human TFIID is a megadalton-sized complex comprising TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). TBP binds to core promoter DNA, recognizing the TATA-box. We identified a ternary complex formed by TBP and the histone fold (HF) domain-containing TFIID subunits TAF11 and TAF13. We demonstrate that TAF11/TAF13 competes for TBP binding with TATA-box DNA, and also with the N-terminal domain of TAF1 previously implicated in TATA-box mimicry. In an integrative approach combining crystal coordinates, biochemical analyses and data from cross-linking mass-spectrometry (CLMS), we determine the architecture of the TAF11/TAF13/TBP complex, revealing TAF11/TAF13 interaction with the DNA binding surface of TBP. We identify a highly conserved C-terminal TBP-binding domain (CTID) in TAF13 which is essential for supporting cell growth. Our results thus have implications for cellular TFIID assembly and suggest a novel regulatory state for TFIID function.

biochemistry↗