Inverted-topology Repeats Research Articles

Crystal structure of an intramembranal phosphatase central to bacterial cell-wall peptidoglycan biosynthesis and lipid recycling

Undecaprenyl pyrophosphate phosphatase (UppP) is an integral membrane protein that recycles the lipid carrier essential to the ongoing biosynthesis of the bacterial cell wall. Individual building blocks of peptidoglycan are assembled in the cytoplasm on undecaprenyl phosphate (C55-P) before being flipped to the periplasmic face, where they are polymerized and transferred to the existing cell wall sacculus, resulting in the side product undecaprenyl pyrophosphate (C55-PP). Interruption of UppP’s regeneration of C55-P from C55-PP leads to the buildup of cell wall intermediates and cell lysis. We present the crystal structure of UppP from Escherichia coli at 2.0 Å resolution, which reveals the mechanistic basis for intramembranal phosphatase action and substrate specificity using an inverted topology repeat. In addition, the observation of key structural motifs common to a variety of cross membrane transporters hints at a potential flippase function in the specific relocalization of the C55-P product back to the cytosolic space.

Asymmetry of inverted-topology repeats in the AE1 anion exchanger suggests an elevator-like mechanism.

The membrane transporter anion exchanger 1 (AE1), or band 3, is a key component in the processes of carbon-dioxide transport in the blood and urinary acidification in the renal collecting duct. In both erythrocytes and the basolateral membrane of the collecting-duct α-intercalated cells, the role of AE1 is to catalyze a one-for-one exchange of chloride for bicarbonate. After decades of biochemical and functional studies, the structure of the transmembrane region of AE1, which catalyzes the anion-exchange reaction, has finally been determined. Each protomer of the AE1 dimer comprises two repeats with inverted transmembrane topologies, but the structures of these repeats differ. This asymmetry causes the putative substrate-binding site to be exposed only to the extracellular space, consistent with the expectation that anion exchange occurs via an alternating-access mechanism. Here, we hypothesize that the unknown, inward-facing conformation results from inversion of this asymmetry, and we propose a model of this state constructed using repeat-swap homology modeling. By comparing this inward-facing model with the outward-facing experimental structure, we predict that the mechanism of AE1 involves an elevator-like motion of the substrate-binding domain relative to the nearly stationary dimerization domain and to the membrane plane. This hypothesis is in qualitative agreement with a wide range of biochemical and functional data, which we review in detail, and suggests new avenues of experimentation.

An Outward-Facing Open Conformational State in a CLC Transporter

As secondary active transporters, CLCs harness energy stored in one ion concentration gradient (Cl- or H+) to pump the other ion against its electrochemical gradient. This occurs through tight coupling of protein conformational changes to ion binding, unbinding, and translocation events. Crystal structures suggest that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Here, we combine spectroscopy, cross-linking studies, crystallography, and computation to evaluate this simple model. Using 19F NMR and double electron-electron resonance (DEER) spectroscopy, we show that as [H+] is increased to enrich the outward-facing state, residues distant from Gluex move. Consistent with the functional relevance of this motion, a cross-link designed to inhibit the motions reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. Together, the results indicate the existence of a previously uncharacterized “outward-facing open” conformational state. A computational model based on the asymmetry exchange hypothesis for transporters with inverted-topology repeats (Forrest et al. 2008, PNAS) predicts such a state. To test the computational model, we monitored H+-induced structural changes using DEER spectroscopy on >20 sets of doubly labeled ClC-ec1 protein samples. Experimental distance distributions are compared with those derived from the ClC-ec1 X-ray structure and the model using restrained-ensemble molecular dynamics simulations (Roux et al. 2013, J Phys Chem B). Preliminary results show H+-dependent structural changes consistent with the computational model that can also be used to refine it. These results highlight the relevance of global structural changes in CLC function and provide the necessary foundation for understanding the Cl-/H+ coupling mechanism.

Sodium-Proton (Na(+)/H(+)) Antiporters: Properties and Roles in Health and Disease.

