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Secretory preproteins of the Sec pathway are targeted post-translationally and cross cellular membranes through translocases. During cytoplasmic transit, mature domains remain non-folded for translocase recognition/translocation. After translocation and signal peptide cleavage, mature domains fold to native states in the bacterial periplasm or traffic further. We sought the structural basis for delayed mature domain folding and how signal peptides regulate it. We compared how evolution diversified a periplasmic peptidyl-prolyl isomerase PpiA mature domain from its structural cytoplasmic PpiB twin. Global and local hydrogen-deuterium exchange mass spectrometry showed that PpiA is a slower folder. We defined at near-residue resolution hierarchical folding initiated by similar foldons in the twins, at different order and rates. PpiA folding is delayed by less hydrophobic native contacts, frustrated residues and a β-turn in the earliest foldon and by signal peptide-mediated disruption of foldon hierarchy. When selected PpiA residues and/or its signal peptide were grafted onto PpiB, they converted it into a slow folder with enhanced in vivo secretion. These structural adaptations in a secretory protein facilitate trafficking. ispartof: EMBO JOURNAL vol:41 issue:23 ispartof: location:England status: published

Neuronal activity causes use-dependent decline in protein function. However, it is unclear how this is coupled to local quality control mechanisms. We show in Drosophila that the endocytic protein Endophilin-A (EndoA) connects activity-induced calcium influx to synaptic autophagy and neuronal survival in a Parkinson disease-relevant fashion. Mutations in the disordered loop, including a Parkinson disease-risk mutation, render EndoA insensitive to neuronal stimulation and affect protein dynamics: when EndoA is more flexible, its mobility in membrane nanodomains increases, making it available for autophagosome formation. Conversely, when EndoA is more rigid, its mobility reduces, blocking stimulation-induced autophagy. Balanced stimulation-induced autophagy is required for dopagminergic neuron survival, and a variant in the human ENDOA1 disordered loop conferring risk to Parkinson disease also blocks nanodomain protein mobility and autophagy both in vivo and in human-induced dopaminergic neurons. Thus, we reveal a mechanism that neurons use to connect neuronal activity to local autophagy and that is critical for neuronal survival. ispartof: NEURON vol:111 issue:9 pages:1402-+ ispartof: location:United States status: published

Sec secretory proteins are distinguished from cytoplasmic ones by N-terminal signal peptides with multiple roles during post-translational translocation. They contribute to preprotein targeting to the translocase by slowing down folding, binding receptors and triggering secretion. While signal peptides get cleaved after translocation, mature domains traffic further and/or fold into functional states. How signal peptides delay folding temporarily, to keep mature domains translocation-competent, remains unclear. We previously reported that the foldon landscape of the periplasmic prolyl-peptidyl isomerase is altered by its signal peptide and mature domain features. Here, we reveal that the dynamics of signal peptides and mature domains crosstalk. This involves the signal peptide's hydrophobic helical core, the short unstructured connector to the mature domain and the flexible rheostat at the mature domain N-terminus. Through this cis mechanism the signal peptide delays the formation of early initial foldons thus altering their hierarchy and delaying mature domain folding. We propose that sequence elements outside a protein's native core exploit their structural dynamics to influence the folding landscape.

Neuronal activity and neurotransmitter release cause use-dependent decline in protein function. However, it is unclear how this is coupled to local protein turnover and quality control mechanisms. Here we show that the endocytic protein Endophilin-A (EndoA/ENDOA1) couples activity-induced calcium influx to synaptic autophagy and neuronal survival. We identify single mutations in the EndoA flexible region that either increases EndoA diffusion and promotes autophagosome formation in the absence of calcium, or immobilizes EndoA and blocks autophagy, even in the presence of calcium. Hence, the EndoA flexible region is a switch that responds to calcium, regulating EndoA nanoscale synaptic organization and association with autophagosomes driving their formation. Interestingly, a pathogenic variant in the human ENDOA1 variable region that confers risk to Parkinson’s disease (PD), also confines ENDOA1 to the synaptic plasma membrane and equally blocks autophagy in flies in vivo and in induced human neurons. Thus, our work reveals a mechanism neurons use to connect neuronal activity to local protein turnover by autophagy, which is critical for neuronal survival.

Secretory preproteins of the Sec pathway bear signal peptides and are targeted post-translationally to cross the plasma membrane or ER through translocases. After translocation and signal peptide cleavage, mature domains fold to native states in the bacterial periplasm or after further trafficking. During cytoplasmic transit, mature domains must remain non-folded for translocase recognition and translocation. Here, we sought the structural basis for the delayed folding mechanism of mature domains and how this is regulated by signal peptides. To address this, we compared how evolution diversified a periplasmic peptidyl-prolyl isomerase PpiA mature domain from its structural twin cytoplasmic PpiB. Using global and local hydrogen deuterium exchange mass spectrometry we showed that PpiA is a slower folder. We defined at near-residue resolution hierarchical folding initiated by similar foldons in the twins, that displayed different order and rates. Folding is delayed in PpiA by less hydrophobic/bulky native contacts, frustrated residues and a critical β -turn in the early folding region and by signal peptide-driven disorder, which disrupts foldon hierarchy. When selected PpiA residues and its signal peptide were grafted onto PpiB they converted it into a slow folder with enhanced in vivo secretion. These data reveal the structural basis of non-folding in a secretory protein, that allows its trafficking.

