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1.
Mol Cell ; 83(11): 1856-1871.e9, 2023 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-37267906

RESUMEN

The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A combination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. Our study provides mechanistic insights into long-distance mRNA transport and demonstrates that the particular architecture of FERRY is closely linked to a previously undescribed mode of RNA binding, involving coiled-coil domains.


Asunto(s)
Proteínas de Transporte Vesicular , Proteínas de Unión al GTP rab5 , Humanos , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/análisis , Proteínas de Unión al GTP rab5/metabolismo , Endosomas/genética , Endosomas/metabolismo
2.
PLoS Pathog ; 18(1): e1010182, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34986192

RESUMEN

The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the structure, function, and activation of many of these effectors remains poorly understood. Here, we present the structures of the T6SS effector RhsA from Pseudomonas protegens and its cognate T6SS spike protein, VgrG1, at 3.3 Å resolution. The structures reveal that the rearrangement hotspot (Rhs) repeats of RhsA assemble into a closed anticlockwise ß-barrel spiral similar to that found in bacterial insecticidal Tc toxins and in metazoan teneurin proteins. We find that the C-terminal toxin domain of RhsA is autoproteolytically cleaved but remains inside the Rhs 'cocoon' where, with the exception of three ordered structural elements, most of the toxin is disordered. The N-terminal 'plug' domain is unique to T6SS Rhs proteins and resembles a champagne cork that seals the Rhs cocoon at one end while also mediating interactions with VgrG1. Interestingly, this domain is also autoproteolytically cleaved inside the cocoon but remains associated with it. We propose that mechanical force is required to remove the cleaved part of the plug, resulting in the release of the toxin domain as it is delivered into a susceptible bacterial cell by the T6SS.


Asunto(s)
Proteínas Bacterianas , Pseudomonas , Sistemas de Secreción Tipo VI
3.
Elife ; 92020 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-33320089

RESUMEN

Type VI secretion systems (T6SSs) deliver antibacterial effector proteins between neighboring bacteria. Many effectors harbor N-terminal transmembrane domains (TMDs) implicated in effector translocation across target cell membranes. However, the distribution of these TMD-containing effectors remains unknown. Here, we discover prePAAR, a conserved motif found in over 6000 putative TMD-containing effectors encoded predominantly by 15 genera of Proteobacteria. Based on differing numbers of TMDs, effectors group into two distinct classes that both require a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMD-chaperone complexes from Salmonella Typhimurium and Pseudomonas aeruginosa, respectively, reveals that Eag chaperones mimic transmembrane helical packing to stabilize effector TMDs. In addition to participating in the chaperone-TMD interface, we find that prePAAR residues mediate effector-VgrG spike interactions. Taken together, our findings reveal mechanisms of chaperone-mediated stabilization and secretion of two distinct families of T6SS membrane protein effectors.


Asunto(s)
Transporte de Proteínas/fisiología , Pseudomonas aeruginosa/metabolismo , Salmonella typhimurium/metabolismo , Sistemas de Secreción Tipo VI/metabolismo , Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Conformación Proteica , Dominios Proteicos
4.
Elife ; 92020 11 25.
Artículo en Inglés | MEDLINE | ID: mdl-33236980

RESUMEN

Canonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca2+ signaling. Inhibition of TRPCs by small molecules was shown to be promising in treating renal diseases. In cells, the channels are regulated by calmodulin (CaM). Molecular details of both CaM and drug binding have remained elusive so far. Here, we report structures of TRPC4 in complex with three pyridazinone-based inhibitors and CaM. The structures reveal that all the inhibitors bind to the same cavity of the voltage-sensing-like domain and allow us to describe how structural changes from the ligand-binding site can be transmitted to the central ion-conducting pore of TRPC4. CaM binds to the rib helix of TRPC4, which results in the ordering of a previously disordered region, fixing the channel in its closed conformation. This represents a novel CaM-induced regulatory mechanism of canonical TRP channels.


Asunto(s)
Calmodulina/metabolismo , Moduladores del Transporte de Membrana/farmacología , Piridazinas/farmacología , Canales Catiónicos TRPC/efectos de los fármacos , Proteínas de Pez Cebra/efectos de los fármacos , Animales , Sitios de Unión , Calmodulina/química , Calmodulina/genética , Células HEK293 , Humanos , Ligandos , Potenciales de la Membrana , Moduladores del Transporte de Membrana/química , Moduladores del Transporte de Membrana/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Piridazinas/química , Piridazinas/metabolismo , Células Sf9 , Relación Estructura-Actividad , Canales Catiónicos TRPC/química , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismo , Xenopus , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
5.
PLoS Pathog ; 16(8): e1008530, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32810181

RESUMEN

Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called α-clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the α-clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the relative position of the N-terminal α-helices in the three LFs determines which factor is translocated first.


