Stefan Köster

Structural basis of Na+-independent and cooperative substrate/product antiport in CaiT

Transport of solutes across biological membranes is performed by specialized secondary transport proteins in the lipid bilayer, and is essential for life. Here we report the structures of the sodium-independent carnitine/butyrobetaine antiporter CaiT from Proteus mirabilis (PmCaiT) at 2.3-Å and from Escherichia coli (EcCaiT) at 3.5-Å resolution. CaiT belongs to the family of betaine/carnitine/choline transporters (BCCT), which are mostly Na+ or H+ dependent, whereas EcCaiT is Na+ and H+ independent. The three-dimensional architecture of CaiT resembles that of the Na+-dependent transporters LeuT and BetP, but in CaiT a methionine sulphur takes the place of the Na+ ion to coordinate the substrate in the central transport site, accounting for Na+-independent transport. Both CaiT structures show the fully open, inward-facing conformation, and thus complete the set of functional states that describe the alternating access mechanism. EcCaiT contains two bound butyrobetaine substrate molecules, one in the central transport site, the other in an extracellular binding pocket. In the structure of PmCaiT, a tryptophan side chain occupies the transport site, and access to the extracellular site is blocked. Binding of both substrates to CaiT reconstituted into proteoliposomes is cooperative, with Hill coefficients up to 1.7, indicating that the extracellular site is regulatory. We propose a mechanism whereby the occupied regulatory site increases the binding affinity of the transport site and initiates substrate translocation.

Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation

Listeriolysin O (LLO) is an essential virulence factor of Listeria monocytogenes that causes listeriosis. Listeria monocytogenes owes its ability to live within cells to the pH- and temperature-dependent pore-forming activity of LLO, which is unique among cholesterol-dependent cytolysins. LLO enables the bacteria to cross the phagosomal membrane and is also involved in activation of cellular processes, including the modulation of gene expression or intracellular Ca(2+) oscillations. Neither the pore-forming mechanism nor the mechanisms triggering the signalling processes in the host cell are known in detail. Here, we report the crystal structure of LLO, in which we identified regions important for oligomerization and pore formation. Mutants were characterized by determining their haemolytic and Ca(2+) uptake activity. We analysed the pore formation of LLO and its variants on erythrocyte ghosts by electron microscopy and show that pore formation requires precise interface interactions during toxin oligomerization on the membrane.

pH-Induced Conformational Change of the β-Barrel-Forming Protein OmpG Reconstituted into Native E. coli Lipids

A gating mechanism of the beta-barrel-forming outer membrane protein G (OmpG) from Escherichia coli was recently presented. The mechanism was based on X-ray structures revealed from crystals grown from solubilized OmpG at both neutral pH and acidic pH. To investigate whether these conformations represent the naturally occurring gating mechanism, we reconstituted OmpG in native E. coli lipids and applied high-resolution atomic force microscopy. The reconstituted OmpG molecules assembled into both monomers and dimers. Single monomeric and dimeric OmpG molecules showed open channel entrances at pH 7.5 and at room temperature. The extracellular loops connecting the beta-strands that form the transmembrane beta-barrel pore exhibited elevated structural flexibility. Upon lowering the pH to 5.0, the conformation of OmpG molecules changed to close the extracellular entrance of their channel. It appears that one or more of the extracellular loops collapsed onto the channel entrance. This conformational change was fully reversible. Our data confirm that the previously reported gating mechanism of OmpG occurs at physiological conditions in E. coli lipid membranes.

