Crystal structures of a polypeptide processing and secretion transporter

Lin, David Yin-wei; Huang, Shuo; Chen, Jue

doi:10.1038/nature14623

Cited by 113 publications

(192 citation statements)

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“…The crystal structures of exporters display varying degrees of NBD disengagement in the absence of nucleotides (Ward et al , 2007; Lin et al , 2015; Perez et al , 2015; Bountra et al , 2017). These data were questioned as a detergent artifact mistaken for dynamic NBDs.…”

Section: Discussion and Outlookmentioning

confidence: 99%

“…DEER data of ABC transporters in liposomes and bicelles show broad lines for apo NBDs (Dong et al , 2005; Borbat et al , 2007; Zou et al , 2009; Bountra et al , 2017), further suggesting that in the absence of nucleotides the NBDs can have different degrees of disengagement. Upon nucleotide binding, the NBDs dimerize and display nearly identical distances across different transporters (Ward et al , 2007; Lin et al , 2015; Perez et al , 2015; Bountra et al , 2017). Our smFRET data in detergent and liposomes revealed that the NBDs of McjD adopt very similar disengagement with E* values of 0.3 and larger E* values of 0.5 upon nucleotide binding.…”

Section: Discussion and Outlookmentioning

confidence: 99%

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Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

et al. 2018

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ABC transporters utilize ATP for export processes to provide cellular resistance against toxins, antibiotics, and harmful metabolites in eukaryotes and prokaryotes. Based on static structure snapshots, it is believed that they use an alternating access mechanism, which couples conformational changes to ATP binding (outward‐open conformation) and hydrolysis (inward‐open) for unidirectional transport driven by ATP. Here, we analyzed the conformational states and dynamics of the antibacterial peptide exporter McjD from Escherichia coli using single‐molecule Förster resonance energy transfer (smFRET). For the first time, we established smFRET for an ABC exporter in a native‐like lipid environment and directly monitor conformational dynamics in both the transmembrane‐ (TMD) and nucleotide‐binding domains (NBD). With this, we unravel the ligand dependences that drive conformational changes in both domains. Furthermore, we observe intrinsic conformational dynamics in the absence of ATP and ligand in the NBDs. ATP binding and hydrolysis on the other hand can be observed via NBD conformational dynamics. We believe that the progress made here in combination with future studies will facilitate full understanding of ABC transport cycles.

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Section: Discussion and Outlookmentioning

confidence: 99%

Section: Discussion and Outlookmentioning

confidence: 99%

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

et al. 2018

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“…The presence of the occluded conformations along the transport cycle probably acts as (i) a shielding mechanism for preventing peptide re‐uptake by McjD and (ii) a mechanism to prime McjD for MccJ25 binding, during its biosynthesis, without building up toxic levels of the peptide (Choudhury et al , 2014). The structures of the peptidase‐containing ATP‐binding cassette transporter (PCAT) PCAT1 from Clostridium thermocellum (Lin et al , 2015) and the lipid‐linked oligosaccharide flippase PglK from Campylobacter jejuni (Perez et al , 2015) have also been determined in a nucleotide‐bound outward‐occluded conformation and without domain intertwining similar to McjD. Therefore, the mechanism we propose for McjD is probably relevant to other ABC transporters that are dedicated to specific substrate export, whose cavity is “immediately shielded” upon substrate release.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD

et al. 2017

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Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self‐immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward‐occluded and a new nucleotide‐bound state, high‐energy outward‐occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross‐linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi‐drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic‐level build‐up.

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“…In ABC transporters, replacing the Glu of the Walker B motif with a Gln has been frequently utilized to eliminate ATPase activity, while preserving the ability to bind ATP (Lee et al, 2014;Lin et al, 2015). Upon co-expression of the corresponding Dd-PrtD-E492Q mutant with PrtE and PrtF, we observe no secretion into the media even after 18 hr of PrtG induction, (Right panel, Fig.…”

Section: Substrate Secretion Is Dependent On Atp Hydrolysismentioning

confidence: 95%

“…The large size of T1SS substrates suggest that the transporter operates in a distinct mechanism from peptide ABC transporters such as microcin exporters and the human antigen transporters, TAP1/2 (Choudhury et al, 2014;Grossmann et al, 2014;Eggensperger and Tampe, 2015;Lin et al, 2015). These transporters appear to operate by the 'alternating access' mechanism whereby the peptide binds from the cytoplasmic side (Rees et al, 2009;Beek et al, 2014;Locher, 2016).…”

Section: Octyl Udp-6-thio-α-d-galactopyranose Disulfide (7)mentioning

confidence: 99%

Structural Studies of Biopolymer Membrane Transport

Morgan¹

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The selective transport of specific substrates across the membrane is an essential part of biology.Organisms have evolved a basic mechanism for the transport of ions and small molecule such as sugars across the membrane. This mechanism is termed 'alternating access' and involves the alternating exposure of a substrate-binding site to either side of the membrane. While the molecular details of 'alternating access' have been revealed for a number of different transporters, this scheme seems infeasible for the transport of long biopolymers such as nucleic acids, proteins, and polysaccharides because 'alternating access' requires that the transporter forms a binding site that is large enough to bind the entire substrate. Here, I present novel data addressing the transport mechanism for two bacterial biopolymer transporter proteins: Bacterial Cellulose Synthase which synthesizes and secretes the polysaccharide, cellulose; and PrtD, an ABC transporter which is involved in the secretion of an extracellular protease.Cellulose is a linear polymer of glucose units that forms a major component of plant cell walls as well as many bacterial biofilms. In bacteria, the components required for cellulose biosynthesis are encoded in a single operon minimally made up of the genes bcsA, bcsB, bcsC, and bcsZ. BcsA is an integral inner-membrane (IM) glycosyltransferase enzyme that is responsible for coupling the synthesis of cellulose with its transport across the IM. BcsB is a periplasmic protein with Cterminal IM anchor, and BcsB forms a complex with BcsA (BcsA-B) that is sufficient for in vitro cellulose synthesis. BcsZ is a periplasmic cellulase enzyme, and BcsC is an outer-membrane β-barrel that presumably forms a pore for the cellulose polymer to cross the outer membrane.iii BcsA-B activity is stimulated by the bacterial signaling molecule, cyclic-di-GMP (c-di-GMP), which is a key regulator of biofilm formation. I present crystal structures of the c-di-GMP-bound BcsA-B complex in the presence and absence of UDP, a competitive inhibitor and substrate mimic. The structures reveal that c-di-GMP releases an auto-inhibited state of the enzyme. A salt bridge stabilizes one of the signature c-di-GMP-binding Arg residues in a position to tether a conserved 'gating loop' in front of the active site. The binding of c-di-GMP releases the tether and allows substrate to access the active site. Additionally, the UDP-bound structure reveals an additional role for the 'gating loop' in coordinating substrate at the active site. Functional experiments confirm the structural interpretation by revealing that disruption of the auto-inhibitory salt bridge by mutagenesis generates a constitutively-active cellulose synthase. The mechanistic insights presented here represent the first examples of how c-di-GMP allosterically modulates enzymatic functions.To address the mechanism by which BcsA-B transports cellulose across the IM, I use in crystallo enzymology with the c-di-GMP-bound BcsA-B crystals. Because crystallized BcsA-B is catalytically a...

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Crystal structures of a polypeptide processing and secretion transporter

Cited by 113 publications

References 53 publications

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD

Structural Studies of Biopolymer Membrane Transport

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