scholarly journals Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dukas Jurėnas ◽  
Leonardo Talachia Rosa ◽  
Martial Rey ◽  
Julia Chamot-Rooke ◽  
Rémi Fronzes ◽  
...  

AbstractBacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins with a modular organization, comprising a C-terminal toxin domain fused to a N-terminal domain that adapts to the delivery apparatus. Polymorphic toxins include bacteriocins, contact-dependent growth inhibition systems, and specialized Hcp, VgrG, PAAR or Rhs Type VI secretion (T6SS) components. We recently described and characterized Tre23, a toxin domain fused to a T6SS-associated Rhs protein in Photorhabdus laumondii, Rhs1. Here, we show that Rhs1 forms a complex with the T6SS spike protein VgrG and the EagR chaperone. Using truncation derivatives and cross-linking mass spectrometry, we demonstrate that VgrG-EagR-Rhs1 complex formation requires the VgrG C-terminal β-helix and the Rhs1 N-terminal region. We then report the cryo-electron-microscopy structure of the Rhs1-EagR complex, demonstrating that the Rhs1 central region forms a β-barrel cage-like structure that encapsulates the C-terminal toxin domain, and provide evidence for processing of the Rhs1 protein through aspartyl autoproteolysis. We propose a model for Rhs1 loading on the T6SS, transport and delivery into the target cell.

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 505
Author(s):  
Marta L. Mendes ◽  
Gunnar Dittmar

The 26S proteasome is a macromolecular complex that degrades proteins maintaining cell homeostasis; thus, determining its structure is a priority to understand its function. Although the 20S proteasome’s structure has been known for some years, the highly dynamic nature of the 19S regulatory particle has presented a challenge to structural biologists. Advances in cryo-electron microscopy (cryo-EM) made it possible to determine the structure of the 19S regulatory particle and showed at least seven different conformational states of the proteasome. However, there are still many questions to be answered. Cross-linking mass spectrometry (CLMS) is now routinely used in integrative structural biology studies, and it promises to take integrative structural biology to the next level, answering some of these questions.


Author(s):  
John Trinickt ◽  
Howard White

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.


2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Young-Jun Park ◽  
Kaitlyn D. Lacourse ◽  
Christian Cambillau ◽  
Frank DiMaio ◽  
Joseph D. Mougous ◽  
...  

Author(s):  
Lucía Quintana-Gallardo ◽  
Moisés Maestro-López ◽  
Jaime Martín-Benito ◽  
Miguel Marcilla ◽  
Daniel Rutz ◽  
...  

2006 ◽  
Vol 12 (S02) ◽  
pp. 658-659
Author(s):  
BK Kaletas ◽  
E Van Duijn ◽  
AJ R Heck ◽  
RB J Geels ◽  
F De Haas ◽  
...  

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2006


2016 ◽  
Vol 23 (10) ◽  
pp. 899-905 ◽  
Author(s):  
Alexandra C Walls ◽  
M Alejandra Tortorici ◽  
Brandon Frenz ◽  
Joost Snijder ◽  
Wentao Li ◽  
...  

2021 ◽  
Author(s):  
Shuo Du ◽  
Pulan Liu ◽  
Zhiying Zhang ◽  
Tianhe Xiao ◽  
Ayijiang Yasimayi ◽  
...  

The spread of the SARS-CoV-2 variants could seriously dampen the global effort to tackle the COVID-19 pandemic. Recently, we investigated the humoral antibody responses of SARS-CoV-2 convalescent patients and vaccinees towards circulating variants, and identified a panel of monoclonal antibodies (mAbs) that could efficiently neutralize the B.1.351 (Beta) variant. Here we investigate how these mAbs target the B.1.351 spike protein using cryo-electron microscopy. In particular, we show that two superpotent mAbs, BD-812 and BD-836, have non-overlapping epitopes on the receptor-binding domain (RBD) of spike. Both block the interaction between RBD and the ACE2 receptor; and importantly, both remain fully efficacious towards the B.1.617.1 (Kappa) and B.1.617.2 (Delta) variants. The BD-812/BD-836 pair could thus serve as an ideal antibody cocktail against the SARS-CoV-2 VOCs.


2020 ◽  
Author(s):  
Alan Rodriguez Carvajal ◽  
Carlos Gomez Diaz ◽  
Antonia Vogel ◽  
Adar Sonn-Segev ◽  
Katrin Schodl ◽  
...  

