Select Publications from the Duong Research Group


Profiling the E. coli Membrane Protein Interactome Captured in Peptidisc Libraries (2019)

Michael Carlson, Greg Stacey, John Young, Irvin Wason, Zhiyu Zhao, David G Rattray, Nichollas Scott, Craig Kerr, Mohan Babu, Leonard J. Foster and Franck Duong

We apply the peptidisc for the capture of the Escherichia coli cell envelope proteome and its high-resolution fractionation in the absence of detergent. Using well-characterized membrane protein systems such as the SecY translocon, the Bam complex and the MetNI transporter, we find the peptidisc library very useful for identifying transient and novel protein interactions. Most of these interactions are largely undetected by standard detergent-based purification. The peptidisc workflow applied to the proteomic field is emerging as a promising novel approach to characterize membrane protein interactions under native expression conditions. 

Investigating the stability of the SecA–SecYEG complex during protein translocation across the bacterial membrane (2019)

John Young and Franck Duong

SecA–SecYEG binding and dissociation events are important for efficient transport of the periplasmic protein proPhoA. A landmark previous study reported that ATP binding to SecA triggers a “power stroke,” resulting in forward movement of a polypeptide segment into the mouth of the SecYEG channel. Following ATP hydrolysis, when ADP is still bound to the enzyme, SecA adopts a conformation allowing the polypeptide to slide across the SecYEG channel until the next ATP- binding event. Instead, SecA rather operates as a “Brownian ratchet,” allowing substrates to passively diffuse within the SecY channel while the enzyme is in an ADP-state.

The Peptidisc, a simple method for stabilizing membrane proteins in detergent-free solution (2018)

Michael Luke Carlson, John William Young, Zhiyu Zhao, Lucien Fabre, Daniel Jun, Jianing Li, Jun Li, Harveer Singh Dhupar, Irvin Wason, Allan T Mills, J Thomas Beatty, John S Klassen, Isabelle Rouiller, and Franck Duong

Obtaining membrane bound proteins in a stable non-aggregated state remains a problem in the scientific community. Previous methods of using detergent micelles (small amphipathic molecules) are quite often detrimental to protein structure and activity, in addition to interfering with downstream analytical methods. However, using multiple copies of an amphipathic bi-helical peptide (here after termed NSPr) wrapping around a target membrane protein allows for its solubilization, retention of 3D structure and protein activity. Therefore allowing membrane proteins to be studied in a solubilized state.

Negative stain single-particle EM of the maltose transporter in nanodiscs reveals asymmetric closure of MalK and catalytic roles of ATP, MalE, and maltose (2017)

Lucien Fabre, Huan Bao, James Innes, Franck Duong and Isabelle Rouiller

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…..

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

Allan Mills and Franck Duong

Nuclease colicins are antibacterial proteins produced by certain strains of E. coli to reduce competition from rival strains. 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 discover that the cytotoxic rRNAse domain of colicin E3 is sufficient to inhibit cell growth. There is high affinity interaction (Kd ~1-2μM) between the rRNAse domain and lipopolysaccharides (LPS) which is important to destabilize the secondary structure of the toxin, which is expectedly crucial for transport through the membrane. 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.

Formation of a chloride-conducting state in the maltose ATP-binding cassette (ABC) transporter (2016)

Michael L. Carlson, Huan Bao and Franck Duong

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. Through the use of a mutant that resides in intermediate conformations close to the transition state, we demonstrate that chloride conductance occurring, to a degree that can be large enough to compromise cell viability. We conclude that ABC transporters must 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 transporter to channel.

Sequential action of MalE and maltose allows coupling ATP Hydrolysis to translocation in the MalFGK2 transporter (2015)

Huan Bao, Kush Dalal, Eric Cytrynbaum and Franck Duong

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.

Sequential action of MalE and maltose allows coupling ATP Hydrolysis to translocation in the MalFGK2 transporter (2015)

Huan Bao, Kush Dalal, Eric Cytrynbaum and Franck Duong

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.

FhuA interactions in a detergent-free nanodisc environment (2014)

Allan Mills, Hai-Tuong Le, James W. Coulton, and Franck Duong

Some metals and vitamins are critical for bacterial cell growth but their availability in the environment is limited. Gram-negative bacteria have evolved a set of outer membrane receptors to efficiently captureand transport these nutrients. Ferricrocin binds to FhuA to trigger the exposure of the TonB box to the periplasm. After recognition by TonB, the plug domain is pulled out of the β-barrel to allow the release of ferricrocin. Reconstitution of FhuA into nanodiscs binds TonB with a much lower affinity (~200 nM) than previously reported in detergent (~20 nM). Significantly, the binding critically depends on ferricrocin. FhuA in nanodiscs also forms a high-affinity binding site for colicin M.