The transmembranal Na(+)/H(+) antiporters transport sodium (or several other monovalent cations) in exchange for H(+) across lipid bilayers in all kingdoms of life. They are critical in pH homeostasis of the cytoplasm and/or organelles. A particularly notable example is the SLC9 gene family, which encodes Na(+)/H(+) exchangers (NHEs) in many species from prokaryotes to eukaryotes. In humans, these proteins are associated with the pathophysiology of various diseases. Yet, the most extensively studied Na(+)/H(+) antiporter is Ec-NhaA, the main Na(+)/H(+) antiporter of Escherichia coli.The crystal structure of down-regulated Ec-NhaA, determined at acidic pH, has provided the first structural insights into the antiport mechanism and pH regulation of an Na(+)/H(+) antiporter. It reveals a unique structural fold (called the NhaA fold) in which transmembrane segments (TMs) are organized in inverted-topology repeats, including two antiparallel unfolded regions that cross each other, forming a delicate electrostatic balance in the middle of the membrane. This unique structural fold (The NhaA fold) contributes to the cation binding site and facilitates the rapid conformational changes expected for Ec-NhaA. The NhaA fold has now been recognized to be shared by four Na(+)/H(+) antiporters (bacterial and archaeal) and a Na(+) symporter. Remarkably, no crystal structure of any of the human Na(+)/H(+) antiporters exists. Nevertheless, the Ec-NhaA crystal structure has enabled the structural modeling of NHE1, NHE9, and NHA2, three human plasmalemmal proteins that are members of the SLC9 family that are involved in human pathophysiology. Moreover, as outlined in this review, developments in the field, including cellular and biophysical methods that enable ion levels and fluxes to be measured in intact cells as well as in knockout mice, have led to striking advances in the identification and characterization of plasma membrane NHEs and NHA.Very little is known about the endomembrane isoforms of NHE. These intracellular exchangers may serve a function in cation homeostasis and/or osmoregulation, and not in pH regulation as is the case for the plasmalemmal isoforms. This intriguing possibility should be borne in mind when designing future studies. Future progress towards gaining an understanding of the SLC9 gene family, including its structure-function relationships and regulatory mechanisms in health and in disease, is likely to include insights into the pathophysiology of multiple diseases.

Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms.

Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to one or other side of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (a)symmetry of these systems has been successfully used as a bioinformatic tool, called “repeat-swap modeling” to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that nucleoside transport also occurs via an elevator-like mechanism.

Gating Topology of the Proton-Coupled Oligopeptide Symporters

SummaryProton-coupled oligopeptide transporters belong to the major facilitator superfamily (MFS) of membrane transporters. Recent crystal structures suggest the MFS fold facilitates transport through rearrangement of their two six-helix bundles around a central ligand binding site; how this is achieved, however, is poorly understood. Using modeling, molecular dynamics, crystallography, functional assays, and site-directed spin labeling combined with double electron-electron resonance (DEER) spectroscopy, we present a detailed study of the transport dynamics of two bacterial oligopeptide transporters, PepTSo and PepTSt. Our results identify several salt bridges that stabilize outward-facing conformations and we show that, for all the current structures of MFS transporters, the first two helices of each of the four inverted-topology repeat units form half of either the periplasmic or cytoplasmic gate and that these function cooperatively in a scissor-like motion to control access to the peptide binding site during transport.

Molecular Dynamics Simulations on the Periplasmic-Open State Lactose Permease

Lactose Permease (LacY) is a secondary active transporter (SAT) that belongs to major facilitator superfamily (MFS). LacY structures of the cytoplasmic-open and more recently occluded-like structure have been determined. The structure of periplasmic-open LacY is important for understanding complete proton/sugar transport of LacY as well as other similar transporters. Though the exact structure of periplasmic-open LacY has not been obtained experimentally, a few molecular models have been determined. Previously, Pendse et al. (JMB, 404: 506-521) obtained a periplasmic-open LacY model through a two-step hybrid implicit-explicit (IM-EX) simulation method. Radestock et al. (JMB, 407: 698-715) proposed two new periplasmic-open LacY models by swapping inverted-topology repeats and adopting the outward L-fucose-proton symporter (fucP) crystal structure. The accuracy of these three periplasmic-open state models are tested by performing MD simulations on LacY-apo and LacY-ββ-(Galp)2. The comparison of the calculated pore radii to the data of the crystal structure indicates that the IM-EX model of LacY remains in periplasmic-open state. However, Radestock et al.'s models close on the periplasmic side and partially close on the cytoplasmic side, which indicates these two outward LacY models are unstable. Compared to the DEER experimental data, the selected residue pair distance changes demonstrates that the repeat-swapped LacY seems to be in the occluded state by 100ns. We will run MD simulations with labeled residues to directly compare with DEER for the IM-EX model to verify the effects of the spin label size and orientation on the accuracy of the distance measurement.

Monitoring Substrate-Driven Structural Changes in a CLC Chloride-Proton Antiporter with Double Electron-Electron Resonance Spectroscopy

ClC-ec1, a prokaryotic Cl-/H+ antiporter in the CLC family, has been crystallized under many conditions. Yet, aside from local structural differences at the chloride binding site, only one major conformation is observed. This failure of X-ray crystallography to reveal the unknown conformational states in the transport cycle motivates the use of alternative approaches. Using 19F-NMR, a previous study demonstrated that a tyrosine residue > 20 A from the chloride-transport pathway undergoes antiport-specific structural changes (Elvington et al. 2009. EMBO J). We combine computational and experimental approaches to further investigate possible protein movements during the antiport cycle. A computational model of ClC-ec1 based on the inverted topology repeat hypothesis (Forrest et al. 2008. PNAS) predicts structural changes that alter the accessibility of pathways to the central Cl- and H+ binding sites. To test the model, we introduced nitroxide paramagnetic spin labels to the ClC-ec1 homodimer via site-directed spin labeling and monitored structural changes using double electron-electron resonance (DEER) spectroscopy. Preliminary results are consistent with the inverted-topology repeat model and suggest that the antiport cycle involves structural changes beyond local movement at the chloride-binding site.

