Structure of the poly-C9 component of the complement membrane attack complex

The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion

Structure of yeast cytochrome c oxidase in a supercomplex with cytochrome bc1

Cytochrome c oxidase (complex IV, CIV) is known in mammals to exist independently or in association with other respiratory proteins to form supercomplexes (SCs). In Saccharomyces cerevisiae, CIV is found solely in a SC with cytochrome bc1 (complex III, CIII). Here, we present the cryo-EM structure of S. cerevisiae CIV in a III2IV2 SC at 3.3 Å resolution. While overall similarity to mammalian homologues is high, we found notable differences in the supernumerary subunits Cox26 and Cox13; the latter exhibits a unique arrangement that precludes CIV dimerization as seen in bovine. A conformational shift in the matrix domain of Cox5A – involved in allosteric inhibition by ATP – may arise from its association with CIII. The CIII–CIV arrangement highlights a conserved interaction interface of CIII, albeit one occupied by complex I in mammalian respirasomes. We discuss our findings in the context of the potential impact of SC formation on CIV regulation

Perforin proteostasis is regulated through its C2 domain: supra-physiological cell death mediated by T431D-perforin

The pore forming, Ca2+-dependent protein, perforin, is essential for the function of cytotoxic lymphocytes, which are at the frontline of immune defence against pathogens and cancer. Perforin is a glycoprotein stored in the secretory granules prior to release into the immune synapse. Congenital perforin deficiency causes fatal immune dysregulation, and is associated with various haematological malignancies. At least 50% of pathological missense mutations in perforin result in protein misfolding and retention in the endoplasmic reticulum. However, the regulation of perforin proteostasis remains unexplored. Using a variety of biochemical assays that assess protein stability and acquisition of complex glycosylation, we demonstrated that the binding of Ca2+ to the C2 domain stabilises perforin and regulates its export from the endoplasmic reticulum to the secretory granules. As perforin is a thermo-labile protein, we hypothesised that by altering its C2 domain it may be possible to improve protein stability. On the basis of the X-ray crystal structure of the perforin C2 domain, we designed a mutation (T431D) in the Ca2+ binding loop. Mutant perforin displayed markedly enhanced thermal stability and lytic function, despite its trafficking from the endoplasmic reticulum remaining unchanged. Furthermore, by introducing the T431D mutation into A90V perforin, a pathogenic mutation, which results in protein misfolding, we corrected the A90V folding defect and completely restored perforin’s cytotoxic function. These results revealed an unexpected role for the Ca2+-dependent C2 domain in maintaining perforin proteostasis and demonstrated the possibility of designing perforin with supra-physiological cytotoxic function through stabilisation of the C2 domain

Conformational changes during pore formation by the perforin-related protein pleurotolysin

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ~70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function

Использование низкобелковых обогащенных крахмаломучных продуктов в диетотерапии больных фенилкетонурией детей в возрасте старше 1 года

