Evolutionary adaptation of the protein folding pathway for secretability.

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 ß-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.

Rare Antagonistic Leptin Variants and Severe, Early-Onset Obesity.

Hormone absence or inactivity is common in congenital disease, but hormone antagonism remains controversial. Here, we characterize two novel homozygous leptin variants that yielded antagonistic proteins in two unrelated children with intense hyperphagia, severe obesity, and high circulating levels of leptin. Both variants bind to the leptin receptor but trigger marginal, if any, signaling. In the presence of nonvariant leptin, the variants act as competitive antagonists. Thus, treatment with recombinant leptin was initiated at high doses, which were gradually lowered. Both patients eventually attained near-normal weight. Antidrug antibodies developed in the patients, although they had no apparent effect on efficacy. No severe adverse events were observed. (Funded by the German Research Foundation and others.).

Structural dynamics in the evolution of a bilobed protein scaffold.

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.

Hierarchical protein targeting and secretion is controlled by an affinity switch in the type III secretion system of enteropathogenic Escherichia coli.

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.

Mechanism of receptor assembly via the pleiotropic adipokine Leptin.

The adipokine Leptin activates its receptor LEP-R in the hypothalamus to regulate body weight and exerts additional pleiotropic functions in immunity, fertility and cancer. However, the structure and mechanism of Leptin-mediated LEP-R assemblies has remained unclear. Intriguingly, the signaling-competent isoform of LEP-R is only lowly abundant amid several inactive short LEP-R isoforms contributing to a mechanistic conundrum. Here we show by X-ray crystallography and cryo-EM that, in contrast to long-standing paradigms, Leptin induces type I cytokine receptor assemblies featuring 3:3 stoichiometry and demonstrate such Leptin-induced trimerization of LEP-R on living cells via single-molecule microscopy. In mediating these assemblies, Leptin undergoes drastic restructuring that activates its site III for binding to the Ig domain of an adjacent LEP-R. These interactions are abolished by mutations linked to obesity. Collectively, our study provides the structural and mechanistic framework for how evolutionarily conserved Leptin:LEP-R assemblies with 3:3 stoichiometry can engage distinct LEP-R isoforms to achieve signaling.

Elevated antimony concentrations in commercial juices.

Antimony concentrations up to a factor of 2.7 above the EU limit for drinking water were found in commercial juices and may either be leached from the packaging material or introduced during manufacturing, pointing out the need for further research on the area.

Structural Basis of the Subcellular Topology Landscape of Escherichia coli.

Cellular proteomes are distributed in multiple compartments: on DNA, ribosomes, on and inside membranes, or they become secreted. Structural properties that allow polypeptides to occupy subcellular niches, particularly to after crossing membranes, remain unclear. We compared intrinsic and extrinsic features in cytoplasmic and secreted polypeptides of the Escherichia coli K-12 proteome. Structural features between the cytoplasmome and secretome are sharply distinct, such that a signal peptide-agnostic machine learning tool distinguishes cytoplasmic from secreted proteins with 95.5% success. Cytoplasmic polypeptides are enriched in aliphatic, aromatic, charged and hydrophobic residues, unique folds and higher early folding propensities. Secretory polypeptides are enriched in polar/small amino acids, ß folds, have higher backbone dynamics, higher disorder and contact order and are more often intrinsically disordered. These non-random distributions and experimental evidence imply that evolutionary pressure selected enhanced secretome flexibility, slow folding and looser structures, placing the secretome in a distinct protein class. These adaptations protect the secretome from premature folding during its cytoplasmic transit, optimize its lipid bilayer crossing and allowed it to acquire cell envelope specific chemistries. The latter may favor promiscuous multi-ligand binding, sensing of stress and cell envelope structure changes. In conclusion, enhanced flexibility, slow folding, looser structures and unique folds differentiate the secretome from the cytoplasmome. These findings have wide implications on the structural diversity and evolution of modern proteomes and the protein folding problem.

Long-Lived Folding Intermediates Predominate the Targeting-Competent Secretome.

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.

Protein export through the bacterial Sec pathway.

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.

Dynamics and ligand-induced conformational changes in human prolyl oligopeptidase analyzed by hydrogen/deuterium exchange mass spectrometry.

Prolyl oligopeptidase (PREP) is conserved in many organisms across life. It is involved in numerous processes including brain function and neuropathology, that require more than its strict proteolytic role. It consists of a seven-bladed ß-propeller juxtaposed to a catalytic α/ß-hydrolase domain. The conformational dynamics of PREP involved in domain motions and the gating mechanism that allows substrate accessibility remain elusive. Here we used Hydrogen Deuterium eXchange Mass Spectrometry (HDX-MS) to derive the first near-residue resolution analysis of global PREP dynamics in the presence or absence of inhibitor bound in the active site. Clear roles are revealed for parts that would be critical for the activation mechanism. In the free state, the inter-domain interface is loose, providing access to the catalytic site. Inhibitor binding "locks" the two domains together exploiting prominent interactions between the loop of the first ß-propeller blade and its proximal helix from the α/ß-hydrolase domain. Loop A, thought to drive gating, is partially stabilized but remains flexible and dynamic. These findings provide a conformational guide for further dissection of the gating mechanism of PREP, that would impact drug development. Moreover, they offer a structural framework against which to study proteolysis-independent interactions with disordered proteins like α-synuclein involved in neurodegenerative disease.

