An ancestral interaction module promotes oligomerization in divergent mitochondrial ATP synthases

Mitochondrial ATP synthase forms stable dimers arranged into oligomeric assemblies that generate the inner-membrane curvature essential for efficient energy conversion. Here, we report cryo-EM structures of the intact ATP synthase dimer from Trypanosoma brucei in ten different rotational states. The model consists of 25 subunits, including nine lineage-specific, as well as 36 lipids. The rotary mechanism is influenced by the divergent peripheral stalk, conferring a greater conformational flexibility. Proton transfer in the lumenal half-channel occurs via a chain of five ordered water molecules. The dimerization interface is formed by subunit-g that is critical for interactions but not for the catalytic activity. Although overall dimer architecture varies among eukaryotes, we find that subunit-g together with subunit-e form an ancestral oligomerization motif, which is shared between the trypanosomal and mammalian lineages. Therefore, our data defines the subunit-g/e module as a structural component determining ATP synthase oligomeric assemblies.

Overactive STAT3 drives accumulation of disease-associated CD21low B cells

Dysregulated STAT3 signalling is correlated with antibody-mediated autoimmunity and B- cell neoplasia, but its effect on B cells is underexplored. Here we address this in children with STAT3 gain-of-function (GOF) syndrome and in mice with STAT3T716M, the most common STAT3 GOF syndrome human mutation, or STAT3K658N, a dimerization interface mutation responsible for STAT3 GOF syndrome in two children. The main B cell consequence of overactive STAT3 was accumulation of CD19high CD21low atypical memory B cells in humans and of CD21low CD23low B cells in mice resembling age-associated B cells expressing T-bet, CD11c and plasma cell differentiation genes. Overactive STAT3 within B cells increased expression of many genes in the B cell receptor and T cell help pathways, increased the tolerogenic receptor CD22, but opposed B cell tolerance checkpoints and increased formation of T-bet+ B cells upon BCR and CD40 stimulation. These results reveal overactive STAT3 as a central driver of a key class of disease-associated B-lymphocytes in humans and mice.

Bacterial Product Template Domain: An Evolutionary Intermediate between Dehydratase and Aldol Cyclase of Type I Polyketide Synthases

The product template (PT) domains act as an aldol cyclase to control the regiospecific aldol cyclization of the extremely reactive poly-β-ketone intermediate assembled by an iterative type I polyketide synthases (PKSs). Up to now, only the structure of fungal PksA PT that mediates the first-ring cyclization via C4-C9 aldol cyclization is available. We describe here the structural and computational characterization of a bacteria PT domain that controls C2-C7 cyclization in orsellinic acid (OSA) synthesis. Mutating the catalytic His949 of the PT abolishes production of OSA and results in a tetraacetic acid lactone (TTL) generated by spontaneous O-C cyclization of the acyl carrier protein (ACP)-bound tetraketide intermediate. Crystal structure of the bacterial PT domain closely resembles dehydrase (DH) domains of modular type I PKSs in the overall fold, dimerization interface and catalytic “His-Asp” dyad organization, but is significantly different from PTs of fungal iterative type I PKSs. QM/MM calculation reveals that the catalytic His949 abstracts a proton from C2 and transfers it to C7 carbonyl to mediate the cyclization reaction. According to the structural similarity to DHs and the functional similarity to fungal PTs, we propose that the bacterial PT represents an evolutionary intermediate between the two tailoring domains of type I PKSs.

Reteplase Fc-fusions produced in N. benthamiana are able to dissolve blood clots ex vivo

Thrombolytic and fibrinolytic therapies are effective treatments to dissolve blood clots in stroke therapy. Thrombolytic drugs activate plasminogen to its cleaved form plasmin, a proteolytic enzyme that breaks the crosslinks between fibrin molecules. The FDA-approved human tissue plasminogen activator Reteplase (rPA) is a non-glycosylated protein produced in E. coli. rPA is a deletion mutant of the wild-type Alteplase that benefits from an extended plasma half-life, reduced fibrin specificity and the ability to better penetrate into blood clots. Different methods have been proposed to improve the production of rPA. Here we show for the first time the transient expression in Nicotiana benthamiana of rPA fused to the immunoglobulin fragment crystallizable (Fc) domain on an IgG1, a strategy commonly used to improve the stability of therapeutic proteins. Despite our success on the expression and purification of dimeric rPA-Fc fusions, protein instability results in high amounts of Fc-derived degradation products. We hypothesize that the “Y”- shape of dimeric Fc fusions cause steric hindrance between protein domains and leads to physical instability. Indeed, mutations of critical residues in the Fc dimerization interface allowed the expression of fully stable rPA monomeric Fc-fusions. The ability of rPA-Fc to convert plasminogen into plasmin was demonstrated by plasminogen zymography and clot lysis assay shows that rPA-Fc is able to dissolve blood clots ex vivo. Finally, we addressed concerns with the plant-specific glycosylation by modulating rPA-Fc glycosylation towards serum-like structures including α2,6-sialylated and α1,6-core fucosylated N-glycans completely devoid of plant core fucose and xylose residues.