One β Hairpin after the Other: Exploring Mechanical Unfolding Pathways of the Transmembrane β‐Barrel Protein OmpG

Single-molecule force spectroscopy (SMFS) is a unique approach to study the mechanical unfolding of proteins. Such forced unfolding experiments yield insight into how interactions stabilize a protein and guide its unfolding pathways. Previous SMFS work has probed the mechanical stability of water-soluble proteins composed of a helices and b strands. A prominent example of unfolding of a b-barrel structure is that of the green fluorescent protein (GFP), the stability of which plays a major role for its application as a marker in modern fluorescence microscopy. In contrast to the variety of water-soluble proteins characterized, only a-helical membrane proteins have been probed by SMFS. It was found that a-helical membrane proteins unfold via many intermediates, which is different to the mostly two-state unfolding process of water-soluble proteins. Upon mechanically pulling the peptide end of a membrane protein, single and grouped a helices and polypeptide loops unfold in steps until the entire protein has unfolded. Whether the a helices and loops unfold individually or cooperatively to form an unfolding intermediate depends on the interactions established within the membrane protein and with the environment. Each of these unfolding events creates an unfolding intermediate with the sequence of intermediates describing the unfolding pathway taken. However, so far, b-barrel-forming membrane proteins have not been characterized by SMFS. For these reasons, we have characterized the interactions and unfolding of the b-barrel-forming outer-membrane protein OmpG from Escherichia coli by SMFS. The structure of OmpG comprises 14 b strands that form a transmembrane b-barrel pore. Six short loops (T1–T6) on the periplasmic side and seven longer loops (L1–L7) on the extracellular side connect the individual b strands. OmpG is gated by loop L6, which controls the flux of small molecules through the pore and the permeability of the bacterial outer membrane in a pH-dependent manner. Being able to withstand rather harsh environmental conditions, OmpG forms a robust pore, which makes it suitable for application as a biosensor. In our SMFS experiments, OmpG reconstituted in E. coli lipid membranes were first imaged by AFM. The AFM tip was then pushed onto the OmpG surface to facilitate the nonspecific attachment of the N or C terminus (Figure 1a).

pH-Dependent Interactions Guide the Folding and Gate the Transmembrane Pore of the β-Barrel Membrane Protein OmpG

The physical interactions that switch the functional state of membrane proteins are poorly understood. Previously, the pH-gating conformations of the beta-barrel forming outer membrane protein G (OmpG) from Escherichia coli have been solved. When the pH changes from neutral to acidic the flexible extracellular loop L6 folds into and closes the OmpG pore. Here, we used single-molecule force spectroscopy to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At acidic pH, we detected a pH-dependent interaction at loop L6. This interaction changed the (un)folding of loop L6 and of beta-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pH-dependent interactions change the folding of a peptide loop to gate the transmembrane pore. They further demonstrate how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore.

Structure of Human Na+/H+ Exchanger NHE1 Regulatory Region in Complex with Calmodulin and Ca2+

Background: The human Na+/H+ exchanger NHE1 is activated through binding of calmodulin. Results: We determined the x-ray structure of the NHE1 regulatory region in complex with calmodulin and calcium. Conclusion: The complex structure serves as a basis for a transport regulatory model. Significance: The complex structure improves our understanding of the medically important NHE1. The ubiquitous mammalian Na+/H+ exchanger NHE1 has critical functions in regulating intracellular pH, salt concentration, and cellular volume. The regulatory C-terminal domain of NHE1 is linked to the ion-translocating N-terminal membrane domain and acts as a scaffold for signaling complexes. A major interaction partner is calmodulin (CaM), which binds to two neighboring regions of NHE1 in a strongly Ca2+-dependent manner. Upon CaM binding, NHE1 is activated by a shift in sensitivity toward alkaline intracellular pH. Here we report the 2.23 Å crystal structure of the NHE1 CaM binding region (NHE1CaMBR) in complex with CaM and Ca2+. The C- and N-lobes of CaM bind the first and second helix of NHE1CaMBR, respectively. Both the NHE1 helices and the Ca2+-bound CaM are elongated, as confirmed by small angle x-ray scattering analysis. Our x-ray structure sheds new light on the molecular mechanisms of the phosphorylation-dependent regulation of NHE1 and enables us to propose a model of how Ca2+ regulates NHE1 activity.