Preprotein Conformational Dynamics Drive Bivalent Translocase Docking and Secretion.

Most bacterial secretory proteins destined beyond the plasma membrane are secreted post-translationally by the Sec translocase. In the first step of translocation, preproteins are targeted for binding to their 2-site receptor SecA, the peripheral ATPase subunit of the translocase. We now reveal that secretory preproteins use a dual-key mechanism to bridge the signal peptide and mature domain receptor sites and cooperatively enhance their affinities. Docking of targeting-competent mature domains requires that their extensive disorder is finely tuned. This is achieved through amino-terminal mature domain regions acting as conformational rheostats. By being linked to the rheostats, signal peptides regulate long-range preprotein disorder. Concomitant conformational changes in SecA sterically adapt its two receptor sites to optimally recognize hundreds of dissimilar preproteins. This novel intramolecular conformational crosstalk in the preprotein chains and the dynamic interaction with their receptor are mechanistically coupled to preprotein engagement in the translocase and essential for secretion.

Preprotein mature domains contain translocase targeting signals that are essential for 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.

Protein folding in the cell envelope of Escherichia coli.

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.

Concurrent quantitative HPLC-mass spectrometry profiling of small selenium species in human serum and urine after ingestion of selenium supplements.

Selenium metabolic patterns in the human body originating from five distinct selenium dietary sources, selenate, selenite, selenomethionine (SeMet), methylselenocysteine (MeSeCys) and selenized yeast, were investigated by performing concurrent HPLC-mass spectrometric analysis of human serum and urine. Total selenium and selenium species time profiles were generated by sampling and analyzing serum and urine from volunteers treated with selenium supplements, up to 5 and 24h following ingestion, respectively. We found that an increase in total serum selenium levels, accompanied by elevated selenium urinary excretion, was the common pattern for all treatments, except for that of selenite supplementation. Selenosugar 1 was a universal serum metabolite in all treatments, indicating that ingested selenium is favorably metabolized to the sugar. Except for selenite and selenized yeast ingestion, these patterns were reflected in the urine time series of the different treatments. Selenosugar 1 was the major selenium species present in urine in all treatments except for the selenate treatment, accounting for about 80% of the identified excreted species within 24h of ingestion. Furthermore, the urinary metabolite trimethylselenonium ion (TMSe) was detected for the first time in human background serum by using HPLC coupled to elemental and molecular mass spectrometry. The concurrent monitoring of non-protein selenium species in both body fluids provides the relation between bioavailability and excretion of the individual ingested species and of their metabolic products, while the combined use of elemental and molecular mass spectrometry enables the accurate quantitation of structurally confirmed species. This successfully applied approach is anticipated to be a useful tool for more extensive future studies into human selenium metabolism.

Bipolar mass spectrometry of labile coordination complexes, redox active inorganic compounds, and proteins using a glass nebulizer for sonic-spray ionization.

In this study, we report on the development of a novel nebulizer configuration for sonic-spray ionization (SSI) mass spectrometry (MS), more specifically for a version of SSI that is referred to as Venturi easy ambient sonic-spray ionization (V-EASI) MS. The developed nebulizer configuration is based on a commercially available pneumatic glass nebulizer that has been used extensively for aerosol formation in atomic spectrometry. In the present study, the nebulizer was modified in order to achieve efficient V-EASI-MS operation. Upon evaluating this system, it has been demonstrated that V-EASI-MS offers some distinct advantages for the analysis of coordination compounds and redox active inorganic compounds over the predominantly used electrospray ionization (ESI) technique. Such advantages, for this type of compounds, are demonstrated here for the first time. More specifically, a series of labile heptanuclear heterometallic [Cu(II) 6Ln(III)] clusters held together with artificial amino acid ligands, in addition to easily oxidized inorganic oxyanions of selenium and arsenic, were analyzed. The observed advantages pertain to V-EASI appearing to be a "milder" ionization source than ESI, not requiring electrical potentials for gas phase ion formation, thus eliminating the possibility of unwanted redox transformations, allowing for the "simultaneous" detection of negative and positive ions (bipolar analysis) without the need to change source ionization conditions, and also not requiring the use of syringes and delivery pumps. Because of such features, especially because of the absence of ionization potentials, EASI can be operated with minimal requirements for source parameter optimization. We observed that source temperature and accelerating voltage do not seem to affect labile compounds to the extent they do in ESI-MS. In addition, bipolar analysis of proteins was demonstrated here by acquiring both positive and negative ion mass spectra from the same protein solutions, without the need to independently adjust solution and source conditions in each mode. Finally, the simple and efficient operation of a dual-nebulizer configuration was demonstrated for V-EASI-MS for the first time.