Nanodisc-Based Proteomics Identify Caj1 as an Hsp40 with Affinity for Phosphatidic Acid Lipids.

Many soluble proteins interact with membranes to perform important biological functions, including signal transduction, regulation, transport, trafficking, and biogenesis. Despite their importance, these protein-membrane interactions are difficult to characterize due to their often-transient nature as well as phospholipids' poor solubility in aqueous solution. Here, we employ nanodiscs-small, water-soluble patches of a lipid bilayer encircled with amphipathic scaffold proteins-along with quantitative proteomics to identify lipid-binding proteins in Saccharomyces cerevisiae. Using nanodiscs reconstituted with yeast total lipid extracts or only phosphatidylethanolamine (PE-nanodiscs), we capture several known membrane-interacting proteins, including the Rab GTPases Sec4 and Ypt1, which play key roles in vesicle trafficking. Utilizing PE-nanodiscs enriched with phosphatidic acid (PEPA-nanodiscs), we specifically capture a member of the Hsp40/J-protein family, Caj1, whose function has recently been linked to membrane protein quality control. We show that the Caj1 interaction with liposomes containing PA is modulated by pH and PE lipids and depends on two patches of positively charged residues near the C-terminus of the protein. The protein Caj1 is the first example of an Hsp40/J-domain protein with affinity for membranes and phosphatidic acid lipid specificity. These findings highlight the utility of combining proteomics with lipid nanodiscs to identify and characterize protein-lipid interactions that may not be evident using other methods. Data are available via ProteomeXchange with the identifier PXD027992.

Investigating the stability of the SecA-SecYEG complex during protein translocation across the bacterial membrane.

During posttranslational translocation in Escherichia coli, polypeptide substrates are driven across the membrane through the SecYEG protein-conducting channel using the ATPase SecA, which binds to SecYEG and couples nucleotide hydrolysis to polypeptide movement. Recent studies suggest that SecA is a highly dynamic enzyme, able to repeatedly bind and dissociate from SecYEG during substrate translocation, but other studies indicate that these dynamics, here referred to as "SecA processivity," are not a requirement for transport. We employ a SecA mutant (PrlD23) that associates more tightly to membranes than WT SecA, in addition to a SecA-SecYEG cross-linked complex, to demonstrate that SecA-SecYEG binding and dissociation events are important for efficient transport of the periplasmic protein proPhoA. Strikingly however, we find that transport of the precursor of the outer membrane protein proOmpA does not depend on SecA processivity. By exchanging signal sequence and protein domains of similar size between PhoA and OmpA, we find that SecA processivity is not influenced by the sequence of the protein substrate. In contrast, using an extended proOmpA variant and a truncated derivative of proPhoA, we show that SecA processivity is affected by substrate length. These findings underscore the importance of the dynamic nature of SecA-SecYEG interactions as a function of the preprotein substrate, features that have not yet been reported using other biophysical or in vivo methods.

Selectively targeting the dimerization interface of human androgen receptor with small-molecules to treat castration-resistant prostate cancer.

Prostate cancer (PCa) is a leading cause of death for men in North America. The androgen receptor (AR) - a hormone inducible transcription factor - drives expression of tumor promoting genes and represents an important therapeutic target in PCa. The AR is activated by steroid recruitment to its ligand binding domain (LBD), followed by receptor nuclear translocation and dimerization via the DNA binding domain (DBD). Clinically used small molecules interfere with steroid recruitment and prevent AR-driven tumor growth, but are rendered ineffective by emergence of LBD mutations or expression of constitutively active variants, such as ARV7, that lack the LBD. Both drug-resistance mechanisms confound treatment of this 'castration resistant' stage of PCa (CRPC), characterized by return of AR signalling. Here, we employ computer-aided drug-design to develop small molecules that block the AR-DBD dimerization interface, an attractive target given its role in AR activation and independence from the LBD. Virtual screening on the AR-DBD structure led to development of prototypical compounds that block AR dimerization, inhibiting AR-transcriptional activity through a LBD-independent mechanism. Such inhibitors may potentially circumvent AR-dependent resistance mechanisms and directly target CRPC tumor growth.

The Peptidisc, a simple method for stabilizing membrane proteins in detergent-free solution.

