Structural basis of pH-dependent activation in a CLC transporter.

CLCs are dimeric chloride channels and anion/proton exchangers that regulate processes such as muscle contraction and endo-lysosome acidification. Common gating controls their activity; its closure simultaneously silences both protomers, and its opening allows them to independently transport ions. Mutations affecting common gating in human CLCs cause dominant genetic disorders. The structural rearrangements underlying common gating are unknown. Here, using single-particle cryo-electron microscopy, we show that the prototypical Escherichia coli CLC-ec1 undergoes large-scale rearrangements in activating conditions. The slow, pH-dependent remodeling of the dimer interface leads to the concerted opening of the intracellular H+ pathways and is required for transport. The more frequent formation of short water wires in the open H+ pathway enables Cl- pore openings. Mutations at disease-causing sites favor CLC-ec1 activation and accelerate common gate opening in the human CLC-7 exchanger. We suggest that the pH activation mechanism of CLC-ec1 is related to the common gating of CLC-7.

Linking function to global and local dynamics in an elevator-type transporter.

Transporters cycle through large structural changes to translocate molecules across biological membranes. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency-yet remain mostly unknown. Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter GltPh We show that the elevator-like movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous, with rates varying by orders of magnitude between individual molecules. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. Hydrogen deuterium exchange coupled to mass spectrometry reveals destabilization of secondary structure around the substrate-binding site, suggesting that increased local dynamics leads to faster rates of global conformational changes and confers gain-of-function properties that set transport rates.

FRET-based Microscopy Assay to Measure Activity of Membrane Amino Acid Transporters with Single-transporter Resolution.

Secondary active transporters reside in cell membranes transporting polar solutes like amino acids against steep concentration gradients, using electrochemical gradients of ions as energy sources. Commonly, ensemble-based measurements of radiolabeled substrate uptakes or transport currents inform on kinetic parameters of transporters. Here we describe a fluorescence-based functional assay for glutamate and aspartate transporters that provides single-transporter, single-transport cycle resolution using an archaeal elevator-type sodium and aspartate symporter GltPh as a model system. We prepare proteo-liposomes containing reconstituted purified GltPh transporters and an encapsulated periplasmic glutamate/aspartate-binding protein, PEB1a, labeled with donor and acceptor fluorophores. We then surface-immobilize the proteo-liposomes and measure transport-dependent Fluorescence Resonance Energy Transfer (FRET) efficiency changes over time using single-molecule Total Internal Reflection Fluorescence (TIRF) microscopy. The assay provides a 10-100 fold increase in temporal resolution compared to radioligand uptake assays. It also allows kinetic characterization of different transport cycle steps and discerns kinetic heterogeneities within the transporter population.

The high-energy transition state of the glutamate transporter homologue GltPh.

Membrane transporters mediate cellular uptake of nutrients, signaling molecules, and drugs. Their overall mechanisms are often well understood, but the structural features setting their rates are mostly unknown. Earlier single-molecule fluorescence imaging of the archaeal model glutamate transporter homologue GltPh from Pyrococcus horikoshii suggested that the slow conformational transition from the outward- to the inward-facing state, when the bound substrate is translocated from the extracellular to the cytoplasmic side of the membrane, is rate limiting to transport. Here, we provide insight into the structure of the high-energy transition state of GltPh that limits the rate of the substrate translocation process. Using bioinformatics, we identified GltPh gain-of-function mutations in the flexible helical hairpin domain HP2 and applied linear free energy relationship analysis to infer that the transition state structurally resembles the inward-facing conformation. Based on these analyses, we propose an approach to search for allosteric modulators for transporters.

Millisecond dynamics of an unlabeled amino acid transporter.