Background. The nutrition of children with phenylketonuria includes specialized starch-based products, the range of which is constantly expanding. Our aim was to study the safety of the composition of starchy flakes enriched with a complex of fat-soluble vitamins, natural fruit and berry additives used in the food of children with phenylketonuria. Methods. The study included children under the age of 14 years who were compliant with the previously conducted hypophenylalanine diet, without acute infectious, severe somatic or neurological diseases. The investigated products (starch-rye, wheat, and wheat fruit flakes with a complex of provitamin A and vitamin E) were prescribed instead of previously used low-protein confectionery products in the amount of 20–25 g/day for children under 6 years, 30–40 g — for children aged 6 years and over. The products were given with the recommendation to use alternately, with a duration of at least 10 days, totally for 30 days of the study. The safety of the products was assessed by phenylalanine concentration in the blood (determined by the fluorimetric method). In addition, we assessed the organoleptic qualities of the products and the dynamics of physical development of children. Results. The study included 15 children, mean age 4.4 ± 1.9 years. The initial concentration of phenylalanine in the blood varied from 1.6 to 3.9 mg%, the median — 2.2 mg% (2.0; 2.8). In 30 days after inclusion of starchy flakes in the diet, the content of phenylalanine in the blood did not change and was 2.5 mg% (2.2; 2.7); p = 0.859. The organoleptic properties of the products were rated «excellent» by all patients and their parents (in children under 6 years, only according to the parents’ assessment). The indicators of physical development did not change. There was no adverse events (allergic reactions, dyspepsia, refusal to take food). Conclusion. Introduction of new functional products — low-protein starchy flakes enriched with a vitamin complex and natural fruit and berry additives — in the diet of children with phenylketonuria allows to maintain the level of phenylalanine in the blood at the level of reference values.Обоснование. В питании детей с фенилкетонурией широко используют специализированные продукты на основе крахмалов, ассортимент которых постоянно расширяется.Цель исследования — изучить безопасность состава хлопьев крахмаломучных, обогащенных комплексом жирорастворимых витаминов, натуральными плодовыми и ягодными добавками, используемых в пище детей с фенилкетонурией.Методы. В исследование включали детей в возрасте до 14 лет, комплаентных к ранее проводимой гипофенилаланиновой диете, без острых инфекционных, тяжелых соматических или неврологических заболеваний. Исследуемые продукты — крахмалоржаные, пшеничные и пшеничные плодово-ягодные хлопья с комплексом провитамина А и витамина Е — назначали взамен применявшихся ранее низкобелковых кондитерских изделий в количестве 20–25 г/сут детям младше 6 лет, по 30–40 г — детям, достигших возраста или старше 6 лет. Продукты выдавали с рекомендацией использовать поочередно, продолжительностью не менее 10 сут, всего на 30 сут исследования. Безопасность продуктов оценивали по концентрации фенилаланина в крови (определяли флюориметрическим методом). Дополнительно оценивали органолептические качества продуктов и динамику физического развития детей.Результаты. В исследование включили 15 детей, средний возраст 4,4±1,9 года. Исходная концентрация фенилаланина в крови варьировала от 1,6 до 3,9 мг%, медиана — 2,2 мг% (2,0; 2,8). Через 30 сут после включения в рацион крахмаломучных хлопьев содержание фенилаланина в крови не изменилось и составило 2,5 мг% (2,2; 2,7); р=0,859. Органолептические свойства продуктов были оценены на «отлично» всеми пациентами и их родителями (у детей в возрасте до 6 лет — только согласно оценке родителей). Показатели физического развития не изменились. Нежелательные явления (аллергические реакции, диспепсии, отказ от приема продуктов) не зафиксированы.Заключение. Введение в рацион детей с фенилкетонурией новых функциональных продуктов — хлопьев крахмаломучных низкобелковых, обогащенных витаминным комплексом и натуральными плодовыми и ягодными добавками, позволяет сохранять уровень фенилаланина в крови на уровне референсных значений.ИСТОЧНИК ФИНАНСИРОВАНИЯ Работа выполнена при поддержке гранта Федерального государственного научного учреждения «Всероссийский научно-исследовательский институт крахмалопродуктов» Федерального агентства научных организаций (Московская область). Для целей исследования использовались продукты, безвозмездно предоставленные производителем (опытное производство ФГНУ «ВНИИК» ФАНО).КОНФЛИКТ ИНТЕРЕСОВ Т.Э. Боровик, Н.Н. Семёнова, О.Л. Лукоянова, Н.Г. Звонкова, Т.В. Бушуева, Т.Н. Степанова, В.А. Скворцова — проведение научно-исследовательских работ при поддержке компаний Heinz, Semper, Хипрока Нутришион Ист Лимитед. И.М. Гусева, Е.А. Рославцева, А.К. Геворкян, С.Т. Быкова, Т.Г. Калинина, С.Г. Калиненкова подтвердили отсутствие конфликта интересов.ВЫРАЖЕНИЕ ПРИЗНАТЕЛЬНОСТИ Выражаем благодарность к.м.н. С.Г. Калиненковой (Московский областной научно-исследовательский клинический институт им. М.Ф. Владимирского) за участие в выполнении лабораторной части данного исследования. 

