ABCH2 transporter mediates deltamethrin uptake and toxicity in the malaria vector Anopheles coluzzii.

Contact insecticides are primarily used for the control of Anopheles malaria vectors. These chemicals penetrate mosquito legs and other appendages; the first barriers to reaching their neuronal targets. An ATP-Binding Cassette transporter from the H family (ABCH2) is highly expressed in Anopheles coluzzii legs, and further induced upon insecticide exposure. RNAi-mediated silencing of the ABCH2 caused a significant increase in deltamethrin mortality compared to control mosquitoes, coincident with a corresponding increase in 14C-deltamethrin penetration. RT-qPCR analysis and immunolocalization revealed ABCH2 to be mainly localized in the legs and head appendages, and more specifically, the apical part of the epidermis, underneath the cuticle. To unravel the molecular mechanism underlying the role of ABCH2 in modulating pyrethroid toxicity, two hypotheses were investigated: An indirect role, based on the orthology with other insect ABCH transporters involved with lipid transport and deposition of CHC lipids in Anopheles legs which may increase cuticle thickness, slowing down the penetration rate of deltamethrin; or the direct pumping of deltamethrin out of the organism. Evaluation of the leg cuticular hydrocarbon (CHC) content showed no affect by ABCH2 silencing, indicating this protein is not associated with the transport of leg CHCs. Homology-based modeling suggested that the ABCH2 half-transporter adopts a physiological homodimeric state, in line with its ability to hydrolyze ATP in vitro when expressed on its own in insect cells. Docking analysis revealed a deltamethrin pocket in the homodimeric transporter. Furthermore, deltamethrin-induced ATP hydrolysis in ABCH2-expressing cell membranes, further supports that deltamethrin is indeed an ABCH2 substrate. Overall, our findings pinpoint ABCH2 participating in deltamethrin toxicity regulation.

Preproteins couple the intrinsic dynamics of SecA to its ATPase cycle to translocate via a catch and release mechanism.

Protein machines undergo conformational motions to interact with and manipulate polymeric substrates. The Sec translocase promiscuously recognizes, becomes activated, and secretes >500 non-folded preprotein clients across bacterial cytoplasmic membranes. Here, we reveal that the intrinsic dynamics of the translocase ATPase, SecA, and of preproteins combine to achieve translocation. SecA possesses an intrinsically dynamic preprotein clamp attached to an equally dynamic ATPase motor. Alternating motor conformations are finely controlled by the γ-phosphate of ATP, while ADP causes motor stalling, independently of clamp motions. Functional preproteins physically bridge these independent dynamics. Their signal peptides promote clamp closing; their mature domain overcomes the rate-limiting ADP release. While repeated ATP cycles shift the motor between unique states, multiple conformationally frustrated prongs in the clamp repeatedly "catch and release" trapped preprotein segments until translocation completion. This universal mechanism allows any preprotein to promiscuously recognize the translocase, usurp its intrinsic dynamics, and become secreted.

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.

A nexus of intrinsic dynamics underlies translocase priming.

The cytoplasmic ATPase SecA and the membrane-embedded SecYEG channel assemble to form the Sec translocase. How this interaction primes and catalytically activates the translocase remains unclear. We show that priming exploits a nexus of intrinsic dynamics in SecA. Using atomistic simulations, smFRET, and HDX-MS, we reveal multiple dynamic islands that cross-talk with domain and quaternary motions. These dynamic elements are functionally important and conserved. Central to the nexus is a slender stem through which rotation of the preprotein clamp of SecA is biased by ATPase domain motions between open and closed clamping states. An H-bonded framework covering most of SecA enables multi-tier dynamics and conformational alterations with minimal energy input. As a result, cognate ligands select preexisting conformations and alter local dynamics to regulate catalytic activity and clamp motions. These events prime the translocase for high-affinity reception of non-folded preprotein clients. Dynamics nexuses are likely universal and essential in multi-liganded proteins.

Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ.

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE-FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE-FRET to monitor protein-protein interactions and conformational states simultaneously.