Significance Proteins conduct numerous complex biological functions by use of tailored structural dynamics. The molecular details of how these emerged from ancestral peptides remains mysterious. How does nature utilize the same repertoire of folds to diversify function? To shed light on this, we analyzed bilobed proteins with a common structural core, which is spread throughout the tree of life and is involved in diverse biological functions such as transcription, enzymatic catalysis, membrane transport, and signaling. We show here that the structural dynamics of the structural core differentiate predominantly via terminal additions during a long-period evolution. This diversifies substrate specificity and, ultimately, biological function., Novel biophysical tools allow the structural dynamics of proteins and the regulation of such dynamics by binding partners to be explored in unprecedented detail. Although this has provided critical insights into protein function, the means by which structural dynamics direct protein evolution remain poorly understood. Here, we investigated how proteins with a bilobed structure, composed of two related domains from the periplasmic-binding protein–like II domain family, have undergone divergent evolution, leading to adaptation of their structural dynamics. We performed a structural analysis on ∼600 bilobed proteins with a common primordial structural core, which we complemented with biophysical studies to explore the structural dynamics of selected examples by single-molecule Förster resonance energy transfer and Hydrogen–Deuterium exchange mass spectrometry. We show that evolutionary modifications of the structural core, largely at its termini, enable distinct structural dynamics, allowing the diversification of these proteins into transcription factors, enzymes, and extracytoplasmic transport-related proteins. Structural embellishments of the core created interdomain interactions that stabilized structural states, reshaping the active site geometry, and ultimately altered substrate specificity. Our findings reveal an as-yet-unrecognized mechanism for the emergence of functional promiscuity during long periods of evolution and are applicable to a large number of domain architectures.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful technique to monitor protein intrinsic dynamics. The technique provides high-resolution information on how protein intrinsic dynamics are altered in response to biological signals, such as ligand binding, oligomerization, or allosteric networks. However, identification, interpretation, and visualization of such events from HDX-MS data sets is challenging as these data sets consist of many individual data points collected across peptides, time points, and experimental conditions. Here, we present PyHDX, an open-source Python package and webserver, that allows the user to batch extract the universal quantity Gibbs free energy at residue levels over multiple protein conditions and homologues. The output is directly visualized on a linear map or 3D structures or is exported as .csv files or PyMOL scripts. ispartof: ANALYTICAL CHEMISTRY vol:93 issue:38 pages:12840-12847 ispartof: location:United States status: published

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca dynamics by controlling IP receptor (IPR) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IPRs and preventing pro-apoptotic Ca release and Bcl-xL sensitizing IPRs to low [IP] and promoting pro-survival Ca oscillations. We here demonstrate that Bcl-xL too inhibits IPR-mediated Ca release by interacting with the same IPR regions as Bcl-2. Via in silico superposition, we previously found that the residue K87 of Bcl-xL spatially resembled K17 of Bcl-2, a residue critical for Bcl-2’s IPR-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IPR and abrogated Bcl-xL’s inhibitory effect on IPRs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xL, suppressed IPR single-channel openings stimulated by sub-maximal and threshold [IP]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IPRs contributes to its anti-apoptotic properties against Ca-driven apoptosis. Staurosporine (STS) elicits long-lasting Ca elevations in wild-type but not in IPR-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca signals and cell death, while Bcl-xL was much less effective in doing so. In the absence of IPRs, Bcl-xL and Bcl-xL were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IPR activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IPR-mediated Ca release and increased the sensitivity towards STS, without altering the ER Ca content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca-signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IPR-mediated Ca release and IPR-driven cell death. Our work further underpins that IPR inhibition is an integral part of Bcl-xL’s anti-apoptotic function., The work was supported by Grants from the Research Foundation—Flanders (FWO) (G.0901.18N), by the Research Council of the KU Leuven (OT14/101, C14/19/099, C14/19/101, and AKUL/19/34), the Interuniversity Attraction Poles Program (Belgian Science Policy; IAP-P7/13), the Central European Leuven Strategic Alliance (CELSA/18/040), and the Canadian Institutes Health Research (FDN143312). NR and HI are recipient of postdoctoral fellowships of the FWO; HI obtained a travel grant from the FWO to perform work in DIY’s laboratory. GB, JBP and DIY are part of the FWO Scientific Research Network CaSign (W0.019.17N). Work in DIY’s lab is supported by NIH (NIDCR) grant DE014756. DWA holds the Tier 1 Canada Research Chair in Membrane Biogenesis. The Switch laboratory was supported by the Flanders institute for Biotechnology (VIB), the University of Leuven, the Fund for Scientific Research Flanders (Hercules Foundation/FWO AKUL/15/34—G0H1716N). NL is funded by the Stichting Alzheimer Onderzoek (SAO-FRA 2020/0013) and is recipient of FWO postdoctoral fellowships (12P0919N and 12P0922N to NL).

The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. The Sec preprotein export pathway is an attractive potential alternative target. It is essential for bacterial viability and includes components that are absent from eukaryotes. Here we used a new high throughputin vivoscreen based on the secretion and activity of alkaline phosphatase (PhoA), a Sec-dependent secreted enzyme that becomes active in the periplasm. The assay was optimized for a luminescence-based substrate and was used to screen a ~240K small molecule compound library. After hit confirmation and analoging, fourteen HTS secretion inhibitors (HSI), belonging to 8 structural classes, were identified (IC50−and Gram+bacterial species (including those from the WHO top pathogens list). Seven of them, HSI#6, 9; HSI#1, 5, 10 and HSI#12, 14 representing three structural families were microbicidals. HSI#6 was the most potent (IC50of 0.4-8.7 μM), against 13 species of both Gram−and Gram+bacteria. HSI#1, 5, 9 and 10 inhibited viability of Gram+bacteria with IC50~6.9-77.8 μM. HSI#9, 12 and 14 inhibited viability ofE. colistrains with IC50E. colistrain missing TolC to improve permeability with IC504-14 μM, indicating their inability to penetrate the outer membrane.In vitroassays revealed that antimicrobial activity was not related to inhibition of the SecA component of the translocase and hence HSI molecules may target new unknown components that affect secretion. The results provide proof of principle for our approach, and new starting compounds for optimization.