Membrane proteins are difficult to work with due to their insolubility in aqueous solution and quite often their poor stability in detergent micelles. Here, we present the peptidisc for their facile capture into water-soluble particles. Unlike the nanodisc, which requires scaffold proteins of different lengths and precise amounts of matching lipids, reconstitution of detergent solubilized proteins in peptidisc only requires a short amphipathic bi-helical peptide (NSPr) and no extra lipids. Multiple copies of the peptide wrap around to shield the membrane-exposed part of the target protein. We demonstrate the effectiveness of this 'one size fits all' method using five different membrane protein assemblies (MalFGK2, FhuA, SecYEG, OmpF, BRC) during 'on-column', 'in-gel', and 'on-bead' reconstitution embedded within the membrane protein purification protocol. The peptidisc method is rapid and cost-effective, and it may emerge as a universal tool for high-throughput stabilization of membrane proteins to advance modern biological studies.

Lipopolysaccharides promote binding and unfolding of the antibacterial colicin E3 rRNAse domain.

Nuclease colicins are antibacterial proteins produced by certain strains of E. coli to reduce competition from rival strains. These colicins are generally organized with an N-terminal transport (T)-domain, a central receptor binding (R)-domain, and a C-terminal cytotoxic nuclease domain. These colicins are always produced in complex with an inhibitory immunity protein, which dissociates prior entrance of the cytotoxic domain in the target cell. How exactly colicins traverse the cell envelope is not understood, yet this knowledge is important for the design of new antibacterial therapies. In this report, we find that the cytotoxic rRNAse domain of colicin E3, lacking both T- and R-domains, is sufficient to inhibit cell growth provided the immunity protein Im3 has been removed. Thus, while the T-domain is needed for dissociation of Im3, the rRNAse alone can associate to the cell surface without R-domain. Accordingly, we find a high affinity interaction (Kd ~1-2µM) between the rRNAse domain and lipopolysaccharides (LPS). Furthermore, we show that binding of ColE3 to LPS destabilizes the secondary structure of the toxin, which is expectedly crucial for transport through the narrow pore of the porin OmpF. The effect of LPS on binding and unfolding of ColE3 may be indicative of a broader role of LPS for transport of colicins in general.

Negative Stain Single-particle EM of the Maltose Transporter in Nanodiscs Reveals Asymmetric Closure of MalK2 and Catalytic Roles of ATP, MalE, and Maltose.

The Escherichia coli MalE-MalFGK2 complex is one of the best characterized members of the large and ubiquitous family of ATP-binding cassette (ABC) transporters. It is composed of a membrane-spanning heterodimer, MalF-MalG; a homodimeric ATPase, MalK2; and a periplasmic maltose receptor, MalE. Opening and closure of MalK2 is coupled to conformational changes in MalF-MalG and the alternate exposition of the substrate-binding site to either side of the membrane. To further define this alternate access mechanism and the impact of ATP, MalE, and maltose on the conformation of the transporter during the transport cycle, we have reconstituted MalFGK2 in nanodiscs and analyzed its conformations under 10 different biochemical conditions using negative stain single-particle EM. EM map results (at 15-25 Å resolution) indicate that binding of ATP to MalK2 promotes an asymmetric, semi-closed conformation in accordance with the low ATPase activity of MalFGK2 In the presence of MalE, the MalK dimer becomes fully closed, gaining the ability to hydrolyze ATP. In the presence of ADP or maltose, MalE·MalFGK2 remains essentially in a semi-closed symmetric conformation, indicating that release of these ligands is required for the return to the initial state. Taken together, this structural information provides a rationale for the stimulation of MalK ATPase activity by MalE as well as by maltose.

TonB-dependent ligand trapping in the BtuB transporter.

