Substrate binding in the multidrug transporter MdfA in detergent solution and in lipid nanodiscs.

MdfA from Escherichia coli is a prototypical secondary multi-drug (Mdr) transporter that exchanges drugs for protons. MdfA-mediated drug efflux is driven by the proton gradient and enabled by conformational changes that accompany the recruitment of drugs and their release. In this work, we applied distance measurements by W-band double electron-electron resonance (DEER) spectroscopy to explore the binding of mito-TEMPO, a nitroxide-labeled substrate analog, to Gd(III)-labeled MdfA. The choice of Gd(III)-nitroxide DEER enabled measurements in the presence of excess of mito-TEMPO, which has a relatively low affinity to MdfA. Distance measurements between mito-TEMPO and MdfA labeled at the periplasmic edges of either of three selected transmembrane helices (TM3101, TM5168, and TM9310) revealed rather similar distance distributions in detergent micelles (n-dodecyl-ß-d-maltopyranoside, DDM)) and in lipid nanodiscs (ND). By grafting the predicted positions of the Gd(III) tag on the inward-facing (If) crystal structure, we looked for binding positions that reproduced the maxima of the distance distributions. The results show that the location of the mito-TEMPO nitroxide in DDM-solubilized or ND-reconstituted MdfA is similar (only 0.4 nm apart). In both cases, we located the nitroxide moiety near the ligand binding pocket in the If structure. However, according to the DEER-derived position, the substrate clashes with TM11, suggesting that for mito-TEMPO-bound MdfA, TM11 should move relative to the If structure. Additional DEER studies with MdfA labeled with Gd(III) at two sites revealed that TM9 also dislocates upon substrate binding. Together with our previous reports, this study demonstrates the utility of Gd(III)-Gd(III) and Gd(III)-nitroxide DEER measurements for studying the conformational behavior of transporters.

Dissection of mechanistic principles of a secondary multidrug efflux protein.

Multidrug transporters are ubiquitous efflux pumps that provide cells with defense against various toxic compounds. In bacteria, which typically harbor numerous multidrug transporter genes, the majority function as secondary multidrug/proton antiporters. Proton-coupled secondary transport is a fundamental process that is not fully understood, largely owing to the obscure nature of proton-transporter interactions. Here we analyzed the substrate/proton coupling mechanism in MdfA, a model multidrug/proton antiporter. By measuring the effect of protons on substrate binding and by directly measuring proton binding and release, we show that substrates and protons compete for binding to MdfA. Our studies strongly suggest that competition is an integral feature of secondary multidrug transport. We identified the proton-binding acidic residue and show that, surprisingly, the substrate binds at a different site. Together, the results suggest an interesting mode of indirect competition as a mechanism of multidrug/proton antiport.

Co-translational membrane association of the Escherichia coli SRP receptor.

The signal recognition particle (SRP) receptor is a major player in the pathway of membrane protein biogenesis in all organisms. The receptor functions as a membrane-bound entity but very little is known about its targeting to the membrane. Here, we demonstrate in vivo that the Escherichia coli SRP receptor targets the membrane co-translationally. This requires emergence from the ribosome of the four-helix-long N-domain of the receptor, of which only helices 2-4 are required for co-translational membrane attachment. The results also suggest that the targeting might be regulated co-translationally. Taken together, our in vivo studies shed light on the biogenesis of the SRP receptor and its hypothetical role in targeting ribosomes to the E. coli membrane.

Is there a twist in the Escherichia coli signal recognition particle pathway?

Integral membrane proteins (IMPs) are usually synthesized by membrane-bound ribosomes, and this process requires proper localization of ribosomes and IMP-encoding transcripts. However, the underlying molecular mechanism of the pathway has not yet been fully established in vivo. The prevailing hypothesis is that ribosomes and transcripts are delivered to the membrane together during IMP translation by the signal recognition particle (SRP) and its receptor. Here, I discuss an alternative hypothesis that posits that ribosomes and transcripts are targeted separately. Ribosome targeting to the membrane might be mediated by the SRP receptor, rather than by SRP, and IMP-encoding transcripts might be targeted to the membrane in a translation-independent manner. According to this scenario, the SRP executes its essential function on the membrane at a later stage of the targeting pathway.

How intramembrane proteases bury hydrolytic reactions in the membrane.