The type 3 secretion system is essential for pathogenesis of several human and animal Gram-negative bacterial pathogens. The T3SS comprises a transmembrane injectisome, providing a conduit from the bacterial cytoplasm to the host cell cytoplasm for the direct delivery of effectors (including toxins). Functional studies of T3SS commonly monitor the extracellular secretion of proteins by SDS-PAGE and western blot analysis, which are slow and semi-quantitative in nature. Here, we describe an enzymatic reporter-based quantitative and rapid in vivo assay for T3SS secretion studies in enteropathogenic E. coli (EPEC). The assay monitors the secretion of the fusion protein SctA-PhoA through the injectisome based on a colorimetric assay that quantifies the activity of alkaline phosphatase. We validated the usage of this reporter system by following the secretion in the absence of various injectisome components, including domains of the gatekeeper essential for T3SS function. This platform can now be used for the isolation of mutations, functional analysis and anti-virulence compound screening. ispartof: MICROORGANISMS vol:8 issue:11 ispartof: location:Switzerland status: published

Hydrogen Deuterium Exchange Mass Spectrometry is a powerful monitor of protein intrinsic dynamics, yet the interpretation, visualization and cross-comparison of HDX-MS datasets is challenging. Here we present PyHDX, an open-source python package and web server, that batch-extracts the universal quantity Gibbs free energy at residue level over multiple protein conditions and homologues. ΔG values relate to protein normal modes and together provide a universal measure of protein flexibility.AvailabilityPyHDX source code is released under the MIT license and can be accessed on GitHub.

Bacterial secretory preproteins are translocated across the inner membrane post-translationally by the SecYEG-SecA translocase. Mature domain features and signal peptides maintain preproteins in kinetically trapped, largely soluble, folding intermediates. Some aggregation-prone preproteins require chaperones, like trigger factor (TF) and SecB, for solubility and/or targeting. TF antagonizes the contribution of SecB to secretion by an unknown molecular mechanism. We reconstituted this interaction in vitro and studied targeting and secretion of the model preprotein pro-OmpA. TF and SecB display distinct, unsuspected roles in secretion. Tightly associating TF:pro-OmpA targets the translocase at SecA, but TF prevents pro-OmpA secretion. In solution, SecB binds TF:pro-OmpA with high affinity. At the membrane, when bound to the SecA C-tail, SecB increases TF and TF:pro-OmpA affinities for the translocase and allows pro-OmpA to resume translocation. Our data reveal that TF, a main cytoplasmic folding pathway chaperone, is also a bona fide post-translational secretory chaperone that directly interacts with both SecB and the translocase to mediate regulated protein secretion. Thus, TF links the cytoplasmic folding and secretion chaperone networks. ispartof: EMBO REPORTS vol:21 issue:6 ispartof: location:England status: published

Type III protein secretion is widespread in Gram-negative pathogens. It comprises the injectisome with a surface-exposed needle and an inner membrane translocase. The translocase contains the SctRSTU export channel enveloped by the export gate subunit SctV that binds chaperone/exported clients and forms a putative ante- chamber. We probed the assembly, function, structure and dynamics of SctV from enteropathogenic E.coli (EPEC). In both EPEC and E.coli lab strains, SctV forms peripheral oligomeric clusters that are detergent-extracted as homo-nonamers. Membrane-embedded SctV9 is necessary and sufficient to act as a receptor for different chaperone/exported protein pairs with distinct C-domain binding sites that are essential for secretion. Negative staining electron microscopy revealed that peptidisc-reconstituted His-SctV9 forms a tripartite particle of ∼22 nm with a N- terminal domain connected by a short linker to a C-domain ring structure with a ∼5 nm-wide inner opening. The isolated C-domain ring was resolved with cryo-EM at 3.1 Å and structurally compared to other SctV homologues. Its four sub-domains undergo a three-stage “pinching” motion. Hydrogen-deuterium exchange mass spectrometry revealed this to involve dynamic and rigid hinges and a hyper-flexible sub-domain that flips out of the ring periphery and binds chaperones on and between adjacent protomers. These motions are coincident with pore surface and ring entry mouth local conformational changes that are also modulated by the ATPase inner stalk. We propose a model that the intrinsic dynamics of the SctV protomer are modulated by chaperones and the ATPase and could affect allosterically the other subunits of the nonameric ring during secretion.

SummaryThe cytoplasmic ATPase SecA and the membrane-embedded SecYEG channel assemble to form the functional Sec translocase. How this interaction primes and catalytically activates the translocase remains unclear. We now show that priming exploits a sophisticated nexus of intrinsic dynamics in SecA. Using atomistic simulations, single molecule FRET and hydrogen/deuterium exchange mass spectrometry we reveal multiple distributed dynamic islands that cross-talk with domain and quaternary motions. These dynamic elements are highly conserved and essential for function. Central to the nexus is a slender Stem through which, motions in the helicase ATPase domain of SecA biases how the preprotein binding domain rotates between open-closed clamping states. Multi-tier dynamics are enabled by an H-bonded framework covering most of the SecA structure and allowing conformational alterations with minimal energy inputs. As a result, dimerization, the channel and nucleotides select pre-existing conformations, and alter local dynamics to restrict or promote catalytic activity and clamp motions. These events prime the translocase for high affinity reception of non-folded preprotein clients. Such dynamics nexuses are likely universal and essential in multi-liganded protein machines.