Asunto(s)
Carbunco/microbiología , Antígenos Bacterianos/química , Bacillus anthracis/fisiología , Toxinas Bacterianas/química , Microscopía por Crioelectrón/métodos , Animales , Humanos , Modelos Moleculares , Conformación Proteica
6.
Commun Biol ; 2: 218, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31240256

RESUMEN

Selecting particles from digital micrographs is an essential step in single-particle electron cryomicroscopy (cryo-EM). As manual selection of complete datasets-typically comprising thousands of particles-is a tedious and time-consuming process, numerous automatic particle pickers have been developed. However, non-ideal datasets pose a challenge to particle picking. Here we present the particle picking software crYOLO which is based on the deep-learning object detection system You Only Look Once (YOLO). After training the network with 200-2500 particles per dataset it automatically recognizes particles with high recall and precision while reaching a speed of up to five micrographs per second. Further, we present a general crYOLO network able to pick from previously unseen datasets, allowing for completely automated on-the-fly cryo-EM data preprocessing during data acquisition. crYOLO is available as a standalone program under http://sphire.mpg.de/ and is distributed as part of the image processing workflow in SPHIRE.


Asunto(s)
Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Conjuntos de Datos como Asunto , Aprendizaje Profundo , Redes Neurales de la Computación
7.
Nat Microbiol ; 3(10): 1142-1152, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30177742

RESUMEN

The type VI secretion system (T6SS) primarily functions to mediate antagonistic interactions between contacting bacterial cells, but also mediates interactions with eukaryotic hosts. This molecular machine secretes antibacterial effector proteins by undergoing cycles of extension and contraction; however, how effectors are loaded into the T6SS and subsequently delivered to target bacteria remains poorly understood. Here, using electron cryomicroscopy, we analysed the structures of the Pseudomonas aeruginosa effector Tse6 loaded onto the T6SS spike protein VgrG1 in solution and embedded in lipid nanodiscs. In the absence of membranes, Tse6 stability requires the chaperone EagT6, two dimers of which interact with the hydrophobic transmembrane domains of Tse6. EagT6 is not directly involved in Tse6 delivery but is crucial for its loading onto VgrG1. VgrG1-loaded Tse6 spontaneously enters membranes and its toxin domain translocates across a lipid bilayer, indicating that effector delivery by the T6SS does not require puncturing of the target cell inner membrane by VgrG1. Eag chaperone family members from diverse Proteobacteria are often encoded adjacent to putative toxins with predicted transmembrane domains and we therefore anticipate that our findings will be generalizable to numerous T6SS-exported membrane-associated effectors.


Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreción Tipo VI/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/ultraestructura , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Membrana Celular/química , Membrana Celular/metabolismo , Microscopía por Crioelectrón , Membrana Dobles de Lípidos/química , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Mutación , Dominios Proteicos , Estabilidad Proteica , Sistemas de Secreción Tipo VI/metabolismo
8.
J Mol Med (Berl) ; 96(6): 483-493, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29730699

RESUMEN

A human cell is a precisely regulated system that relies on the complex interaction of molecules. Structural insights into the cellular machinery at the atomic level allow us to understand the underlying regulatory mechanism and provide us with a roadmap for the development of novel drugs to fight diseases. Facilitated by recent technological breakthroughs, the Nobel prize-winning technique electron cryomicroscopy (cryo-EM) has become a versatile and extremely powerful tool to solve routinely near-atomic resolution three-dimensional protein structures. Consequently, it has become the focus of attention for structure-based drug design. In this review, we describe the basics of cryo-EM and highlight its growing role in biomedical research. Furthermore, we discuss latest developments as well as future perspectives.


Asunto(s)
Microscopía por Crioelectrón , Investigación Biomédica , Proteínas
9.
Cell ; 163(3): 607-19, 2015 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-26456113

RESUMEN

Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD(+) and NADP(+). Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). These proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.


Asunto(s)
Toxinas Bacterianas/metabolismo , NAD+ Nucleosidasa/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreción Tipo VI/química , ADP Ribosa Transferasas/metabolismo , Toxinas Bacterianas/química , Modelos Moleculares , NAD/metabolismo , NAD+ Nucleosidasa/química , NADP/metabolismo , Factor Tu de Elongación Peptídica/química , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/enzimología , Sistemas de Secreción Tipo VI/metabolismo
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