Single-molecule studies of conformational states and dynamics in the ABC importer OpuA.

The current model of active transport via ABC importers is mostly based on structural, biochemical and genetic data. We here establish single-molecule Förster resonance energy transfer (smFRET) assays to monitor the conformational states and heterogeneity of the osmoregulatory type I ABC importer OpuA from Lactococcus lactis. We present data probing both intradomain distances that elucidate conformational changes within the substrate-binding domain (SBD) OpuAC, and interdomain distances between SBDs or transmembrane domains. Using this methodology, we studied ligand-binding mechanisms, as well as ATP and glycine betaine dependences of conformational changes. Our work expands the scope of smFRET investigations towards a class of so far unstudied ABC importers, and paves the way for a full understanding of their transport cycle in the future.

Current Blockades of Proteins inside Nanopores for Real-Time Metabolome Analysis.

Biological nanopores are emerging as powerful and low-cost sensors for real-time analysis of biological samples. Proteins can be incorporated inside the nanopore, and ligand binding to the protein adaptor yields changes in nanopore conductance. In order to understand the origin of these conductance changes and develop sensors for detecting metabolites, we tested the signal originating from 13 different protein adaptors. We found that the quality of the protein signal depended on both the size and charge of the protein. The engineering of a dipole within the surface of the adaptor reduced the current noise by slowing the protein dynamics within the nanopore. Further, the charge of the ligand and the induced conformational changes of the adaptor defined the conductance changes upon metabolite binding, suggesting that the protein resides in an electrokinetic minimum within the nanopore, the position of which is altered by the ligand. These results represent an important step toward understanding the dynamics of the electrophoretic trapping of proteins inside nanopores and will allow developing next-generation sensors for metabolome analysis.

Single-Molecule Observation of Ligand Binding and Conformational Changes in FeuA.

The specific binding of ligands by proteins and the coupling of this process to conformational changes is fundamental to protein function. We designed a fluorescence-based single-molecule assay and data analysis procedure that allows the simultaneous real-time observation of ligand binding and conformational changes in FeuA. The substrate-binding protein FeuA binds the ligand ferri-bacillibactin and delivers it to the ATP-binding cassette importer FeuBC, which is involved in bacterial iron uptake. The conformational dynamics of FeuA was assessed via Förster resonance energy transfer, whereas the presence of the ligand was probed by fluorophore quenching. We reveal that ligand binding shifts the conformational equilibrium of FeuA from an open to a closed conformation. Ligand binding occurs via an induced-fit mechanism, i.e., the ligand binds to the open state and subsequently triggers a rapid closing of the protein. However, FeuA also rarely samples the closed conformation without the involvement of the ligand. This shows that ligand interactions are not required for conformational changes in FeuA. However, ligand interactions accelerate the conformational change 10,000-fold and temporally stabilize the formed conformation 250-fold.

ABCE1 Controls Ribosome Recycling by an Asymmetric Dynamic Conformational Equilibrium.

The twin-ATPase ABCE1 has a vital function in mRNA translation by recycling terminated or stalled ribosomes. As for other functionally distinct ATP-binding cassette (ABC) proteins, the mechanochemical coupling of ATP hydrolysis to conformational changes remains elusive. Here, we use an integrated biophysical approach allowing direct observation of conformational dynamics and ribosome association of ABCE1 at the single-molecule level. Our results from FRET experiments show that the current static two-state model of ABC proteins has to be expanded because the two ATP sites of ABCE1 are in dynamic equilibrium across three distinct conformational states: open, intermediate, and closed. The interaction of ABCE1 with ribosomes influences the conformational dynamics of both ATP sites asymmetrically and creates a complex network of conformational states. Our findings suggest a paradigm shift to redefine the understanding of the mechanochemical coupling in ABC proteins: from structure-based deterministic models to dynamic-based systems.

Conformational and dynamic plasticity in substrate-binding proteins underlies selective transport in ABC importers.

