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.

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.

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.

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.

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.

A urea channel from Bacillus cereus reveals a novel hexameric structure.

Urea is exploited as a nitrogen source by bacteria, and its breakdown products, ammonia and bicarbonate, are employed to counteract stomach acidity in pathogens such as Helicobacter pylori. Uptake in the latter is mediated by UreI, a UAC (urea amide channel) family member. In the present paper, we describe the structure and function of UACBc, a homologue from Bacillus cereus. The purified channel was found to be permeable not only to urea, but also to other small amides. CD and IR spectroscopy revealed a structure comprising mainly α-helices, oriented approximately perpendicular to the membrane. Consistent with this finding, site-directed fluorescent labelling indicated the presence of seven TM (transmembrane) helices, with a cytoplasmic C-terminus. In detergent, UACBc exists largely as a hexamer, as demonstrated by both cross-linking and size-exclusion chromatography. A 9 Å (1 Å=0.1 nm) resolution projection map obtained by cryo-electron microscopy of two-dimensional crystals shows that the six protomers are arranged in a planar hexameric ring. Each exhibits six density features attributable to TM helices, surrounding a putative central channel, while an additional helix is peripherally located. Bioinformatic analyses allowed individual TM regions to be tentatively assigned to the density features, with the resultant model enabling identification of residues likely to contribute to channel function.

The transition state for folding of an outer membrane protein.

Inspired by the seminal work of Anfinsen, investigations of the folding of small water-soluble proteins have culminated in detailed insights into how these molecules attain and stabilize their native folds. In contrast, despite their overwhelming importance in biology, progress in understanding the folding and stability of membrane proteins remains relatively limited. Here we use mutational analysis to describe the transition state involved in the reversible folding of the beta-barrel membrane protein PhoPQ-activated gene P (PagP) from a highly disordered state in 10 M urea to a native protein embedded in a lipid bilayer. Analysis of the equilibrium stability and unfolding kinetics of 19 variants that span all eight beta-strands of this 163-residue protein revealed that the transition-state structure is a highly polarized, partly formed beta-barrel. The results provide unique and detailed insights into the transition-state structure for beta-barrel membrane protein folding into a lipid bilayer and are consistent with a model for outer membrane protein folding via a tilted insertion mechanism.

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.

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.

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.

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."

The N-terminal helix is a post-assembly clamp in the bacterial outer membrane protein PagP.

The Escherichia coli outer membrane beta-barrel enzyme PagP and its homologues are unique in that the eight-stranded barrel is tilted by about 25 degrees with respect to the membrane normal and is preceded by a 19-residue amphipathic alpha-helix. To investigate the role of this helix in the folding and stability of PagP, mutants were generated in which the helix was deleted (Delta(1-19)), or in which residues predicted to be involved in helix-barrel interactions were altered (W17A or R59L). The ability of the variants to insert into detergent micelles or liposomes was studied in vitro using circular dichroism, fluorescence, Fourier transform infrared spectroscopy, electrophoretic mobility and gain of enzyme activity. The data show that PagP, initially unfolded in 5% (w/v) perfluoro-octanoic acid or 6 M guanidinium chloride, inserts spontaneously and folds quantitatively to an active conformation into detergent micelles of cyclofos-7 or into large vesicles of diC(12:0)-phosphatidylcholine (diC(12:0)PC), respectively, the latter in the presence of 7 M urea. Successful refolding of all variants into both micelles and liposomes ruled out an essential role for the helix or helix-barrel interactions in folding and membrane insertion. Measurements of thermal stability indicated that the variants R59L, W17A/R59L and Delta(1-19) were destabilised substantially compared with wild-type PagP. However, in contrast to the other variants, destabilisation of the W17A variant relative to wild-type PagP was much greater in liposomes than in micelles. Analysis of the kinetics of folding and unfolding of all variants in diC(12:0)PC liposomes suggested that this destabilisation arises predominantly from an increased dissociation of the refolded variant proteins from the lipid-inserted state. The data support the view that the helix of PagP is not required for folding and assembly, but instead acts as a clamp, stabilising membrane-inserted PagP after folding and docking with the membrane are complete.

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.

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.

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.

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.

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.

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.