TonB-dependent transporters are ß-barrel outer membrane proteins occluded by a plug domain. Upon ligand binding, these transporters extend a periplasmic motif termed the TonB box. The TonB box permits the recruitment of the inner membrane protein complex TonB-ExbB-ExbD, which drives import of ligands in the cell periplasm. It is unknown precisely how the plug domain is moved aside during transport nor have the intermediate states between TonB recruitment and plug domain movement been characterized biochemically. Here we employ nanodiscs, native gel electrophoresis, and scintillation proximity assays to determine the binding kinetics of vitamin B12 to BtuB. The results show that ligand-bound BtuB recruits a monomer of TonB (TonB∆1-31), which in turn increases retention of vitamin B12 within the transporter. The TonB box and the extracellular residue valine 90 that forms part of the vitamin B12 binding site are essential for this event. These results identify a novel step in the TonB-dependent transport process. They show that TonB binding to BtuB trap the ligand, possibly until the ExbB-ExbD complex is activated or recruited to ensure subsequent transport.

Salmonella Rapidly Regulates Membrane Permeability To Survive Oxidative Stress.

UNLABELLED: The outer membrane (OM) of Gram-negative bacteria provides protection against toxic molecules, including reactive oxygen species (ROS). Decreased OM permeability can promote bacterial survival under harsh circumstances and protects against antibiotics. To better understand the regulation of OM permeability, we studied the real-time influx of hydrogen peroxide in Salmonella bacteria and discovered two novel mechanisms by which they rapidly control OM permeability. We found that pores in two major OM proteins, OmpA and OmpC, could be rapidly opened or closed when oxidative stress is encountered and that the underlying mechanisms rely on the formation of disulfide bonds in the periplasmic domain of OmpA and TrxA, respectively. Additionally, we found that a Salmonella mutant showing increased OM permeability was killed more effectively by treatment with antibiotics. Together, these results demonstrate that Gram-negative bacteria regulate the influx of ROS for defense against oxidative stress and reveal novel targets that can be therapeutically targeted to increase bacterial killing by conventional antibiotics. IMPORTANCE: Pathogenic bacteria have evolved ways to circumvent inflammatory immune responses. A decrease in bacterial outer membrane permeability during infection helps protect bacteria from toxic molecules produced by the host immune system and allows for effective colonization of the host. In this report, we reveal molecular mechanisms that rapidly alter outer membrane pores and their permeability in response to hydrogen peroxide and oxidative stress. These mechanisms are the first examples of pores that are rapidly opened or closed in response to reactive oxygen species. Moreover, one of these mechanisms can be targeted to artificially increase membrane permeability and thereby increase bacterial killing by the antibiotic cefotaxime during in vitro experiments and in a mouse model of infection. We envision that a better understanding of the regulation of membrane permeability will lead to new targets and treatment options for multidrug-resistant infections.

Formation of a Chloride-conducting State in the Maltose ATP-binding Cassette (ABC) Transporter.

ATP-binding cassette transporters use an alternating access mechanism to move substrates across cellular membranes. This mode of transport ensures the selective passage of molecules while preserving membrane impermeability. The crystal structures of MalFGK2, inward- and outward-facing, show that the transporter is sealed against ions and small molecules. It has yet to be determined whether membrane impermeability is maintained when MalFGK2 cycles between these two conformations. Through the use of a mutant that resides in intermediate conformations close to the transition state, we demonstrate that not only is chloride conductance occurring, but also to a degree large enough to compromise cell viability. Introduction of mutations in the periplasmic gate lead to the formation of a channel that is quasi-permanently open. MalFGK2 must therefore stay away from these ion-conducting conformations to preserve the membrane barrier; otherwise, a few mutations that increase access to the ion-conducting states are enough to convert an ATP-binding cassette transporter into a channel.

Sequential Action of MalE and Maltose Allows Coupling ATP Hydrolysis to Translocation in the MalFGK2 Transporter.

ATP-binding cassette (ABC) transporters have evolved an ATP-dependent alternating-access mechanism to transport substrates across membranes. Despite important progress, especially in their structural analysis, it is still unknown how the substrate stimulates ATP hydrolysis, the hallmark of ABC transporters. In this study, we measure the ATP turnover cycle of MalFGK2 in steady and pre-steady state conditions. We show that (i) the basal ATPase activity of MalFGK2 is very low because the cleavage of ATP is rate-limiting, (ii) the binding of open-state MalE to the transporter induces ATP cleavage but leaves release of Pi limiting, and (iii) the additional presence of maltose stimulates release of Pi, and therefore increases the overall ATP turnover cycle. We conclude that open-state MalE stabilizes MalFGK2 in the outward-facing conformation until maltose triggers return to the inward-facing state for substrate and Pi release. This concerted action explains why ATPase activity of MalFGK2 depends on maltose, and why MalE is essential for transport.