Friend or foe: the same fold for attack and defense

Two recent crystal structures of membrane attack complex/perforin (MACPF) domains found in the complement and perforin families unexpectedly reveal that some proteins of the immune system share a common core fold with their bacterial targets. Although a relationship between MACPF proteins and the previously characterized bacterial cholesterol-dependent cytolysins (CDCs) is not detectable by sequence analysis, the MACPF structures show that eukaryotic defense and bacterial CDC attack share a common mechanism of membrane insertion and pore formation

3D Reconstruction of Mammalian Septin Filaments

Mammalian septins are a family of guanosine triphosphate-binding proteins thought to play a role in a number of key cellular processes, such as cytokinesis, protein scaffolding and vesicle trafficking. Although their precise functions remain to be determined, electron microscopy has shown septin filament formation in vitro and a role as a cytoskeletal polymer has been proposed. Here, we present a 3D reconstruction of septin filaments determined using electron microscopy of negatively stained specimens and single-particle image processing. Septin was isolated from rat brain as an approximately 240-kDa complex, from which immunoblotting and N-terminal sequencing identified the major components as septins 3, 5 and 7. Electron microscopy and single-particle analysis indicated that the majority of the septin filaments were ∼ 27 nm long. A comparison of 3D volumes obtained using two independent starting models (a row of spheres or a helix) and projection matching techniques revealed no major differences at the final resolution of 27 Å, and this structure was highly reproducible when the entire procedure was repeated several times. The reconstruction revealed three apparent subunits, each separated by a cleft; these subunits were similar, but not identical, possibly indicating multiple isoforms within each filament. In some views a smaller cleft appeared to separate the subunits into two smaller regions, perhaps reflecting the presence of septin dimers. This is the first 3D reconstruction of the native septin assembly, and appears compatible with the hypothesis that the septin complex is a hexamer consisting of dimers or heterotrimers. Further investigations are necessary to confirm how the structure of the filaments determined in the present study correlates with the roles of septins in vivo

The pore conformation of lymphocyte perforin

Perforin is a pore-forming protein that facilitates rapid killing of pathogen-infected or cancerous cells by the immune system. Perforin is released from cytotoxic lymphocytes, together with proapoptotic granzymes, to bind to a target cell membrane where it oligomerizes and forms pores. The pores allow granzyme entry, which rapidly triggers the apoptotic death of the target cell. Here, we present a 4-Å resolution cryo–electron microscopy structure of the perforin pore, revealing previously unidentified inter- and intramolecular interactions stabilizing the assembly. During pore formation, the helix-turn-helix motif moves away from the bend in the central β sheet to form an intermolecular contact. Cryo–electron tomography shows that prepores form on the membrane surface with minimal conformational changes. Our findings suggest the sequence of conformational changes underlying oligomerization and membrane insertion, and explain how several pathogenic mutations affect function

CryoEM Structure and Assembly Mechanism of a Bacterial Virus Genome Gatekeeper

International audienceNumerous viruses package their dsDNA genome into preformed capsids through a portal gatekeeper that is subsequently closed. We report the structure of the DNA gatekeeper complex of bacteriophage SPP1 (gp612gp1512gp166) in the post-DNA packaging state at 2.7 Å resolution obtained by single particle cryo-electron microscopy. Comparison of the native SPP1 complex with assembly-naïve structures of individual components uncovered the complex program of conformational changes leading to its assembly. After DNA packaging, gp15 binds via its C-terminus to the gp6 oligomer positioning gp15 subunits for oligomerization. Gp15 refolds its inner loops creating an intersubunit β-barrel that establishes different types of contacts with six gp16 subunits. Gp16 binding and oligomerization is accompanied by folding of helices that close the portal channel to keep the viral genome inside the capsid. This mechanism of assembly has broad functional and evolutionary implications for viruses of the prokaryotic tailed viruses-herpesviruses lineage