Substrate-binding proteins (SBPs) are associated with ATP-binding cassette importers and switch from an open to a closed conformation upon substrate binding, providing specificity for transport. We investigated the effect of substrates on the conformational dynamics of six SBPs and the impact on transport. Using single-molecule FRET, we reveal an unrecognized diversity of plasticity in SBPs. We show that a unique closed SBP conformation does not exist for transported substrates. Instead, SBPs sample a range of conformations that activate transport. Certain non-transported ligands leave the structure largely unaltered or trigger a conformation distinct from that of transported substrates. Intriguingly, in some cases, similar SBP conformations are formed by both transported and non-transported ligands. In this case, the inability for transport arises from slow opening of the SBP or the selectivity provided by the translocator. Our results reveal the complex interplay between ligand-SBP interactions, SBP conformational dynamics and substrate transport.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Author Correction: A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization.

This corrects the article DOI: 10.1038/ncomms10144.

Corrigendum: A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization.

This corrects the article DOI: 10.1038/ncomms10144.

Corrigendum: A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization.

This corrects the article DOI: 10.1038/ncomms10144.

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.

Watching conformational dynamics of ABC transporters with single-molecule tools.

ATP-binding cassette (ABC) transporters play crucial roles in cellular processes, such as nutrient uptake, drug resistance, cell-volume regulation and others. Despite their importance, all proposed molecular models for transport are based on indirect evidence, i.e. functional interpretation of static crystal structures and ensemble measurements of function and structure. Thus, classical biophysical and biochemical techniques do not readily visualize dynamic structural changes. We recently started to use single-molecule fluorescence techniques to study conformational states and changes of ABC transporters in vitro, in order to observe directly how the different steps during transport are coordinated. This review summarizes our scientific strategy and some of the key experimental advances that allowed the substrate-binding mechanism of prokaryotic ABC importers and the transport cycle to be explored. The conformational states and transitions of ABC-associated substrate-binding domains (SBDs) were visualized with single-molecule FRET, permitting a direct correlation of structural and kinetic information of SBDs. We also delineated the different steps of the transport cycle. Since information in such assays are restricted by proper labelling of proteins with fluorescent dyes, we present a simple approach to increase the amount of protein with FRET information based on non-specific interactions between a dye and the size-exclusion chromatography (SEC) column material used for final purification.

Conformational dynamics in substrate-binding domains influences transport in the ABC importer GlnPQ.

The conformational dynamics in ABC transporters is largely elusive. The ABC importer GlnPQ from Lactococcus lactis has different covalently linked substrate-binding domains (SBDs), thus making it an excellent model system to elucidate the dynamics and role of the SBDs in transport. We demonstrate by single-molecule spectroscopy that the two SBDs intrinsically transit from open to closed ligand-free conformation, and the proteins capture their amino acid ligands via an induced-fit mechanism. High-affinity ligands elicit transitions without changing the closed-state lifetime, whereas low-affinity ligands dramatically shorten it. We show that SBDs in the closed state compete for docking onto the translocator, but remarkably the effect is strongest without ligand. We find that the rate-determining steps depend on the SBD and the amino acid transported. We conclude that the lifetime of the closed conformation controls both SBD docking to the translocator and substrate release.

Quaternary dynamics of the SecA motor drive translocase catalysis.

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.

In vitro assays to analyze translocation of the model secretory preprotein alkaline phosphatase.

Almost one-third of the proteins synthesized in the cytosol of cells ends up in membranes or outside the cell. Secretory polypeptides are synthesized as precursor proteins that carry N-terminal signal sequences. Secretion is catalyzed by the "translocase" that comprises a channel-clamp protein and an ATPase motor. Translocase activities have been fully reconstituted in vitro. This provided powerful tools to examine the role of each component in the reaction. Here we describe protocols for the purification of the secretory preprotein alkaline phosphatase and a series of in vitro assays developed in order to examine the binding of alkaline phosphatase to the translocase, its ability to stimulate ATP hydrolysis, and finally its transfer across the membrane. The assays are applicable to any similar study of secretory preproteins.