B-cell lymphoma 2 (Bcl-2) protein is the archetype apoptosis suppressor protein. The N-terminal Bcl-2-homology 4 (BH4) domain of Bcl-2 is required for the anti-apoptotic function of this protein at the mitochondria and endoplasmic reticulum (ER). The involvement of the BH4 domain in Bcl-2's anti-apoptotic functions has been proposed based on Gly-based substitutions of the Ile14/Val15 amino acids, two hydrophobic residues located in the centre of Bcl-2's BH4 domain. Following this strategy, we recently showed that a BH4-domain-derived peptide in which Ile14 and Val15 have been replaced by Gly residues, was unable to dampen pro-apoptotic Ca(2+) -release events from the ER. Here, we investigated the impact of these mutations on the overall structure, stability and function of full-length Bcl-2 as a regulator of Ca(2+) signalling and cell death. Our results indicate that full-length Bcl-2 I14G/V15G, in contrast to wild-type Bcl-2, (i) displayed severely reduced structural stability and a shortened protein half-life; (ii) failed to interact with BAX, to inhibit the inositol 1,4,5-trisphosphate receptor (IP3 R) and to protect against Ca(2+) -mediated apoptosis. We conclude that the hydrophobic face of Bcl-2's BH4 domain (Ile14, Val15) is an important structural regulatory element by affecting protein stability and turnover, thereby likely reducing Bcl-2's ability to modulate the function of its targets, like IP3 R and BAX. Therefore, Bcl-2 structure/function studies require pre-emptive and reliable determination of protein stability upon introduction of point mutations at the level of the BH4 domain. This article is protected by copyright. All rights reserved. ispartof: FEBS Journal vol:285 issue:1 pages:127-145 ispartof: location:England status: published

The inner membrane of Gram-negative bacteria is a ~6 nm thick phospholipid bilayer. It forms a semi-permeable barrier between the cytoplasm and periplasm allowing only regulated export and import of ions, sugar polymers, DNA and proteins. Inner membrane proteins, embedded via hydrophobic transmembrane α-helices, play an essential role in this regulated trafficking: they mediate insertion into the membrane (insertases) or complete crossing of the membrane (translocases) or both. The Gram-negative inner membrane is equipped with a variety of different insertases and translocases. Many of them are specialized, taking care of the export of only a few protein substrates, while others have more general roles. Here, we focus on the three general export/insertion pathways, the secretory (Sec) pathway, YidC and the twin-arginine translocation (TAT) pathway, focusing closely on the Escherichia coli (E. coli) paradigm. We only briefly mention dedicated export pathways found in different Gram-negative bacteria. The Sec system deals with the majority of exported proteins and functions both as a translocase for secretory proteins and an insertase for membrane proteins. The insertase YidC assists the Sec system or operates independently on membrane protein clients. Sec and YidC, in common with most export pathways, require their protein clients to be in soluble non-folded states to fit through the translocation channels and grooves. The TAT pathway is an exception, as it translocates folded proteins, some loaded with prosthetic groups.

More than a third of all bacterial polypeptides, comprising the 'exportome', are transported to extracytoplasmic locations. Most of the exportome is targeted and inserts into ('membranome') or crosses ('secretome') the plasma membrane. The membranome and secretome use distinct targeting signals and factors, and driving forces, but both use the ubiquitous and essential Sec translocase and its SecYEG protein-conducting channel. Membranome export is co-translational and uses highly hydrophobic N-terminal signal anchor sequences recognized by the signal recognition particle on the ribosome, that also targets C-tail anchor sequences. Translating ribosomes drive movement of these polypeptides through the lateral gate of SecY into the inner membrane. On the other hand, secretome export is post-translational and carries two types of targeting signals: cleavable N-terminal signal peptides and multiple short hydrophobic targeting signals in their mature domains. Secretome proteins remain translocation competent due to occupying loosely folded to completely non-folded states during targeting. This is accomplished mainly by the intrinsic properties of mature domains and assisted by signal peptides and/or chaperones. Secretome proteins bind to the dimeric SecA subunit of the translocase. SecA converts from a dimeric preprotein receptor to a monomeric ATPase motor and drives vectorial crossing of chains through SecY aided by the proton motive force. Signal peptides are removed by signal peptidases and translocated chains fold or follow subsequent trafficking.

Protein secretion is a central biological process in all organisms. Most studies dissecting bacterial secretion mechanisms have focused on Gram-negative cell envelopes such as that of Escherichia coli However, proteomics analyses in Gram negatives is hampered by their outer membrane. Here we studied protein secretion in the Gram-positive bacterium Streptomyces lividans TK24, in which most of the secretome is released in the growth medium. We monitored changes of the secretome as a function of growth phase and medium. We determined distinct protein classes of "house-keeping" secreted proteins that do not change their appearance or abundance in the various media and growth phases. These comprise mainly enzymes involved in cell wall maintenance and basic transport. In addition, we detected significant abundance and content changes to a sub-set of the proteome, as a function of growth in the different media. These did not depend on the media being minimal or rich. Transcriptional regulation but not changes in export machinery components can explain some of these changes. However, additional downstream mechanisms must be important for selective secretome funneling. These observations lay the foundations of using S. lividans as a model organism to study how metabolism is linked to optimal secretion and help develop rational optimization of heterologous protein production. ispartof: MOLECULAR & CELLULAR PROTEOMICS vol:18 issue:3 pages:423-436 ispartof: location:United States status: published

