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Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

Coudray, Nicolas; Isom, Georgia L; MacRae, Mark R; Saiduddin, Mariyah N; Bhabha, Gira; Ekiert, Damian C
In double-membraned bacteria, phospholipid transport across the cell envelope is critical to maintain the outer membrane barrier, which plays a key role in virulence and antibiotic resistance. An MCE transport system called Mla has been implicated in phospholipid trafficking and outer membrane integrity, and includes an ABC transporter, MlaFEDB. The transmembrane subunit, MlaE, has minimal sequence similarity to other transporters, and the structure of the entire inner-membrane MlaFEDB complex remains unknown. Here we report the cryo-EM structure of MlaFEDB at 3.05 Ã… resolution, revealing distant relationships to the LPS and MacAB transporters, as well as the eukaryotic ABCA/ABCG families. A continuous transport pathway extends from the MlaE substrate-binding site, through the channel of MlaD, and into the periplasm. Unexpectedly, two phospholipids are bound to MlaFEDB, suggesting that multiple lipid substrates may be transported each cycle. Our structure provides mechanistic insight into substrate recognition and transport by MlaFEDB.
PMID: 33236984
ISSN: 2050-084x
CID: 4680732

3-Dimensional organization and dynamics of the microsporidian polar tube invasion machinery

Jaroenlak, Pattana; Cammer, Michael; Davydov, Alina; Sall, Joseph; Usmani, Mahrukh; Liang, Feng-Xia; Ekiert, Damian C; Bhabha, Gira
Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process of three human-infecting microsporidia species in vitro: Anncaliia algerae, Encephalitozoon hellem and E. intestinalis. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 μm⋅s-1, and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is at least 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection.
PMID: 32946515
ISSN: 1553-7374
CID: 4593522

Structure of MlaFB uncovers novel mechanisms of ABC transporter regulation

Kolich, Ljuvica R; Chang, Ya-Ting; Coudray, Nicolas; Giacometti, Sabrina I; MacRae, Mark R; Isom, Georgia L; Teran, Evelyn M; Bhabha, Gira; Ekiert, Damian C
ABC transporters facilitate the movement of diverse molecules across cellular membranes, but how their activity is regulated post-translationally is not well understood. Here we report the crystal structure of MlaFB from E. coli, the cytoplasmic portion of the larger MlaFEDB ABC transporter complex, which drives phospholipid trafficking across the bacterial envelope to maintain outer membrane integrity. MlaB, a STAS domain protein, binds the ABC nucleotide binding domain, MlaF, and is required for its stability. Our structure also implicates a unique C-terminal tail of MlaF in self-dimerization. Both the C-terminal tail of MlaF and the interaction with MlaB are required for the proper assembly of the MlaFEDB complex and its function in cells. This work leads to a new model for how an important bacterial lipid transporter may be regulated by small proteins, and raises the possibility that similar regulatory mechanisms may exist more broadly across the ABC transporter family.
PMID: 32602838
ISSN: 2050-084x
CID: 4504052

LetB Structure Reveals a Tunnel for Lipid Transport across the Bacterial Envelope

Isom, Georgia L; Coudray, Nicolas; MacRae, Mark R; McManus, Collin T; Ekiert, Damian C; Bhabha, Gira
Gram-negative bacteria are surrounded by an outer membrane composed of phospholipids and lipopolysaccharide, which acts as a barrier and contributes to antibiotic resistance. The systems that mediate phospholipid trafficking across the periplasm, such as MCE (Mammalian Cell Entry) transporters, have not been well characterized. Our ~3.5 Å cryo-EM structure of the E. coli MCE protein LetB reveals an ~0.6 megadalton complex that consists of seven stacked rings, with a central hydrophobic tunnel sufficiently long to span the periplasm. Lipids bind inside the tunnel, suggesting that it functions as a pathway for lipid transport. Cryo-EM structures in the open and closed states reveal a dynamic tunnel lining, with implications for gating or substrate translocation. Our results support a model in which LetB establishes a physical link between the two membranes and creates a hydrophobic pathway for the translocation of lipids across the periplasm.
PMID: 32359438
ISSN: 1097-4172
CID: 4415712

Architectures of Lipid Transport Systems for the Bacterial Outer Membrane

Ekiert, Damian C; Bhabha, Gira; Isom, Georgia L; Greenan, Garrett; Ovchinnikov, Sergey; Henderson, Ian R; Cox, Jeffery S; Vale, Ronald D
How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles.
PMID: 28388411
ISSN: 1097-4172
CID: 2530792

Structure of a PE-PPE-EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion

Ekiert, Damian C; Cox, Jeffery S
Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed "type VII." How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), which adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE-PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Moreover, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways.
PMID: 25275011
ISSN: 1091-6490
CID: 2291272