Excitatory amino acid transporters (EAATs) are important in many physiological processes and crucial for the removal of excitatory amino acids from the synaptic cleft. Here, we develop and apply high-speed atomic force microscopy line-scanning (HS-AFM-LS) combined with automated state assignment and transition analysis for the determination of transport dynamics of unlabeled membrane-reconstituted GltPh, a prokaryotic EAAT homologue, with millisecond temporal resolution. We find that GltPh transporters can operate much faster than previously reported, with state dwell-times in the 50 ms range, and report the kinetics of an intermediate transport state with height between the outward- and inward-facing states. Transport domains stochastically probe transmembrane motion, and reversible unsuccessful excursions to the intermediate state occur. The presented approach and analysis methodology are generally applicable to study transporter kinetics at system-relevant temporal resolution.

Single-molecule transport kinetics of a glutamate transporter homolog shows static disorder.

Kinetic properties of membrane transporters are typically poorly defined because high-resolution functional assays analogous to single-channel recordings are lacking. Here, we measure single-molecule transport kinetics of a glutamate transporter homolog from Pyrococcus horikoshii, GltPh, using fluorescently labeled periplasmic amino acid binding protein as a fluorescence resonance energy transfer-based sensor. We show that individual transporters can function at rates varying by at least two orders of magnitude that persist for multiple turnovers. A gain-of-function mutant shows increased population of the fast-acting transporters, leading to a 10-fold increase in the mean transport rate. These findings, which are broadly consistent with earlier single-molecule measurements of GltPh conformational dynamics, suggest that GltPh transport is defined by kinetically distinct populations that exhibit long-lasting "molecular memory."

Use of paramagnetic 19F NMR to monitor domain movement in a glutamate transporter homolog.

In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homolog with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified one inward- and two outward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.

Determining the Free Energies of Outer Membrane Proteins in Lipid Bilayers.

The thermodynamic stabilities of membrane proteins are of fundamental interest to provide a biophysical description of their structure-function relationships because energy determines conformational populations. In addition, structure-energy relationships can be exploited in membrane protein design and in synthetic biology. To determine the thermodynamic stability of a membrane protein, it is not sufficient to be able to unfold and refold the molecule: establishing path independence of this reaction is essential. Here we describe the procedures required to measure and verify path independence for the folding of outer membrane proteins in large unilamellar vesicles.

Molecular basis for the folding of ß-helical autotransporter passenger domains.

Bacterial autotransporters comprise a C-terminal ß-barrel domain, which must be correctly folded and inserted into the outer membrane to facilitate translocation of the N-terminal passenger domain to the cell exterior. Once at the surface, the passenger domains of most autotransporters are folded into an elongated ß-helix. In a cellular context, key molecules catalyze the assembly of the autotransporter ß-barrel domain. However, how the passenger domain folds into its functional form is poorly understood. Here we use mutational analysis on the autotransporter Pet to show that the ß-hairpin structure of the fifth extracellular loop of the ß-barrel domain has a crucial role for passenger domain folding into a ß-helix. Bioinformatics and structural analyses, and mutagenesis of a homologous autotransporter, suggest that this function is conserved among autotransporter proteins with ß-helical passenger domains. We propose that the autotransporter ß-barrel domain is a folding vector that nucleates folding of the passenger domain.

Prepore Stability Controls Productive Folding of the BAM-independent Multimeric Outer Membrane Secretin PulD.

Members of a group of multimeric secretion pores that assemble independently of any known membrane-embedded insertase in Gram-negative bacteria fold into a prepore before membrane-insertion occurs. The mechanisms and the energetics that drive the folding of these proteins are poorly understood. Here, equilibrium unfolding and hydrogen/deuterium exchange monitored by mass spectrometry indicated that a loss of 4-5 kJ/mol/protomer in the N3 domain that is peripheral to the membrane-spanning C domain in the dodecameric secretin PulD, the founding member of this class, prevents pore formation by destabilizing the prepore into a poorly structured dodecamer as visualized by electron microscopy. Formation of native PulD-multimers by mixing protomers that differ in N3 domain stability, suggested that the N3 domain forms a thermodynamic seal onto the prepore. This highlights the role of modest free energy changes in the folding of pre-integration forms of a hyperstable outer membrane complex and reveals a key driving force for assembly independently of the ß-barrel assembly machinery.

Folding outer membrane proteins independently of the ß-barrel assembly machinery: an assembly pathway for multimeric complexes?