Bcl-2 is an important regulator of inositol 1,4,5-trisphosphate receptor (IP3R) activity and, thus of Ca2+ release from internal stores and associated processes, including cellular proliferation and death. The presence of multiple regulatory domains in both proteins suggests a complex interaction. Earlier reports have shown the involvement of both the BH4 and the transmembrane domains of Bcl-2 in regulating IP3R activity, while the Bcl-2 hydrophobic cleft was associated primarily with its anti-apoptotic, IP3R-independent, action at the mitochondria. Here, we show that occlusion of Bcl-2’s hydrophobic cleft by the drug ABT-199 finely modulates IP3R gating in the low open probability regime, characteristic of the basal IP3R activity in non-excited cells. Complementary MD simulations allowed us to propose a model of this modulation, involving an allosteric interaction with the BH4 domain on the opposite side of Bcl-2.

SecA converts ATP energy to protein translocation work. Together with the membrane-embedded SecY channel it forms the bacterial protein translocase. How secretory proteins bind to SecA and drive conformational cascades to promote their secretion remains unknown. To address this, we focus on the preprotein binding domain (PBD) of SecA. PBD crystalizes in three distinct states, swiveling around its narrow stem. Here, we examined whether PBD displays intrinsic dynamics in solution using single-molecule Forster resonance energy transfer (smFRET). Unique cysteinyl pairs on PBD and apposed domains were labeled with donor/acceptor dyes. Derivatives were analyzed using pulsed interleaved excitation and multi-parameter fluorescence detection. The PBD undergoes significant rotational motions, occupying at least three distinct states in dimeric and four in monomeric soluble SecA. Nucleotides do not affect smFRET-detectable PBD dynamics. These findings lay the foundations for single-molecule dissection of translocase mechanics and suggest models for possible PBD involvement during catalysis. We are grateful to G. Gouridis (Rega Institute, KU Leuven) for discussions. Our research was funded through the Research Foundation Flanders (FWO), grant #G.0B49.15 (to J. Hofkens, J. Hendrix, and S.K.), grants #G0683.15 and #G0A5817.N (to J. Hofkens), large infrastructure grant ZW15_09 GOH6316N (to J. Hofkens and J. Hendrix); grant #G0C6814N RiMembR (to A.E.); the Flemish government through long-term structural funding Methusalem CASAS2, grant #Meth/15/04 (to J. Hofkens); the FWO/F.R.S. - FNRS Excellence of Science-EOS program grant #30550343 (to A.E.); the KU Leuven grant C14/16/053 (to J. Hendrix), the EU (FP7 KBBE. 2013.3.6-02: Synthetic Biology towards applications; #613877 StrepSynth to A.E.), and RUN (#RUN/16/001 KU Leuven to A.E.). N.V. acknowledges the Agency for Innovation by Science and Technology in Flanders (IWT) for a doctoral scholarship. V.Z. is an FWO postdoctoral fellow.

The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. Protein export pathways are attractive potential targets. The Sec pathway is essential for bacterial viability and includes components that are absent from eukaryotes. Here, we used a new high-throughput in vivo screen based on the secretion and activity of alkaline phosphatase (PhoA), a Sec-dependent secreted enzyme that becomes active in the periplasm. The assay was optimized for a luminescence-based substrate and was used to screen a ~240K small molecule compound library. After hit confirmation and analoging, 14 HTS secretion inhibitors (HSI), belonging to eight structural classes, were identified with IC50 &lt, 60 µM. The inhibitors were evaluated as antibacterials against 19 Gram-negative and Gram-positive bacterial species (including those from the WHO’s top pathogens list). Seven of them—HSI#6, 9, HSI#1, 5, 10, and HSI#12, 14—representing three structural families, were bacteriocidal. HSI#6 was the most potent hit against 13 species of both Gram-negative and Gram-positive bacteria with IC50 of 0.4 to 8.7 μM. HSI#1, 5, 9 and 10 inhibited the viability of Gram-positive bacteria with IC50 ~6.9–77.8 μM. HSI#9, 12, and 14 inhibited the viability of E. coli strains with IC50 &lt, 65 μM. Moreover, HSI#1, 5 and 10 inhibited the viability of an E. coli strain missing TolC to improve permeability with IC50 4 to 14 μM, indicating their inability to penetrate the outer membrane. The antimicrobial activity was not related to the inhibition of the SecA component of the translocase in vitro, and hence, HSI molecules may target new unknown components that directly or indirectly affect protein secretion. The results provided proof of the principle that the new broad HTS approach can yield attractive nanomolar inhibitors that have potential as new starting compounds for optimization to derive potential antibiotics.

The general secretory (Sec) pathway comprises an essential, ubiquitous and universal export machinery for most proteins that integrate into, or translocate through, the plasma membrane. Sec exportome polypeptides are synthesized as pre-proteins that have cleavable signal peptides fused to the exported mature domains. Recent advances have re-evaluated the interaction networks of pre-proteins with chaperones that are involved in pre-protein targeting from the ribosome to the SecYEG channel and have identified conformational signals as checkpoints for high-fidelity targeting and translocation. The recent structural and mechanistic insights into the channel and its ATPase motor SecA are important steps towards the elucidation of the allosteric crosstalk that mediates secretion. In this Review, we discuss recent biochemical, structural and mechanistic insights into the consecutive steps of the Sec pathway - sorting and targeting, translocation and release - in both co-translational and post-translational modes of export. The architecture and conformational dynamics of the SecYEG channel and its regulation by ribosomes, SecA and pre-proteins are highlighted. Moreover, we present conceptual models of the mechanisms and energetics of the Sec-pathway dependent secretion process in bacteria.