Antibody recognition of a highly conserved influenza virus epitope

Ekiert, Damian C; Bhabha, Gira; Elsliger, Marc-Andre; Friesen, Robert H E; Jongeneelen, Mandy; Throsby, Mark; Goudsmit, Jaap; Wilson, Ian A
Influenza virus presents an important and persistent threat to public health worldwide, and current vaccines provide immunity to viral isolates similar to the vaccine strain. High-affinity antibodies against a conserved epitope could provide immunity to the diverse influenza subtypes and protection against future pandemic viruses. Cocrystal structures were determined at 2.2 and 2.7 angstrom resolutions for broadly neutralizing human antibody CR6261 Fab in complexes with the major surface antigen (hemagglutinin, HA) from viruses responsible for the 1918 H1N1 influenza pandemic and a recent lethal case of H5N1 avian influenza. In contrast to other structurally characterized influenza antibodies, CR6261 recognizes a highly conserved helical region in the membrane-proximal stem of HA1 and HA2. The antibody neutralizes the virus by blocking conformational rearrangements associated with membrane fusion. The CR6261 epitope identified here should accelerate the design and implementation of improved vaccines that can elicit CR6261-like antibodies, as well as antibody-based therapies for the treatment of influenza.
PMID: 19251591
ISSN: 1095-9203
CID: 2291482

Protein target highlights in CASP15: Analysis of models by structure providers

Alexander, Leila T; Durairaj, Janani; Kryshtafovych, Andriy; Abriata, Luciano A; Bayo, Yusupha; Bhabha, Gira; Breyton, Cécile; Caulton, Simon G; Chen, James; Degroux, Séraphine; Ekiert, Damian C; Erlandsen, Benedikte S; Freddolino, Peter L; Gilzer, Dominic; Greening, Chris; Grimes, Jonathan M; Grinter, Rhys; Gurusaran, Manickam; Hartmann, Marcus D; Hitchman, Charlie J; Keown, Jeremy R; Kropp, Ashleigh; Kursula, Petri; Lovering, Andrew L; Lemaitre, Bruno; Lia, Andrea; Liu, Shiheng; Logotheti, Maria; Lu, Shuze; Markússon, Sigurbjörn; Miller, Mitchell D; Minasov, George; Niemann, Hartmut H; Opazo, Felipe; Phillips, George N; Davies, Owen R; Rommelaere, Samuel; Rosas-Lemus, Monica; Roversi, Pietro; Satchell, Karla; Smith, Nathan; Wilson, Mark A; Wu, Kuan-Lin; Xia, Xian; Xiao, Han; Zhang, Wenhua; Zhou, Z Hong; Fidelis, Krzysztof; Topf, Maya; Moult, John; Schwede, Torsten
We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.
PMID: 37493353
ISSN: 1097-0134
CID: 5575182

3D reconstructions of parasite development and the intracellular niche of the microsporidian pathogen Encephalitozoon intestinalis

Antao, Noelle V; Lam, Cherry; Davydov, Ari; Riggi, Margot; Sall, Joseph; Petzold, Christopher; Liang, Feng-Xia; Iwasa, Janet H; Ekiert, Damian C; Bhabha, Gira
Microsporidia are an early-diverging group of fungal pathogens with a wide host range. Several microsporidian species cause opportunistic infections in humans that can be fatal. As obligate intracellular parasites with highly reduced genomes, microsporidia are dependent on host metabolites for successful replication and development. Our knowledge of microsporidian intracellular development remains rudimentary, and our understanding of the intracellular niche occupied by microsporidia has relied on 2D TEM images and light microscopy. Here, we use serial block-face scanning electron microscopy (SBF-SEM) to capture 3D snapshots of the human-infecting species, Encephalitozoon intestinalis, within host cells. We track E. intestinalis development through its life cycle, which allows us to propose a model for how its infection organelle, the polar tube, is assembled de novo in developing spores. 3D reconstructions of parasite-infected cells provide insights into the physical interactions between host cell organelles and parasitophorous vacuoles, which contain the developing parasites. The host cell mitochondrial network is substantially remodeled during E. intestinalis infection, leading to mitochondrial fragmentation. SBF-SEM analysis shows changes in mitochondrial morphology in infected cells, and live-cell imaging provides insights into mitochondrial dynamics during infection. Our data provide insights into parasite development, polar tube assembly, and microsporidia-induced host mitochondria remodeling.
PMID: 37996434
ISSN: 2041-1723
CID: 5576302

Structure of an endogenous mycobacterial MCE lipid transporter

Chen, James; Fruhauf, Alice; Fan, Catherine; Ponce, Jackeline; Ueberheide, Beatrix; Bhabha, Gira; Ekiert, Damian C
To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host1,2. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis1,3, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids4-7 and cholesterol1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.
PMID: 37495693
ISSN: 1476-4687
CID: 5560192