Since the discovery of the essential role of the ß-barrel assembly machinery (BAM) for the membrane insertion of outer membrane proteins (OMPs) that are unrelated in sequence, members of this universally conserved family dominate discussions on OMP assembly in bacteria, mitochondria and chloroplasts. However, several multimeric bacterial OMPs assemble independently of the catalyzing BAM-component BamA. Recent progress on this alternative pathway is reviewed here, and a model for BAM-independent assembly for multimeric OMPs is proposed in which monomer delivery to the membrane and stable prepore formation are key steps towards productive membrane insertion.

Structural Basis of Pullulanase Membrane Binding and Secretion Revealed by X-Ray Crystallography, Molecular Dynamics and Biochemical Analysis.

The Klebsiella lipoprotein pullulanase (PulA) is exported to the periplasm, triacylated, and anchored via lipids in the inner membrane (IM) prior to its transport to the bacterial surface through a type II secretion system (T2SS). X-Ray crystallography and atomistic molecular dynamics (MD) simulations of PulA in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) model membrane provided an unprecedented molecular view of an N-terminal unstructured tether and the IM lipoprotein retention signal, and revealed novel interactions with the IM via N-terminal immunoglobulin-like domains in PulA. An efficiently secreted nonacylated variant (PulANA) showed similar peripheral membrane association during MD simulations, consistent with the binding of purified PulANA to liposomes. Remarkably, combined X-ray, MD, and functional studies identified a novel subdomain, Ins, inserted in the α-amylase domain, which is required for PulA secretion. Available data support a model in which PulA binding to the IM promotes interactions with the T2SS, possibly via the Ins subdomain.

A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters.

Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.

Lipids assist the membrane insertion of a BAM-independent outer membrane protein.

Like several other large, multimeric bacterial outer membrane proteins (OMPs), the assembly of the Klebsiella oxytoca OMP PulD does not rely on the universally conserved ß-barrel assembly machinery (BAM) that catalyses outer membrane insertion. The only other factor known to interact with PulD prior to or during outer membrane targeting and assembly is the cognate chaperone PulS. Here, in vitro translation-transcription coupled PulD folding demonstrated that PulS does not act during the membrane insertion of PulD, and engineered in vivo site-specific cross-linking between PulD and PulS showed that PulS binding does not prevent membrane insertion. In vitro folding kinetics revealed that PulD is atypical compared to BAM-dependent OMPs by inserting more rapidly into membranes containing E. coli phospholipids than into membranes containing lecithin. PulD folding was fast in diC14:0-phosphatidylethanolamine liposomes but not diC14:0-phosphatidylglycerol liposomes, and in diC18:1-phosphatidylcholine liposomes but not in diC14:1-phosphatidylcholine liposomes. These results suggest that PulD efficiently exploits the membrane composition to complete final steps in insertion and explain how PulD can assemble independently of any protein-assembly machinery. Lipid-assisted assembly in this manner might apply to other large OMPs whose assembly is BAM-independent.

A mortise-tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes.

Bacterial autotransporters comprise a 12-stranded membrane-embedded ß-barrel domain, which must be folded in a process that entraps segments of an N-terminal passenger domain. This first stage of autotransporter folding determines whether subsequent translocation can deliver the N-terminal domain to its functional form on the bacterial cell surface. Here, paired glycine-aromatic 'mortise and tenon' motifs are shown to join neighbouring ß-strands in the C-terminal barrel domain, and mutations within these motifs slow the rate and extent of passenger domain translocation to the surface of bacterial cells. In line with this, biophysical studies of the autotransporter Pet show that the conserved residues significantly quicken completion of the folding reaction and promote stability of the autotransporter barrel domain. Comparative genomics demonstrate conservation of glycine-aromatic residue pairings through evolution as a previously unrecognized feature of all autotransporter proteins.

Independent domain assembly in a trapped folding intermediate of multimeric outer membrane secretins.