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP.

Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodium-binding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuT-like (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent K(m) and K(d) for sodium, as measured by betaine uptake, tryptophan fluorescence, and (22)Na(+) binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.

The Alternating-Access Mechanism of MFS Transporters Arises from Inverted-Topology Repeats

Lactose permease (LacY) is the prototype of the major facilitator superfamily (MFS) of secondary transporters. Available structures of LacY reveal a state in which the substrate is exposed to the cytoplasm but is occluded from the periplasm. However, the alternating-access transport mechanism requires the existence of a periplasm-facing state. We recently showed that inverted-topology structural repeats provide the foundation for the mechanisms of two transporter families with folds distinct from the MFS. Here, we generated a structural model of LacY by swapping the conformations of inverted-topology repeats identified in its two domains. The model exhibits all required properties of an outward-facing conformation, i.e., closure of the binding site to the cytoplasm and exposure to the periplasm. Furthermore, the model agrees with double electron–electron resonance distance changes, accessibility to cysteine-modifying reagents, cysteine cross-linking data, and a recent structure of a distantly related transporter. Analysis of the intradomain differences between the two states suggests a role for conserved sequence motifs in occluding the central pathway through kinking of the pore-lining helices. In addition, predicted re-pairing of critical salt-bridging residues in the binding sites agrees remarkably well with previous proposals, allowing a description of the proton/sugar transport mechanism. More fundamentally, our model demonstrates that inverted-topology repeats provide the foundation for the alternating-access mechanisms of MFS transporters.

Symmetry in the Structure of the Glutamate Transporter GltPh Suggests Conformation of an Alternate State

Glutamate transporters regulate synaptic concentrations of L-glutamate to prevent excitotoxicity in nerve cells. Current crystal structures of GltPh, an archeal homologue of the Glutamate transporters, have an extracellular-facing binding site. The alternating access theory implies that a cytoplasm-facing state also exists. In order to model this state, we have identified two distinct sets of inverted-topology repeats, and used these repeats to model an inward-facing conformation of the protein. Specifically, we modeled the sequence of each repeat on the structure of its partner. In this model, a portion of the protein containing two transmembrane helices (TM7 and 8) and two helical hairpins (HP1 and HP2) is displaced relative to the crystal structure so that the binding site is exposed to the cytoplasm. In order to validate our model, pairs of cysteines were introduced into the neuronal glutamate transporter EAAC1 at positions that were greater than 27 Angstroms apart in the outward-facing crystal structure, but closer to 10 Angstroms apart in our model. Transport in these mutants was activated by pretreatment with the reducing agent dithithreitol. Once treated with the oxidizing agent copper(II)(1,10-phenantroline)3, however, activity ceased. Importantly, this inhibition was potentiated under conditions expected to promote the inward-facing conformation. This suggests that during the transport cycle these cysteines come within the range necessary to crosslink, as predicted by our inverted-topology repeat model of the cytoplasm-facing state. Previously, an alternative conformational state of the LeuT transporter was also modeled using inverted-topology repeats, suggesting that inverted-topology repeats may provide a general and elegant solution to the requirement for two symmetry-related states in a single protein.

Inward-facing conformation of glutamate transporters as revealed by their inverted-topology structural repeats

Glutamate transporters regulate synaptic concentrations of this neurotransmitter by coupling its flux to that of sodium and other cations. Available crystal structures of an archeal homologue of these transporters, GltPh, resemble an extracellular-facing state, in which the bound substrate is occluded only by a small helical hairpin segment called HP2. However, a pathway to the cytoplasmic side of the membrane is not clearly apparent. We previously modeled an alternate state of a transporter from the neurotransmitter:sodium symporter family, which has an entirely different fold, solely on the presence of inverted-topology structural repeats. In GltPh, we identified two distinct sets of inverted-topology repeats and used these repeats to model an inward-facing conformation of the protein. To test this model, we introduced pairs of cysteines into the neuronal glutamate transporter EAAC1, at positions that are >27 A apart in the crystal structures of GltPh, but approximately = 10 A apart in the inward-facing model. Transport by these mutants was activated by pretreatment with the reducing agent dithithreitol. Subsequent treatment with the oxidizing agent copper(II)(1,10-phenantroline)(3) abolished this activation. The inhibition of transport was potentiated under conditions thought to promote the inward-facing conformation of the transporter. By contrast, the inhibition was reduced in the presence of the nontransportable substrate analogue D,L-threo-beta-benzyloxyaspartate, which favors the outward-facing conformation. Other conformation-sensitive accessibility measurements are also accommodated by our inward-facing model. These results suggest that the inclusion of inverted-topology repeats in transporters may provide a general solution to the requirement for two symmetry-related states in a single protein.