Structure and Function of the Feob G-Domain from Methanococcus Jannaschii

FeoB in bacteria and archaea is involved in the uptake of ferrous iron (Fe(2+)), an important cofactor in biological electron transfer and catalysis. Unlike any other known prokaryotic membrane protein, FeoB contains a GTP-binding domain at its N-terminus. We determined high-resolution X-ray structures of the FeoB G-domain from Methanococcus jannaschii with and without bound GDP or Mg(2+)-GppNHp. The G-domain forms the same dimer in all three structures, with the nucleotide-binding pockets at the dimer interface, as in the ATP-binding domain of ABC transporters. The G-domain follows the typical fold of nucleotide-binding proteins, with a beta-strand inserted in switch I that becomes partially disordered upon GTP binding. Switch II does not contact the nucleotide directly and does not change its conformation in response to the bound nucleotide. Release of the nucleotide causes a rearrangement of loop L6, which we identified as the G5 region of FeoB. Together with the C-terminal helix, this loop may transmit the information about the nucleotide-bound state from the G-domain to the transmembrane region of FeoB.

One β Hairpin Follows the Other: Exploring Refolding Pathways and Kinetics of the Transmembrane β-Barrel Protein OmpG

Despite their enormous relevance to cellular vitality, the folding mechanisms of only a few transmembrane proteins have been studied. From these studies, only a handful of bstranded membrane proteins were characterized. Current models describe that transmembrane b barrels fold into the lipid membrane in two major steps. Firstly, the unfolded polypeptide interacts with the lipid surface where it folds, tilts, and then inserts into the membrane. Consequently, it is thought that single b strands and b hairpins form unstable units, and that b-barrel proteins (pre-)fold prior to their insertion into the cellular membrane. Experiments studying the (un-)folding of membrane proteins are conventionally carried out by using thermal or chemical denaturation. In most cases, membrane proteins that were solubilized in detergent and/or exposed to approximately 4–10m urea were studied. In vivo membrane proteins fold under different conditions. Thus, the folding pathways studied may be different from those that occur in nature. Single-molecule force spectroscopy (SMFS) represents a unique approach to studying the refolding of membrane proteins into the lipid membrane. SMFS is used to unfold and refold membrane proteins under conditions typical for their physiological environment such as pH, electrolytes, temperature, and, importantly in the absence of any chemical denaturant or detergent. In such experiments, a single membrane protein is first mechanically unfolded and its polypeptide is fully stretched. Then this unfolded polypeptide is relaxed to allow refolding into the membrane bilayer. Repeated mechanical unfolding is used to determine which structural regions of the membrane protein are refolded. Allowing the polypeptide different refolding times addresses the refolding kinetics of structural regions. Thus, SMFS can be used to detect the mechanical unfolding pathways and the equilibrium refolding pathways of a membrane protein. In previous SMFS work, the mechanical unfolding and refolding of many different water-soluble proteins have been investigated. However, compared to the variety of water-soluble proteins that were characterized, SMFS of membrane proteins reveals much more detailed unfolding and folding pathways. To date, the refolding of b-barrel membrane proteins into a lipid membrane has never been addressed by SMFS. Herein we report the application of SMFS to unfold and refold the outer membrane protein G (OmpG) from Escherichia coli (Figure 1). The structure of OmpG comprises 14 b strands that form a transmembrane b-barrel pore. Six short turns connect individual b strands on the periplasmic side and seven longer loops (L1–L7) on the extracellular side. In vitro experiments show that OmpG is gated by loop L6, which controls the permeability of the pore in a pHdependent manner. In previous SMFS studies, we found that the b barrel of OmpG unfolds via many intermediates. The main unfolding pathway described the stepwise unfolding of single b hairpins. This unfolding pathway was much more detailed than that detected for the water-soluble b-barrel green fluorescent protein (GFP), which mainly unfolds in one step when a sufficiently high pulling force was applied. In our refolding experiments, OmpG that had been reconstituted in native E. coli lipid membranes was first imaged by AFM. Then, the AFM tip was pushed onto the OmpG surface to facilitate the nonspecific attachment of the N terminus (Figure 1). Withdrawal of the AFM tip stretched the terminus and induced the unfolding of OmpG. Force– distance (F–D) curves recorded the force peaks that reflect the unfolding steps of a single OmpG (Figure 1). Each unfolding step represents that of a b hairpin of the transmembrane b barrel. To refold the partially unfolded OmpG, we stopped withdrawal before unfolding the last b hairpin VII. Then, we relaxed the unfolded polypeptide by approaching the AFM tip close to the membrane (ca. 5 nm). After a given time to allow the polypeptide to refold, the protein was unfolded again to probe which structural regions refolded into the lipid membrane (see Figure S1 in the Supporting Information). Individual F–D curves of the refolding polypeptide showed a series of force peaks that varied in occurrence (Figure 1). These force peaks were detected at similar positions as upon initial unfolding of OmpG. If b hairpins had folded without inserting or had attached to themembrane surface, the force peaks would have been detected at shifted positions (see the Supporting Information, Part 2). Similarly, force peaks which are characteristic for the folding of membrane proteins, would have changed their position if misfolding events had occurred. Thus, the unfolded OmpG polypeptide folded and inserted single b hairpins into the native E. coli lipid membrane. Probing the content of refolding in dependence of different refolding times (0.1–5 s) [*] M. Damaghi, Dr. C. A. Bippes, Prof. Dr. D. J. M ller ETH Z rich, Dept. of Biosystems Science and Engineering 4058 Basel (Switzerland) Fax: (+41)61-387-3994 E-mail: daniel.mueller@bsse.ethz.ch