Bacteria are of tremendous importance in the pharma- and bio-industry as producers of a broad range of economically interesting metabolites and proteins. Gram-positive bacteria are valuable hosts for the production of heterologous proteins for obvious reasons. Contrary to Gram-negative bacteria, Gram-positive bacteria release their secreted proteins immediately into the spent culture broth as they are not hindered by an outer membrane. Secretory protein production also avoids the formation of inclusion bodies, hence facilitating downstream processing. Eight protein secretion pathways have been described in Gram-positive bacteria, but solely the general secretion or Sec pathway and, to a lesser extent, the Twin-arginine pathway, are used for the recombinant protein production. This process is not always successful, but might be hampered by inefficient secretion, misfolding of the recombinant protein, its degradation by proteases and metabolic burden by the host hindering proper growth and diminishing product yield. In this review, the different protein export avenues will be briefly discussed, and the potential means to optimize protein secretion and yields for the Streptomyces lividans model presented. The proposed approaches are largely applicable for other Streptomyces host systems.

Background Members of the genus Streptomyces are Gram-positive bacteria that are used as important cell factories to produce secondary metabolites and secrete heterologous proteins. They possess some of the largest bacterial genomes and thus proteomes. Understanding their complex proteomes and metabolic regulation will improve any genetic engineering approach. Results Here, we performed a comprehensive annotation of the subcellular localization of the proteome of Streptomyces lividans TK24 and developed the Subcellular Topology of Polypeptides in Streptomyces database (SToPSdb) to make this information widely accessible. We first introduced a uniform, improved nomenclature that re-annotated the names of ~ 4000 proteins based on functional and structural information. Then protein localization was assigned de novo using prediction tools and edited by manual curation for 7494 proteins, including information for 183 proteins that resulted from a recent genome re-annotation and are not available in current databases. The S. lividans proteome was also linked with those of other model bacterial strains including Streptomyces coelicolor A3(2) and Escherichia coli K-12, based on protein homology, and can be accessed through an open web interface. Finally, experimental data derived from proteomics experiments have been incorporated and provide validation for protein existence or topology for 579 proteins. Proteomics also reveals proteins released from vesicles that bleb off the membrane. All export systems known in S. lividans are also presented and exported proteins assigned export routes, where known. Conclusions SToPSdb provides an updated and comprehensive protein localization annotation resource for S. lividans and other streptomycetes. It forms the basis for future linking to databases containing experimental data of proteomics, genomics and metabolomics studies for this organism. Electronic supplementary material The online version of this article (10.1186/s12934-018-0892-0) contains supplementary material, which is available to authorized users.

The type 3 secretion system (T3SS) is a protein export pathway common to Gram-negative pathogens. It comprises a trans-envelope syringe, the injectisome, with a cytoplasm-facing translocase channel. In enteropathogenic Escherichia coli, exported substrates are chaperone-delivered to the major translocase component, EscV, and cross the membrane in strict hierarchical manner, e.g. first 'translocators', then 'effectors'. The in vitro dissection of the T3SS and the determination of its structure are hampered by the low numbers of the injectisomes per cell. We have now defined an optimal M9 minimal medium and established that the per transcriptional regulator enhances the number of filamented cells, the number of injectisomes per cell and the secretion of T3S substrates. Our findings provide a valuable tool for further biochemical and biophysical analysis of the T3SS and suggest that additional improvement to maximize injectisome production is possible in future efforts.

Searching for new antiviral agents, we focused our interest on the influenza PA-Nter endonuclease. Therefore, we developed a three-dimensional pharmacophore model which contains the binding features addressed to the metal-chelating active site. The obtained hypothesis has been fruitfully employed to select three "hit compounds" through an in silico screening campaign on our in-house database of small molecules. We studied the binding poses of these hit compounds using molecular docking, and subjected them to an enzymatic assay with recombinant PA-Nter endonuclease. Compound 20 proved the most active inhibitor of the endonucleolytic cleavage reaction, with an IC50 value of 12 μM. ispartof: BIOORGANIC & MEDICINAL CHEMISTRY vol:26 issue:15 pages:4544-4550 ispartof: location:England status: published

Background The Gram-positive Streptomyces lividans TK24 is an attractive host for heterologous protein production because of its high capability to secrete proteins—which favors correct folding and facilitates downstream processing—as well as its acceptance of methylated DNA and its low endogeneous protease activity. However, current inconsistencies in protein yields urge for a deeper understanding of the burden of heterologous protein production on the cell. In the current study, transcriptomics and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{13}\hbox {C}$$\end{document}13C-based fluxomics were exploited to uncover gene expression and metabolic flux changes associated with heterologous protein production. The Rhodothermus marinus thermostable cellulase A (CelA)—previously shown to be successfully overexpressed in S. lividans—was taken as an example protein. Results RNA-seq and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{13}\hbox {C}$$\end{document}13C-based metabolic flux analysis were performed on a CelA-producing and an empty-plasmid strain under the same conditions. Differential gene expression, followed by cluster analysis based on co-expression and co-localization, identified transcriptomic responses related to secretion-induced stress and DNA damage. Furthermore, the OsdR regulon (previously associated with hypoxia, oxidative stress, intercellular signaling, and morphological development) was consistently upregulated in the CelA-producing strain and exhibited co-expression with isoenzymes from the pentose phosphate pathway linked to secondary metabolism. Increased expression of these isoenzymes matches to increased fluxes in the pentose phosphate pathway. Additionally, flux maps of the central carbon metabolism show increased flux through the tricarboxylic acid cycle in the CelA-producing strain. Redirection of fluxes in the CelA-producing strain leads to higher production of NADPH, which can only partly be attributed to increased secretion. Conclusions Transcriptomic and fluxomic changes uncover potential new leads for targeted strain improvement strategies which may ease the secretion stress and metabolic burden associated with heterologous protein synthesis and secretion, and may help create a more consistently performing S. lividans strain. Yet, links to secondary metabolism and redox balancing should be further investigated to fully understand the S. lividans metabolome under heterologous protein production. Electronic supplementary material The online version of this article (10.1186/s12934-018-1040-6) contains supplementary material, which is available to authorized users.