Intramembrane proteolysis is increasingly seen as a regulatory step in a range of diverse processes, including development, organelle shaping, metabolism, pathogenicity and degenerative disease. Initial scepticism over the existence of intramembrane proteases was soon replaced by intense exploration of their catalytic mechanisms, substrate specificities, regulation and structures. Crystal structures of metal-dependent and serine intramembrane proteases have revealed active sites embedded in the plane of the membrane but accessible by water, a requirement for hydrolytic reactions. Efforts to understand how these membrane-bound proteases carry out their reactions have started to yield results.

Manipulating the drug/proton antiport stoichiometry of the secondary multidrug transporter MdfA.

Multidrug transporters are integral membrane proteins that use cellular energy to actively extrude antibiotics and other toxic compounds from cells. The multidrug/proton antiporter MdfA from Escherichia coli exchanges monovalent cationic substrates for protons with a stoichiometry of 1, meaning that it translocates only one proton per antiport cycle. This may explain why transport of divalent cationic drugs by MdfA is energetically unfavorable. Remarkably, however, we show that MdfA can be easily converted into a divalent cationic drug/≥ 2 proton-antiporter, either by random mutagenesis or by rational design. The results suggest that exchange of divalent cationi c drugs with two (or more) protons requires an additional acidic residue in the multidrug recognition pocket of MdfA. This outcome further illustrates the exceptional promiscuous capabilities of multidrug transporters.

Early targeting events during membrane protein biogenesis in Escherichia coli.

All living cells have co-translational pathways for targeting membrane proteins. Co-translation pathways for secretory proteins also exist but mostly in eukaryotes. Unlike secretory proteins, the biosynthetic pathway of most membrane proteins is conserved through evolution and these proteins are usually synthesized by membrane-bound ribosomes. Translation on the membrane requires that both the ribosomes and the mRNAs be properly localized. Theoretically, this can be achieved by several means. (i) The current view is that the targeting of cytosolic mRNA-ribosome-nascent chain complexes (RNCs) to the membrane is initiated by information in the emerging hydrophobic nascent polypeptides. (ii) The alternative model suggests that ribosomes may be targeted to the membrane also constitutively, whereas the appropriate mRNAs may be carried on small ribosomal subunits or targeted by other cellular factors to the membrane-bound ribosomes. Importantly, the available experimental data do not rule out the possibility that cells may also utilize both pathways in parallel. In any case, it is well documented that a major player in the targeting pathway is the signal recognition particle (SRP) system composed of the SRP and its receptor (SR). Although the functional core of the SRP system is evolutionarily conserved, its composition and biological practice come with different flavors in various organisms. This review is dedicated mainly to the Escherichia (E.) coli SRP, where the biochemical and structural properties of components of the SRP system have been relatively characterized, yielding essential information about various aspects of the pathway. In addition, several cellular interactions of the SRP and its receptor have been described in E. coli, providing insights into their spatial function. Collectively, these in vitro studies have led to the current view of the targeting pathway [see (i) above]. Interestingly, however, in vivo studies of the role of the SRP and its receptor, with emphasis on the temporal progress of the pathway, elicited an alternative hypothesis [see (ii) above]. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Studying membrane proteins through the eyes of the genetic code revealed a strong uracil bias in their coding mRNAs.

Posttranscriptional processes often involve specific signals in mRNAs. Because mRNAs of integral membrane proteins across evolution are usually translated at distinct locations, we searched for universally conserved specific features in this group of mRNAs. Our analysis revealed that codons of very hydrophobic amino acids, highly represented in integral membrane proteins, are composed of 50% uracils (U). As expected from such a strong U bias, the calculated U profiles of mRNAs closely resemble the hydrophobicity profiles of their encoded proteins and may designate genes encoding integral membrane proteins, even in the absence of information on ORFs. We also show that, unexpectedly, the U-richness phenomenon is not merely a consequence of the codon composition of very hydrophobic amino acids, because counterintuitively, the relatively hydrophilic serine and tyrosine, also encoded by U-rich codons, are overrepresented in integral membrane proteins. Interestingly, although the U-richness phenomenon is conserved, there is an evolutionary trend that minimizes usage of U-rich codons. Taken together, the results suggest that U-richness is an evolutionarily ancient feature of mRNAs encoding integral membrane proteins, which might serve as a physiologically relevant distinctive signature to this group of mRNAs.

Co-Translational Membrane Targeting and Holo-Translocon Docking of Ribosomes Translating the SRP Receptor.