Nucleotide-free MalK drives the transition of the maltose transporter to the inward-facing conformation.

The complex MalFGK2 hydrolyzes ATP and alternates between inward- and outward-facing conformations during maltose transport. It has been shown that ATP promotes closure of MalK2 and opening of MalFG toward the periplasm. Yet, why the transporter rests in a conformation facing the cytosol in the absence of nucleotide and how it returns to this state after hydrolysis of ATP is unknown. The membrane domain MalFG may be naturally stable in the inward-facing conformation, or the ABC domain may catalyze the transition. We address this question by analyzing the conformation of MalFG in nanodiscs and in proteoliposomes. We find that MalFG alone exists in an intermediate state until MalK binds and converts the membrane domain to the inward-facing state. We also find that MalK, if overly-bound to MalFG, blocks the transition of the transporter, whereas suppressor mutations that weaken this association restore transport. MalK therefore exploits hydrolysis of ATP to reverse the conformation of MalFG to the inward-facing conformation, a step essential for release of maltose in the cytosol.

FhuA interactions in a detergent-free nanodisc environment.

TonB-dependent membrane receptors from bacteria have been analyzed in detergent-containing solution, an environment that may influence the role of ligand in inducing downstream interactions. We report reconstitution of FhuA into a membrane mimetic: nanodiscs. In contrast to previous results in detergent, we show that binding of TonB to FhuA in nanodiscs depends strongly on ferricrocin. The stoichiometry of interaction is 1:1 and the binding constant KD is ~200nM; an equilibrium affinity that is ten-fold lower than reported in detergent. FhuA in nanodiscs also forms a high-affinity binding site for colicin M (KD ~3.5nM), while ferricrocin renders FhuA refractory to colicin binding. Together, these results demonstrate the importance of the ligand in regulating receptor interactions and the advantages of nanodiscs to study ß-barrel membrane proteins in a membrane-like environment.

Phosphatidylglycerol directs binding and inhibitory action of EIIAGlc protein on the maltose transporter.

The signal-transducing protein EIIA(Glc) belongs to the phosphoenolpyruvate carbohydrate phosphotransferase system. In its dephosphorylated state, EIIA(Glc) is a negative regulator for several permeases, including the maltose transporter MalFGK2. How EIIA(Glc) is targeted to the membrane, how it interacts with the transporter, and how it inhibits sugar uptake remain obscure. We show here that acidic phospholipids together with the N-terminal tail of EIIA(Glc) are essential for the high affinity binding of the protein to the transporter. Using protein docking prediction and chemical cross-linking, we demonstrate that EIIA(Glc) binds to the MalK dimer, interacting with both the nucleotide-binding and the C-terminal regulatory domains. Dissection of the ATPase cycle reveals that EIIA(Glc) does not affect the binding of ATP but rather inhibits the capacity of MalK to cleave ATP. We propose a mechanism of maltose transport inhibition by this central amphitropic regulatory protein.

The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity.

The coupling between ATP hydrolysis and substrate transport remains a key question in the understanding of ABC-mediated transport. We show using the MalFGK2 complex reconstituted into nanodiscs, that membrane lipids participate directly to the coupling reaction by stabilizing the transporter in a low energy conformation. When surrounded by short acyl chain phospholipids, the transporter is unstable and hydrolyzes large amounts of ATP without inducing maltose. The presence of long acyl chain phospholipids stabilizes the conformational dynamics of the transporter, reduces its ATPase activity and restores dependence on maltose. Membrane lipids therefore play an essential allosteric function, they restrict the transporter ATPase activity to increase coupling to the substrate. In support to the notion, we show that increasing the conformational dynamics of MalFGK2 with mutations in MalF increases the transporter ATPase activity but decreases the maltose transport efficiency.

SecYEG activates GTPases to drive the completion of cotranslational protein targeting.