Biological membranes define cells and cellular compartments and are essential in regulating bidirectional flow of chemicals and signals. Characterizing their protein content therefore is required to determine their function, nevertheless, the comprehensive determination of membrane-embedded sub-proteomes remains challenging. Here, we experimentally characterized the inner membrane proteome (IMP) of the model organism E. coli BL21(DE3). We took advantage of the recent extensive re-annotation of the theoretical E. coli IMP regarding the sub-cellular localization of all its proteins. Using surface proteolysis of IMVs with variable chemical treatments followed by nanoLC-MS/MS analysis, we experimentally identified ∼45% of the expressed IMP in wild type E. coli BL21(DE3) with 242 proteins reported here for the first time. Using modified label-free approaches we quantified 220 IM proteins. Finally, we compared protein levels between wild type cells and those over-synthesizing the membrane-embedded translocation channel SecYEG proteins. We propose that this proteomics pipeline will be generally applicable to the determination of IMP from other bacteria.

Secretory preproteins carry signal peptides fused amino-terminally to mature domains. They are post-translationally targeted to cross the plasma membrane in non-folded states with the help of translocases, and fold only at their final destinations. The mechanism of this process of postponed folding is unknown, but is generally attributed to signal peptides and chaperones. We herein demonstrate that, during targeting, most mature domains maintain loosely packed folding intermediates. These largely soluble states are signal peptide independent and essential for translocase recognition. These intermediates are promoted by mature domain features: residue composition, elevated disorder, and reduced hydrophobicity. Consequently, a mature domain folds slower than its cytoplasmic structural homolog. Some mature domains could not evolve stable, loose intermediates, and hence depend on signal peptides for slow folding to the detriment of solubility. These unique features of secretory proteins impact our understanding of protein trafficking, folding, and aggregation, and thus place them in a distinct class. Tsirigotaki et al. demonstrate that secretory mature domains inhabit a rich landscape of loosely packed folding intermediates essential for translocase recognition. Mature domains have elevated disorder and reduced hydrophobicity, and fold more slowly than cytoplasmic structural homologs. Some loose intermediates require signal peptides. Preproteins constitute a distinct protein class.

Background The gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening. CelA is a protein of 260 aminoacyl residues with a 28-residue amino-terminal signal peptide. Mature CelA was poorly synthesized in some Escherichia coli strains and not at all in others. Here we present an alternative approach for its heterologous production as a secreted polypeptide in Streptomyces. Results CelA was successfully over-expressed as a secreted polypeptide in Streptomyces lividans TK24. To this end, CelA was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (Sianidis et al. in J Biotechnol. 121: 498–507, 2006) from Streptomyces venezuelae and a new cloning strategy developed. Optimal growth media and conditions that stall biomass production promote excessive CelA secretion. Under optimal growth conditions in nutrient broth medium, significant amounts of mature CelA (50–90 mg/L or 100–120 mg/g of dry cell weight) are secreted in the spent growth media after 7 days. A protocol to rapidly purify CelA to homogeneity from culture supernatants was developed and specific anti-sera raised against it. Biophysical, biochemical and immmuno-detection analyses indicate that the enzyme is intact, stable and fully functional. CelA is the most thermostable heterologous polypeptide shown to be secreted from S. lividans. Conclusion This study further validates and extends the use of the S. lividans platform for production of heterologous enzymes of industrial importance and extends it to active thermostable enzymes. This study contributes to developing a platform for poly-omics analysis of protein secretion in S. lividans. Electronic supplementary material The online version of this article (10.1186/s12934-017-0847-x) contains supplementary material, which is available to authorized users.

Type III secretion (T3S), a protein export pathway common to Gram-negative pathogens, comprises a trans-envelope syringe, the injectisome, with a cytoplasm-facing translocase channel. Exported substrates are chaperone-delivered to the translocase, EscV in enteropathogenic Escherichia coli, and cross it in strict hierarchical manner, for example, first "translocators", then "effectors". We dissected T3S substrate targeting and hierarchical switching by reconstituting them in vitro using inverted inner membrane vesicles. EscV recruits and conformationally activates the tightly membrane-associated pseudo-effector SepL and its chaperone SepD. This renders SepL a high-affinity receptor for translocator/chaperone pairs, recognizing specific chaperone signals. In a second, SepD-coupled step, translocators docked on SepL become secreted. During translocator secretion, SepL/SepD suppress effector/chaperone binding to EscV and prevent premature effector secretion. Disengagement of the SepL/SepD switch directs EscV to dedicated effector export. These findings advance molecular understanding of T3S and reveal a novel mechanism for hierarchical trafficking regulation in protein secretion channels. ispartof: EMBO Journal vol:36 issue:23 pages:3517-3531 ispartof: location:England status: published