AbstractThe linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase that generates linear/Met1-linked ubiquitin chains. One of the LUBAC components, HOIL-1L, was recently shown to catalyse oxyester bond formation between the C-terminus of ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. We present the first 3D reconstruction of LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that addition of the oxyester-bound branches depends on HOIL-1L catalytic activity. We suggest a coordinated ubiquitin relay mechanism between the HOIP and HOIL-1L ligases supported by cross-linking mass spectrometry data, which show proximity between the catalytic RBR domains. Mutations in the linear ubiquitin chain-binding NZF domain of HOIL-1L reduces chain branching confirming its role in the process. In cells, these heterotypic chains were induced by TNF. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains with linear and oxyester-linked branches by the concerted action of HOIP and HOIL-1L.


2020 ◽  
Author(s):  
Raudatun Hasanah

Rumah sakit sebagai sarana pelayanan kesehatan pada dasarnya adalah untuk menyelamatkan pasien, keselamatan pasien merupakan prioritas bagi pelaksanaan lima isu penting tentang keselamatan di rumah sakit, karena masalah keselamatan pasien berkaitan erat dengan kualitas dan citra rumah sakit itu sendiri. Perkembangan ilmu pengetahuan dan tekhnologi yang sedemikian pesat menyebabkan pelayanan kesehatan di rumah sakit menjadi sangat kompleks sehingga jika tidak dilakukan dengan benar dan hati-hati akan berpotensi untuk terjadinya Insiden Keselamatan Pasien (IKP) yang terdiri dari Kejadian Tidak Diharapkan (KTD), Kejadian Nyaris Cedera (KNC), Kejadian Tidak Cedera (KTC) dan Kondisi Potensial Cedera (KPC) (Depkes,2006).Setiap rumah sakit sudah diwajibkan untuk melakukan gerakan keselamatan pasien, apalagi PERSI sudah menerbitkan buku tentang pelaksanan keselamatan pasien di rumah sakit dan dalam waktu mendatang keselamatan pasien termasuk dalam penilaian akreditasi rumah sakit. Pada prinsipnya pelaksaan keselamatan pasien sesuai dengan standar departemen kesehatan, namun didalam pelaksanaan keselamatan pasien hampir sama pada setiap rumah sakit.Diawal tahun ini keselamatan pasien harus semakin ditingkatkan disebabkan munculnya virus covid 19 yang awalnya terjadi di Wuhan, provinsi Hubei, China dan dikaitkan dengan pasar binatang. Dalam rentang waktu satu bulan terjadi peningkatan kasus yang signifikan dan meluas ke beberapa provinsi di China, bahkan ke Jepang, Thailand dan Korea Selatan.satu Penyebaran penyakit yang begitu cepat serta meluas ke beberapa negara menyebabkan World Health Organization (WHO) akhirnya mengumumkan COVID-19 sebagai pandemi pada 12 Maret 2020. Virus corona berbentuk bulat dengan diameter sekitar 125 nm seperti yang digambarkan dalam penelitian menggunakan cryo-electron microscopy. Partikel virus corona mengandung empat protein struktural utama, yaitu protein S (spike protein) yang berbentuk seperti paku, protein M (membrane protein), protein E (envelope protein), dan protein N (nucleocapside protein). Protein S (~150 kda), protein M (~25– 30 kda), protein E (~8–12 kda) sedangkan protein N terdapat di dalam nukleokapsid.Virus corona merupakan zoonosis, sehingga terdapat kemungkinkan virus berasal dari hewan dan ditularkan ke manusia. Pada COVID-19 belum diketahui dengan pasti proses penularan dari Perkembangan data selanjutnya menunjukkan penularan antar manusia (human to human), yaitu diprediksi melalui droplet dan kontak dengan virus yang dikeluarkan dalam droplet. Penularan ini terjadi umumnya melalui droplet dan kontak dengan virus kemudian virus dapat masuk ke dalam mukosa yang terbuka. Suatu analisis mencoba mengukur laju penularan berdasarkan masa inkubasi, gejala dan durasi antara gejala dengan pasien yang diisolasi. Analisis tersebut mendapatkan hasil penularan dari 1 pasien ke sekitar 3 orang di sekitarnya, tetapi kemungkinan penularan di masa inkubasi menyebabkan masa kontak pasien ke orang sekitar lebih lama sehingga risiko jumlah kontak tertular dari 1 pasien mungkin dapat lebih besar.


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