Correlation between the Ompg Secondary Structure and its Ph-Dependent Alterations Monitored by Ftir.

The channel activity of the outer-membrane protein G (OmpG) from Escherichia coli is pH-dependent. To investigate the role of the histidine pair His231/His261 in triggering channel opening and closing, we mutated both histidines to alanines and cysteines. Fourier transform infrared spectra revealed that the OmpG mutants stay-independent of pH-in an open conformation. Temperature ramp experiments indicate that the mutants are as stable as the open state of wild-type OmpG. The X-ray structure of the alanine-substituted OmpG mutant obtained at pH 6.5 confirms the constitutively open conformation. Compared to previous structures of the wild-type protein in the open and closed conformation, the mutant structure shows a difference in the extracellular loop L6 connecting beta-strands S12 and S13. A deletion of amino acids 220-228, which are thought to block the channel at low pH in wild-type OmpG, indicates conformational changes, which might be triggered by His231/His261.

The Role of Lipids for the Functional Integrity of Porin: An FTIR Study Using Lipid and Protein Reporter Groups

We have investigated the temperature-dependent interaction of the porins OmpF from Paracoccus denitrificans and OmpG from Escherichia coli with lipid molecules after reconstitution in lecithin. Effects of incubation at increased temperatures on activity were tested by functional experiments for OmpG and compared with previously published results of OmpF in order to understand the activity loss of OmpF with monomerization. Protein-lipid interaction was monitored by different reporter groups both from lipid molecules and from protein. OmpF loses its activity by approximately 90% at 50 degrees C while OmpG does not show a temperature-dependent change in activity between room temperature and 50 degrees C. The interaction between OmpF and lipid molecules is severely altered in a two-step mechanism at 55 and approximately 75 degrees C for OmpF. The first step is attributed to changes in the degree of interaction between the aromatic girdle of OmpF and the interfacial region of the lipid bilayer, leading to monomerization of this trimeric porin. The second step at 75 degrees C is attributed to the changes in lipid-porin monomer interaction. Around 90 degrees C, reconstituted porin aggregates. For OmpG, changes in lipid-protein interaction were observed starting from approximately 80 degrees C because of temperature-induced breakdown of its folding. This study provides deeper understanding of porin-lipid bilayer interaction as a function of temperature and can explain the functional breakdown by monomerization while porin secondary structure is still preserved.