Nucleotide-free MalK drives the transition of the maltose transporter to the inward-facing conformation (2014)

Huan Bao and Franck Duong

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.

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SecYEG activates GTPases to drive the completion of cotranslational protein targeting (2013)

David Akopian, Kush Dalal, Kuang Shen, Franck Duong, and Shu-ou Shan

A conserved protein-conducting channel, SecYEG in bacteria or Sec61p in eukaryotes, is the point of convergence of post- and co-translational protein targeting pathways and mediates the translocation or integration of newly synthesized proteins. Extensive work on the Sec pathway showed that SecYEG and pre-proteins stimulate SecA’s ATPase activity and activate it to drive the translocation of pre-proteins into the periplasm. During targeting, SRP and FtsY form a heterodimer in which both proteins undergo a series of conformational changes, including the early, closed, and activated states, that culminate in reciprocal GTPase activation.

ATP alone triggers the outward-facing conformation of the maltose ABC transporter (2013)

Huan Bao and Franck Duong

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.

Nanodiscs and SILAC-based mass spectrometry to identify a membrane protein interactome (2012)

Xiao X. Zhang, Catherine S. Chan, Huan Bao, Yuan Fang, Leonard J. Foster and Franck Duong

Integral membrane proteins are challenging to work with biochemically given their insoluble nature; the nanodisc circumvents the difficulty by stabilizing them in small patches of lipid bilayer. Here, we show that nanodiscs combined with SILAC-based quantitative proteomics can be used to identify the soluble interacting partners of virtually any membrane protein. As a proof of principle, we applied the method to the bacterial SecYEG protein-conducting channel, the maltose transporter MalFGK(2) and the membrane integrase YidC. In contrast to the detergent micelles, which tend to destabilize interactions, the nanodisc was able to capture out of a complex whole cell extract the proteins SecA, Syd, and MalE with a high degree of confidence and specificity. The method was sensitive enough to isolate these interactors as a function of the lipid composition in the disc and the culture conditions.

Two copies of the SecY channel and acidic lipids are necessary to activate the SecA translocation ATPase (2012).

Kush Dalal, Catherine S. Chan, Stephen G. Sligar and Franck Duong

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.

The SecY complex: conducting the orchestra of protein translocation (2011)

Kush Dalal and Franck Duong

Like the conductor of an orchestra, the Sec protein translocation channel is the platform needed to bring together the many different players required for the constitutive and obligatory process of protein transport. This conserved membrane channel, termed SecY in bacteria and Sec61 in eukaryotes, creates a ubiquitous protein-conducting pathway by which thousands of newly synthesized polypeptides make their way through the lipid bilayer. The channel is not a simple passive pore, however; it displays remarkable complexity by interacting with numerous soluble partners, including SecA, Syd, FtsY and the ribosome in bacteria.

The SecY complex forms a channel capable of ionic discrimination (2009)

Kush Dalal and Franck Duong

Protein translocation across the bacterial membrane occurs at the SecY complex or channel. The resting SecY channel is impermeable to small molecules owing to a plug domain that creates a seal. Here, we report that a channel loosely sealed, or with a plug locked open, does not, however, lead to general membrane permeability. Instead, strong selectivity towards small monovalent anions, especially chloride, is observed. Mutations in the pore ring-structure increase both the translocation activity of the channel and its ionic conductance, however the selectivity is maintained. The same ionic specificity also occurs at the onset of protein translocation and across the archaeal SecY complex. Thus, the ion-conducting characteristic of the channel seems to be conserved as a normal consequence of protein translocation. We propose that the pore ring-structure forms a selectivity filter, allowing cells to tolerate channels with imperfect plugs.

Previous Publications

Bao H, Dalal K, Cytrynbaum E, Duong F. (2015) Sequential action of MalE and maltose allows coupling ATP Hydrolysis to translocation in the MalFGK2 transporter. J Biol Chem. jbc.M115.671826.

Duong F (2014). Capturing the bacterial holo-complex. Proc Natl Acad Sci U S A. 111:4739-40. 41.