Molecular architecture of nucleosome remodeling and deacetylase sub-complexes by integrative structure determination

The Nucleosome Remodeling and Deacetylase (NuRD) complex is a chromatin-modifying assembly that regulates gene expression and DNA damage repair. Despite its importance, limited structural information is available on the complex and a detailed understanding of its mechanism is lacking. We investigated the molecular architecture of three NuRD sub-complexes: MTA1-HDAC1-RBBP4 (MHR), MTA1N-HDAC1-MBD3GATAD2CC (MHM), and MTA1-HDAC1-RBBP4-MBD3-GATAD2 (NuDe) using Bayesian integrative structure determination with IMP (Integrative Modeling Platform), drawing on information from SEC-MALLS, DIA-MS, XLMS, negative stain EM, X-ray crystallography, NMR spectroscopy, secondary structure and homology predictions. The structures were corroborated by independent cryo-EM maps, biochemical assays, and known cancer-associated mutations. Our integrative structure of the 2:2:2 MHM complex shows asymmetric binding of MBD3, whereas our structure of the NuDe complex shows MBD3 localized precisely to a single position distant from the MTA1 dimerization interface. Our models suggest a possible mechanism by which asymmetry is introduced in NuRD, and indicate three previously unrecognized subunit interfaces in NuDe: HDAC1C-MTA1BAH, MTA1BAH-MBD3, and HDAC160-100-MBD3. We observed that a significant number of cancer-associated mutations mapped to protein-protein interfaces in NuDe. Our approach also allows us to localize regions of unknown structure, such as HDAC1C and MBD3IDR, thereby resulting in the most complete structural characterization of these NuRD sub-complexes so far.

Structural basis of membrane recognition of Toxoplasma gondii vacuole by Irgb6

The p47 immunity-related GTPase (IRG) Irgb6 plays a pioneering role in host defense against Toxoplasma gondii infection. Irgb6 is recruited to the parasitophorous vacuole membrane (PVM) formed by T. gondii and disrupts it. Despite the importance of this process, the molecular mechanisms accounting for PVM recognition by Irgb6 remain elusive because of lack of structural information on Irgb6. Here we report the crystal structures of mouse Irgb6 in the GTP-bound and nucleotide-free forms. Irgb6 exhibits a similar overall architecture to other IRGs in which GTP binding induces conformational changes in both the dimerization interface and the membrane-binding interface. The membrane-binding interface of Irgb6 assumes a unique conformation, composed of N- and C-terminal helical regions forming a phospholipid binding site. In silico docking of phospholipids further revealed membrane-binding residues that were validated through mutagenesis and cell-based assays. Collectively, these data demonstrate a novel structural basis for Irgb6 to recognize T. gondii PVM in a manner distinct from other IRGs.

An ancestral interaction module promotes oligomerisation in divergent mitochondrial ATP synthases

Mitochondrial ATP synthase forms stable dimers arranged into oligomeric assemblies that generate the inner-membrane curvature essential for efficient energy conversion. Here, we report cryo EM structures of the intact ATP synthase dimer from trypanosomes in 10 different rotational states. The model consists of 25 subunits, including 11 lineage-specific, as well as 36 lipids. The rotary mechanism is influenced by the divergent peripheral stalk, conferring a greater conformational flexibility. Proton transfer in the lumenal half-channel occurs via a chain of five ordered water molecules. The dimerization interface is formed by subunit-g that is critical for interactions but not for the catalytic activity. Although overall dimer architecture varies among eukaryotes, we find that subunit-g and -e form a common ancestral oligomerisation motif, which is shared between the trypanosomal and mammalian lineages. Therefore, our data defines the subunit-g/e module as a structural component determining ATP synthase oligomeric assemblies.

Divergence in dimerization and activity of primate APOBEC3C

The APOBEC3 (A3) family of single-stranded DNA cytidine deaminases are host restriction factors that inhibit lentiviruses, such as HIV-1, in the absence of the Vif protein that causes their degradation. Deamination of cytidine in HIV-1 (-)DNA forms uracil that causes inactivating mutations when uracil is used as a template for (+)DNA synthesis. For APOBEC3C (A3C), the chimpanzee and gorilla orthologues are more active than human A3C, and the Old World Monkey A3C from rhesus macaque (rh) is not active against HIV-1. Multiple integrated analyses determined why rhA3C was not active against HIV-1 and how to increase this activity. Biochemical, virological, and coevolutionary analyses combined with molecular dynamics simulations showed that the key amino acids needed to promote rhA3C antiviral activity also promoted dimerization. Although rhA3C shares a similar dimer interface with hominid A3C, the key amino acid contacts were different. Overall, our results determine the basis for why rhA3C is less active than human A3C, establish the amino acid network for dimerization and increased activity, and track the loss and gain of A3C antiviral activity in primates. The coevolutionary analysis of the A3C dimerization interface provides a basis from which to analyze dimerization interfaces of other A3 family members.