Many integral membrane proteins are produced by translocon-associated ribosomes. The assembly of ribosomes translating membrane proteins on the translocons is mediated by a conserved system, composed of the signal recognition particle and its receptor (FtsY in Escherichia coli). FtsY is a peripheral membrane protein, and its role late during membrane protein targeting involves interactions with the translocon. However, earlier stages in the pathway have remained obscure, namely, how FtsY targets the membrane in vivo and where it initially docks. Our previous studies have demonstrated co-translational membrane-targeting of FtsY translation intermediates and identified a nascent FtsY targeting-peptide. Here, in a set of in vivo experiments, we utilized tightly stalled FtsY translation intermediates, pull-down assays and site-directed cross-linking, which revealed FtsY-nascent chain-associated proteins in the cytosol and on the membrane. Our results demonstrate interactions between the FtsY-translating ribosomes and cytosolic chaperones, which are followed by directly docking on the translocon. In support of this conclusion, we show that translocon over-expression increases dramatically the amount of membrane associated FtsY-translating ribosomes. The co-translational contacts of the FtsY nascent chains with the translocon differ from its post-translational contacts, suggesting a major structural maturation process. The identified interactions led us to propose a model for how FtsY may target the membrane co-translationally. On top of our past observations, the current results may add another tier to the hypothesis that FtsY acts stoichiometrically in targeting ribosomes to the membrane in a constitutive manner.

Genetic evidence for functional interaction of the Escherichia coli signal recognition particle receptor with acidic lipids in vivo.

The mechanism underlying the interaction of the Escherichia coli signal recognition particle receptor FtsY with the cytoplasmic membrane has been studied in detail. Recently, we proposed that FtsY requires functional interaction with inner membrane lipids at a late stage of the signal recognition particle pathway. In addition, an essential lipid-binding α-helix was identified in FtsY of various origins. Theoretical considerations and in vitro studies have suggested that it interacts with acidic lipids, but this notion is not yet fully supported by in vivo experimental evidence. Here, we present an unbiased genetic clue, obtained by serendipity, supporting the involvement of acidic lipids. Utilizing a dominant negative mutant of FtsY (termed NG), which is defective in its functional interaction with lipids, we screened for E. coli genes that suppress the negative dominant phenotype. In addition to several unrelated phenotype-suppressor genes, we identified pgsA, which encodes the enzyme phosphatidylglycerophosphate synthase (PgsA). PgsA is an integral membrane protein that catalyzes the committed step to acidic phospholipid synthesis, and we show that its overexpression increases the contents of cardiolipin and phosphatidylglycerol. Remarkably, expression of PgsA also stabilizes NG and restores its biological function. Collectively, our results strongly support the notion that FtsY functionally interacts with acidic lipids.

A promiscuous conformational switch in the secondary multidrug transporter MdfA.

Multidrug (Mdr) transporters are membrane proteins that actively export structurally dissimilar drugs from the cell, thereby rendering the cell resistant to toxic compounds. Similar to substrate-specific transporters, Mdr transporters also undergo substrate-induced conformational changes. However, the mechanism by which a variety of dissimilar substrates are able to induce similar transport-compatible conformational responses in a single transporter remains unclear. To address this major aspect of Mdr transport, we studied the conformational behavior of the Escherichia coli Mdr transporter MdfA. Our results show that indeed, different substrates induce similar conformational changes in the transporter. Intriguingly, in addition, we observed that compounds other than substrates are able to confer similar conformational changes when covalently attached at the putative Mdr recognition pocket of MdfA. Taken together, the results suggest that the Mdr-binding pocket of MdfA is conformationally sensitive. We speculate that the same conformational switch that usually drives active transport is triggered promiscuously by merely occupying the Mdr-binding site.

Bacterial multidrug transport through the lens of the major facilitator superfamily.

Multidrug transporters are membrane proteins that expel a wide spectrum of cytotoxic compounds from the cell. Through this function, they render cells resistant to multiple drugs. These transporters are found in many different families of transport proteins, of which the largest is the major facilitator superfamily. Multidrug transporters from this family are highly represented in bacteria and studies of them have provided important insight into the mechanism underlying multidrug transport. This review summarizes the work carried out on these interesting proteins and underscores the differences and similarities to other transport systems.

The bioterrorism threat and dual-use biotechnological research: an Israeli perspective.