Bao H, Duong F (2014). Nucleotide-free MalK drives the transition of the maltose transporter to the inward-facing conformation. J Biol Chem. 289:9844-51.

Mills A, Le HT, Coulton JW, Duong F (2014). Evaluating the FhuA interactions in a detergent-free nanodisc environment. Biochim Biophys Acta. 1838:364-71.

Bao H, Duong F (2013). Phosphatidylglycerol directs binding and inhibitory action of EIIAGlc on the maltose transporter. J Biol Chem. 288:23666-74.

Bao H, Dalal K, Wang V, Rouiller I, Duong F (2013). The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity. BBA Biomembranes. 1828:1723-1730.

Akopian D, Dalal K, Shen K, Duong F, Shan S-o (2013). SecYEG activates GTPases to drive the completion of cotranslational protein targeting. J Cell Biol. 200:397-405.

Bao H, Duong F (2013) ATP alone triggers the outward-facing conformation of the maltose ABC transporter. J Biol Chem. 288:3439-48.

Bao H, Duong F, Chan CS (2012). A step-by-step method for the reconstitution of an ABC transporter into nanodisc lipid particles. J Vis Exp, e3910. *corresponding author.

Bao H, Duong F (2012). Discovery of an auto-regulation mechanism for the maltose ABC transporter MalFGK2. PloS ONE 7:e34836. Important new findings for an otherwise well known transporter .

Dalal K, Chan CS, Sligar SG, Duong F (2012). Two copies of the SecY channel and acidic lipids are necessary to activate the SecA translocation ATPase. Proc Natl Acad Sci U S A 109:4104-4109. This work is a succesful the application of the nanodiscs to isolate and study membrane protein oligomers.

Fonseca BD, Diering GH, Bidinosti MA, Dalal K, Alain T, Balgi AD, Forestieri R, Nodwell M, Rajadurai CV, Gunaratnam C, Tee AR, Duong F, Andersen RJ, Orlowski J, Numata M, Sonenberg N, Roberge M (2012). Structure-activity analysis of niclosamide reveals potential role for cytoplasmic pH in control of mammalian target of rapamycin complex 1 (mTORC1) signaling. J Biol Chem 287:17530-45.

Zhang XX, Chan CS, Bao H, Fang Y, Foster LJ, Duong F (2012). Nanodiscs and SILAC-based mass spectrometry to identify a membrane protein interactome. J Proteome Res 11:1454-1459.

Dalal K, Duong F (2011). The SecY complex: conducting the orchestra of protein translocation. Trends in Cell Biology 21:506-514. Review.

Gold VA, Robson A, Bao H, Romantsov T, Duong F, Collinson I (2010). The action of cardiolipin on the bacterial translocon. Proc Natl Acad Sci U S A 107:10044-10049.

Dalal K, Bao H, Duong F (2010). Modulation of the SecY channel permeability by pore mutations and trivalent cations. Channels 4:83-96.

Dalal K, Duong F (2009). Reconstitution of the SecY complex in Nanodiscs. In Protein Secretion. Ed. E. Economou, Methods in Molecular Biology 619:145-156.

Dalal K, Duong F (2009). The SecY complex forms a channel capable of ionic discrimination. The EMBO Rep 10:762-768.

Dalal K, Nguyen N, Alami M, Tan J, Moraes TF, Lee WC, Maurus R, Sligar SS, Brayer GD, Duong F (2009). Binding, structure and activity of Syd, a SecY interacting protein. J Biol Chem 284:7897-7902. The details of the work were highlighted by the editor of Nature Structural & Molecular Biology 16, pp106 (2009).

Duong F (2007). “ Cell biology: Fraternal twins ” Nature News & Views, 446:741-743. Commentary.

Gold V, Duong F, Collinson I (2007). The bacterial protein translocation reaction. Mol Membr Biol 24:387-394. Review.

Alami M, Dalal K, Lelj-Garolla B, Sligar S and Duong F (2007). Nanodiscs unravel the stoichiometry of the interaction between the SecYEG channel and its cytosolic partner SecA. EMBO J 26:1995-2004. The principal discovery was highlighted by the editor of Science 316, pp174 (2007).

Maillard A, Lalani S, Silva F, Belin D, Duong F (2007). Deregulation of the SecYEG translocation channel upon removal of the plug domain. J Biol Chem 282:1281-1287.

Tam P, Maillard A, Chan K, Duong F (2005). Investigating the SecY-plug movement at the SecYEG translocation channel. EMBO J 24:3380-3388.