Secretory preproteins contain a mature domain fused to a signal peptide that targets the protein to the translocase, which mediates secretion. In this study, the authors show that the mature domains bear independent targeting signals (MTS) that consist of multiple, degenerate, interchangeable, linear or 3D hydrophobic stretches that are essential for proper secretion., Secretory proteins are only temporary cytoplasmic residents. They are typically synthesized as preproteins, carrying signal peptides N-terminally fused to their mature domains. In bacteria secretion largely occurs posttranslationally through the membrane-embedded SecA-SecYEG translocase. Upon crossing the plasma membrane, signal peptides are cleaved off and mature domains reach their destinations and fold. Targeting to the translocase is mediated by signal peptides. The role of mature domains in targeting and secretion is unclear. We now reveal that mature domains harbor their own independent targeting signals (mature domain targeting signals [MTSs]). These are multiple, degenerate, interchangeable, linear or 3D hydrophobic stretches that become available because of the unstructured states of targeting-competent preproteins. Their receptor site on the cytoplasmic face of the SecYEG-bound SecA is also of hydrophobic nature and is located adjacent to the signal peptide cleft. Both the preprotein MTSs and their receptor site on SecA are essential for protein secretion. Evidently, mature domains have their own previously unsuspected distinct roles in preprotein targeting and secretion.

More than 30years of research have revealed that the dynamic nanomotor SecA is a central player in bacterial protein secretion. SecA associates with the SecYEG channel and transports polypeptides post-translationally to the trans side of the cytoplasmic membrane. It comprises a helicase-like ATPase core coupled to two domains that provide specificity for preprotein translocation. Apart from SecYEG, SecA associates with multiple ligands like ribosomes, nucleotides, lipids, chaperones and preproteins. It exerts its essential contribution in two phases. First, SecA, alone or in concert with chaperones, helps mediate the targeting of the secretory proteins from the ribosome to the membrane. Next, at the membrane it converts chemical energy to mechanical work and translocates preproteins through the SecYEG channel. SecA is a highly dynamic enzyme, it exploits disorder–order kinetics, swiveling and dissociation of domains and dimer to monomer transformations that are tightly coupled with its catalytic function. Preprotein signal sequences and mature domains exploit these dynamics to manipulate the nanomotor and thus achieve their export at the expense of metabolic energy. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Most secretory preproteins exit bacterial cells through the protein translocase, comprising the SecYEG channel and the dimeric peripheral ATPase motor SecA. Energetic coupling to work remains elusive. We now demonstrate that translocation is driven by unusually dynamic quaternary changes in SecA. The dimer occupies several successive states with distinct protomer arrangements. SecA docks on SecYEG as a dimer and becomes functionally asymmetric. Docking occurs via only one protomer. The second protomer allosterically regulates downstream steps. Binding of one preprotein signal peptide to the SecYEG-docked SecA protomer elongates the SecA dimer and triggers the translocase holoenzyme to obtain a lower activation energy conformation. ATP hydrolysis monomerizes the triggered SecA dimer, causing mature chain trapping and processive translocation. This is a unique example of one protein exploiting quaternary dynamics to become a substrate receptor, a "loading clamp," and a "processive motor." This mechanism has widespread implications on protein translocases, chaperones, and motors.

While the entire proteome is synthesized on cytoplasmic ribosomes, almost half associates with, localizes in or crosses the bacterial cell envelope. In Escherichia coli a variety of mechanisms are important for taking these polypeptides into or across the plasma membrane, maintaining them in soluble form, trafficking them to their correct cell envelope locations and then folding them into the right structures. The fidelity of these processes must be maintained under various environmental conditions including during stress; if this fails, proteases are called in to degrade mislocalized or aggregated proteins. Various soluble, diffusible chaperones (acting as holdases, foldases or pilotins) and folding catalysts are also utilized to restore proteostasis. These responses can be general, dealing with multiple polypeptides, with functional overlaps and operating within redundant networks. Other chaperones are specialized factors, dealing only with a few exported proteins. Several complex machineries have evolved to deal with binding to, integration in and crossing of the outer membrane. This complex protein network is responsible for fundamental cellular processes such as cell wall biogenesis; cell division; the export, uptake and degradation of molecules; and resistance against exogenous toxic factors. The underlying processes, contributing to our fundamental understanding of proteostasis, are a treasure trove for the development of novel antibiotics, biopharmaceuticals and vaccines.

A hydrogel based on sulfobetaine methacrylate monomer N-(methacryloyloxyethyl)-N,N-dimethyl-N-(3-sulfopropyl)ammonium betaine and N,N-bis(methacryloyloxyethyl)-N-methyl-N-(3-sulfopropyl)ammonium betaine used as a crosslinker was investigated as a potential material for biosensor applications. The glucose diffusion coefficient of 1.2 × 10−10 m2 s−1 was determined from the glucose release experiment. Inverse size-exclusion chromatography was performed to determine the molecular weight cut-off of the hydrogel to be 8 kDa with respect to pullulans that corresponds to a viscosity radius of 2.1 nm. The narrow pore-size distribution suggests that using the sulfobetaine crosslinker suppresses the composition drift and results in a homogeneous hydrogel network. Furthermore, a glucose biosensor construct comprising the periplasmic glucose-binding protein of Escherichia coli fused to cyan and yellow fluorescent proteins was effectively entrapped in the hydrogel exhibiting no leakage for at least 7 days. The glucose-binding protein showed stability of its secondary structure and sensitivity to glucose as assessed by circular dichroism and Förster (fluorescence) resonance energy transfer measurements under physiological conditions and a physiological range of glucose concentration, respectively. Scopus