Signal recognition particle (SRP) and its receptor (SR) comprise a highly conserved cellular machine that cotranslationally targets proteins to a protein-conducting channel, the bacterial SecYEG or eukaryotic Sec61p complex, at the target membrane. Whether SecYEG is a passive recipient of the translating ribosome or actively regulates this targeting machinery remains unclear. Here we show that SecYEG drives conformational changes in the cargo-loaded SRP-SR targeting complex that activate it for GTP hydrolysis and for handover of the translating ribosome. These results provide the first evidence that SecYEG actively drives the efficient delivery and unloading of translating ribosomes at the target membrane.

ATP alone triggers the outward facing conformation of the maltose ATP-binding cassette transporter.

The maltose transporter MalFGK(2) is a study prototype for ABC importers. During catalysis, the MalFG membrane domain alternates between inward and outward facing conformations when the MalK dimer closes and hydrolyzes ATP. Because a rapid ATP hydrolysis depends on MalE and maltose, it has been proposed that closed liganded MalE facilitates the transition to the outward facing conformation. Here we find that, in contrast to the expected, ATP is sufficient for the closure of MalK and for the conversion of MalFG to the outward facing state. The outward facing transporter binds MalE with nanomolar affinity, yet neither MalE nor maltose is necessary or facilitates the transition. Thus, the rapid hydrolysis of ATP observed in the presence of MalE and maltose is not because closed liganded MalE accelerates the formation of the outward facing conformation. These findings have fundamental implications for the description of the transport reaction.

A step-by-step method for the reconstitution of an ABC transporter into nanodisc lipid particles.

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.

Discovery of an auto-regulation mechanism for the maltose ABC transporter MalFGK2.

The maltose transporter MalFGK(2), together with the substrate-binding protein MalE, is one of the best-characterized ABC transporters. In the conventional model, MalE captures maltose in the periplasm and delivers the sugar to the transporter. Here, using nanodiscs and proteoliposomes, we instead find that MalE is bound with high-affinity to MalFGK2 to facilitate the acquisition of the sugar. When the maltose concentration exceeds the transport capacity, MalE captures maltose and dissociates from the transporter. This mechanism explains why the transport rate is high when MalE has low affinity for maltose, and low when MalE has high affinity for maltose. Transporter-bound MalE facilitates the acquisition of the sugar at low concentrations, but also captures and dissociates from the transporter past a threshold maltose concentration. In vivo, this maltose-forced dissociation limits the rate of transport. Given the conservation of the substrate-binding proteins, this mode of allosteric regulation may be universal to ABC importers.

Structure-activity analysis of niclosamide reveals potential role for cytoplasmic pH in control of mammalian target of rapamycin complex 1 (mTORC1) signaling.

Mammalian target of rapamycin complex 1 (mTORC1) signaling is frequently dysregulated in cancer. Inhibition of mTORC1 is thus regarded as a promising strategy in the treatment of tumors with elevated mTORC1 activity. We have recently identified niclosamide (a Food and Drug Administration-approved antihelminthic drug) as an inhibitor of mTORC1 signaling. In the present study, we explored possible mechanisms by which niclosamide may inhibit mTORC1 signaling. We tested whether niclosamide interferes with signaling cascades upstream of mTORC1, the catalytic activity of mTOR, or mTORC1 assembly. We found that niclosamide does not impair PI3K/Akt signaling, nor does it inhibit mTORC1 kinase activity. We also found that niclosamide does not interfere with mTORC1 assembly. Previous studies in helminths suggest that niclosamide disrupts pH homeostasis of the parasite. This prompted us to investigate whether niclosamide affects the pH balance of cancer cells. Experiments in both breast cancer cells and cell-free systems demonstrated that niclosamide possesses protonophoric activity in cells and in vitro. In cells, niclosamide dissipated protons (down their concentration gradient) from lysosomes to the cytosol, effectively lowering cytoplasmic pH. Notably, analysis of five niclosamide analogs revealed that the structural features of niclosamide required for protonophoric activity are also essential for mTORC1 inhibition. Furthermore, lowering cytoplasmic pH by means other than niclosamide treatment (e.g. incubation with propionic acid or bicarbonate withdrawal) recapitulated the inhibitory effects of niclosamide on mTORC1 signaling, lending support to a possible role for cytoplasmic pH in the control of mTORC1. Our data illustrate a potential mechanism for chemical inhibition of mTORC1 signaling involving modulation of cytoplasmic pH.