Israel has a long history of concern with chemical and biological threats, since several hostile states in the Middle East are likely to possess such weapons. The Twin-Tower terrorist attacks and Anthrax envelope scares of 2001 were a watershed for public perceptions of the threat of unconventional terror in general and of biological terror in particular. New advances in biotechnology will only increase the ability of terrorists to exploit the burgeoning availability of related information to develop ever-more destructive bioweapons. Many areas of modern biological research are unavoidably dual-use by nature. They thus have a great potential for both help and harm; and facilitating the former while preventing the latter remains a serious challenge to researchers and governments alike. This article addresses how Israel might best (1) prevent hostile elements from obtaining, from Israel's biological research system, materials, information and technologies that might facilitate their carrying out a biological attack, while (2) continuing to promote academic openness, excellence and other hallmarks of that system. This important and sensitive issue was assessed by a special national committee, and their recommendations are presented and discussed. One particularly innovative element is the restructuring and use of Israel's extensive biosafety system to also address biosecurity goals, with minimal disruption or delay.

The Multidrug Transporter MdfA Deviates from the Canonical Model of Alternating Access of MFS Transporters.

The prototypic multidrug (Mdr) transporter MdfA from Escherichia coli efflux chemically- dissimilar substrates in exchange for protons. Similar to other transporters, MdfA purportedly functions by alternating access of a central substrate binding pocket to either side of the membrane. Accordingly, MdfA should open at the cytoplasmic side and/or laterally toward the membrane to enable access of drugs into its pocket. At the end of the cycle, the periplasmic side is expected to open to release drugs. Two distinct conformations of MdfA have been captured by X-ray crystallography: An outward open (Oo) conformation, stabilized by a Fab fragment, and a ligand-bound inward-facing (If) conformation, possibly stabilized by a mutation (Q131R). Here, we investigated how these structures relate to ligand-dependent conformational dynamics of MdfA in lipid bilayers. For this purpose, we combined distances measured by double electron-electron resonance (DEER) between pairs of spin labels in MdfA, reconstituted in nanodiscs, with cysteine cross-linking of natively expressed membrane-embedded MdfA variants. Our results suggest that in a membrane environment, MdfA assumes a relatively flexible, outward-closed/inward-closed (Oc/Ic) conformation. Unexpectedly, our data show that neither the substrate TPP nor protonation induces large-scale conformational changes. Rather, we identified a substrate-responsive lateral gate, which is open toward the inner leaflet of the membrane but closes upon drug binding. Together, our results suggest a modified model for the functional conformational cycle of MdfA that does not invoke canonical elements of alternating access.

Cleavage of a multispanning membrane protein by an intramembrane serine protease.

All intramembrane proteases are known to cleave membrane proteins with a single transmembrane helix. Such cleavages often release anchored soluble domains, which play a role in physiologically important inter- and intracellular processes. However, in many cases the physiological roles/substrates of intramembrane proteases are not known. It is interesting that no multispanning substrates were identified so far, despite the fact that intramembrane proteases have promiscuous substrate recognition and cleavage capabilities. Here we determined whether, in a synthetic experimental system, intramembrane proteases have the capability to interact with and cleave multispanning membrane proteins. We utilized the Escherichia coli rhomboid GlpG, an intramembrane serine protease, and truncated versions of the E. coli multidrug transporter MdfA as model multispanning membrane proteins. On the basis of in vivo and in vitro studies on the association of GlpG with various MdfA constructs and their cleavage, we conclude that GlpG is able to recognize and cleave truncated forms of MdfA but not the intact protein. We propose that GlpG has the capacity to act on unfolded multispanning membrane proteins, thus providing an incentive for investigating possible physiological consequences.

Accumulation of endoplasmic membranes and novel membrane-bound ribosome-signal recognition particle receptor complexes in Escherichia coli.

In Escherichia coli, ribosomes must interact with translocons on the membrane for the proper integration of newly synthesized membrane proteins, cotranslationally. Previous in vivo studies indicated that unlike the E. coli signal recognition particle (SRP), the SRP receptor FtsY is required for membrane targeting of ribosomes. Accordingly, a putative SRP-independent, FtsY-mediated ribosomal targeting pathway has been suggested (Herskovits, A.A., E.S. Bochkareva, and E. Bibi. 2000. Mol. Microbiol. 38:927-939). However, the nature of the early contact of ribosomes with the membrane, and the involvement of FtsY in this interaction are unknown. Here we show that in cells depleted of the SRP protein, Ffh or the translocon component SecE, the ribosomal targeting pathway is blocked downstream and unprecedented, membrane-bound FtsY-ribosomal complexes are captured. Concurrently, under these conditions, novel, ribosome-loaded intracellular membrane structures are formed. We propose that in the absence of a functional SRP or translocon, ribosomes remain jammed at their primary membrane docking site, whereas FtsY-dependent ribosomal targeting to the membrane continues. The accumulation of FtsY-ribosome complexes induces the formation of intracellular membranes needed for their quantitative accommodation. Our results with E. coli, in conjunction with recent observations made with the yeast Saccharomyces cerevisiae, raise the possibility that the SRP receptor-mediated formation of intracellular membrane networks is governed by evolutionarily conserved principles.