The outer membrane portal of the Klebsiella oxytoca type II secretion system, PulD, is a prototype of a family of proteins, the secretins, which are essential components of many bacterial secretion and pilus assembly machines. PulD is a homododecamer with a periplasmic vestibule and an outer chamber on either side of a membrane-spanning region that is poorly resolved by electron microscopy. Membrane insertion involves the formation of a dodecameric membrane-embedded intermediate. Here, we describe an amino acid substitution in PulD that blocks its assembly at this intermediate "prepore" stage. Electron microscopy indicated that the prepore has an apparently normal periplasmic vestibule but a poorly organized outer chamber. A peptide loop around this amino acid appears to be important for the formation/stability of the fully folded complex. A similar assembly intermediate results from creation of the same amino acid substitution in the Pseudomonas aeruginosa secretin XcpQ.

Bacterial secretins form constitutively open pores akin to general porins.

Proteins called secretins form large multimeric complexes that are essential for macromolecular transit across the outer membrane of Gram-negative bacteria. Evidence suggests that the channels formed by some secretin complexes are not tightly closed, but their permeability properties have not been well characterized. Here, we used cell-free synthesis coupled with spontaneous insertion into liposomes to investigate the permeability of the secretin PulD. Leakage assays using preloaded liposomes indicated that PulD allows the efflux of small fluorescent molecules with a permeation cutoff similar to that of general porins. Other secretins were also found to form similar pores. To define the polypeptide region involved in determining the pore size, we analyzed a collection of PulD variants and studied the roles of gates 1 and 2, which were previously reported to affect the pore size of filamentous phage f1 secretin pIV, in assembly and pore formation. Liposome leakage and a novel in vivo assay showed that replacement of the conserved proline residue at position 443 in PulD by leucine increased the apparent size of the pore. The in vitro approach described here could be used to study the pore properties of membrane proteins whose production in vivo is toxic.

Sequential steps in the assembly of the multimeric outer membrane secretin PulD.

Investigations into protein folding are largely dominated by studies on monomeric proteins. However, the transmembrane domain of an important group of membrane proteins is only formed upon multimerization. Here, we use in vitro translation-coupled folding and insertion into artificial liposomes to investigate kinetic steps in the assembly of one such protein, the outer membrane secretin PulD of the bacterial type II secretion system. Analysis of the folding kinetics, measured by the acquisition of distinct determinants of the native state, provides unprecedented evidence for a sequential multistep process initiated by membrane-driven oligomerization. The effects of varying the lipid composition of the liposomes indicate that PulD first forms a "prepore" structure that attains the native state via a conformational switch.

Dissecting the effects of periplasmic chaperones on the in vitro folding of the outer membrane protein PagP.

Although many periplasmic folding factors have been identified, the mechanisms by which they interact with unfolded outer membrane proteins (OMPs) to promote correct folding and membrane insertion remain poorly understood. Here, we have investigated the effect of two chaperones, Skp and SurA, on the folding kinetics of the OMP, PagP. Folding kinetics of PagP into both zwitterionic diC12:0PC (1,2-dilauroyl-sn-glycero-3-phosphocholine) liposomes and negatively charged 80:20 diC12:0PC:diC12:0PG [1,2-dilauroyl-sn-glycero-3-phospho-(1'-rac-glycerol)] liposomes were investigated using a combination of spectroscopic and SDS-PAGE assays. The results indicate that Skp modulates the observed rate of PagP folding in a manner that is dependent on the composition of the membrane and the ionic strength of the buffer used. These data suggest that electrostatic interactions play an important role in Skp-assisted substrate delivery to the membrane. In contrast, SurA showed no effect on the observed folding rates of PagP, consistent with the view that these chaperones act by distinct mechanisms in partially redundant parallel chaperone pathways that facilitate OMP assembly. In addition to delivery of the substrate protein to the membrane, the ability of Skp to prevent OMP aggregation was investigated. The results show that folding and membrane insertion of PagP can be restored, in part, by Skp in conditions that strongly favour PagP aggregation. These results illustrate the utility of in vitro systems for dissecting the complex folding environment encountered by OMPs in the periplasm and demonstrate the key role of Skp in holding aggregation-prone OMPs prior to their direct or indirect delivery to the membrane.