Probing the solution structure of the E. coli multidrug transporter MdfA using DEER distance measurements with nitroxide and Gd(III) spin labels.

Methodological and technological advances in EPR spectroscopy have enabled novel insight into the structural and dynamic aspects of integral membrane proteins. In addition to an extensive toolkit of EPR methods, multiple spin labels have been developed and utilized, among them Gd(III)-chelates which offer high sensitivity at high magnetic fields. Here, we applied a dual labeling approach, employing nitroxide and Gd(III) spin labels, in conjunction with Q-band and W-band double electron-electron resonance (DEER) measurements to characterize the solution structure of the detergent-solubilized multidrug transporter MdfA from E. coli. Our results identify highly flexible regions of MdfA, which may play an important role in its functional dynamics. Comparison of distance distribution of spin label pairs on the periplasm with those calculated using inward- and outward-facing crystal structures of MdfA, show that in detergent micelles, the protein adopts a predominantly outward-facing conformation, although more closed than the crystal structure. The cytoplasmic pairs suggest a small preference to the outward-facing crystal structure, with a somewhat more open conformation than the crystal structure. Parallel DEER measurements with the two types of labels led to similar distance distributions, demonstrating the feasibility of using W-band spectroscopy with a Gd(III) label for investigation of the structural dynamics of membrane proteins.

Do physiological roles foster persistence of drug/multidrug-efflux transporters? A case study.

Drug and multidrug resistance have greatly compromised the compounds that were once the mainstays of antibiotic therapy. This resistance often persists despite reductions in the use of antibiotics, indicating that the proteins encoded by antibiotic-resistance genes have alternative physiological roles that can foster such persistence in the absence of selective pressure by antibiotics. The recent observations that Tet(L), a tetracycline-efflux transporter, and MdfA, a multidrug-efflux transporter, both confer alkali tolerance offer a striking case study in support of this hypothesis.

The fascinating but mysterious mechanistic aspects of multidrug transport by MdfA from Escherichia coli.

MdfA is an interesting member of a large group of secondary multidrug (Mdr) transporters. Through genetic, biochemical and biophysical studies of MdfA, many challenging aspects of the multidrug transport phenomenon have been addressed. This includes its ability to interact with chemically unrelated drugs and how it utilizes energy to drive efflux of compounds that are not only structurally, but also electrically, different. Admittedly, however, despite all efforts and a recent pioneering structural contribution, several important mechanistic issues of the promiscuous capabilities of MdfA still seek better molecular and dynamic understanding.

Co-translational Folding Intermediate Dictates Membrane Targeting of the Signal Recognition Particle Receptor.

Much of our knowledge on the function of proteins is deduced from their mature, folded states. However, it is unknown whether partially synthesized nascent protein segments can execute biological functions during translation and whether their premature folding states matter. A recent observation that a nascent chain performs a distinct function, co-translational targeting in vivo, has been made with the Escherichia coli signal recognition particle receptor FtsY, a major player in the conserved pathway of membrane protein biogenesis. FtsY functions as a membrane-associated entity, but very little is known about the mode of its targeting to the membrane. Here we investigated the underlying structural mechanism of the co-translational FtsY targeting to the membrane. Our results show that helices N2-4, which mediate membrane targeting, form a stable folding intermediate co-translationally that greatly differs from its fold in the mature FtsY. These results thus resolve a long-standing mystery of how the receptor targets the membrane even when deleted of its alleged membrane targeting sequence. The structurally distinct targeting determinant of FtsY exists only co-translationally. Our studies will facilitate further efforts to seek cellular factors required for proper targeting and association of FtsY with the membrane. Moreover, the results offer a hallmark example for how co-translational nascent intermediates may dictate biological functions.