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Abstract Bacterial AB toxins are secreted key virulence factors that are internalized by target cells through receptor-mediated endocytosis, translocating their enzymatic domain to the cytosol from endosomes (short-trip) or the endoplasmic reticulum (long-trip). To accomplish this, bacterial AB toxins evolved a multidomain structure organized into either a single polypeptide chain or non-covalently associated polypeptide chains. The prototypical short-trip single-chain toxin is characterized by a receptor-binding domain that confers cellular specificity and a translocation domain responsible for pore formation whereby the catalytic domain translocates to the cytosol in an endosomal acidification-dependent way. In this work, the determination of the three-dimensional structure of AIP56 shows that, instead of a two-domain organization suggested by previous studies, AIP56 has three-domains: a non-LEE encoded effector C (NleC)-like catalytic domain associated with a small middle domain that contains the linker-peptide, followed by the receptor-binding domain. In contrast to prototypical single-chain AB toxins, AIP56 does not comprise a typical structurally complex translocation domain; instead, the elements involved in translocation are scattered across its domains. Thus, the catalytic domain contains a helical hairpin that serves as a molecular switch for triggering the conformational changes necessary for membrane insertion only upon endosomal acidification, whereas the middle and receptor-binding domains are required for pore formation.

The mechanism of methylmannose polysaccharide (MMP) biogenesis in nontuberculous mycobacteria (NTM) is characterized, which involves an MMP-specific hydrolase (MmpH) and a rare mannosyltransferase (ManT). MmpH and ManT structures are presented.

ABSTRACT An important feature associated with Candida albicans pathogenicity is its ability to switch between yeast and hyphal forms, a process in which CaRas1 plays a key role. CaRas1 is activated by the guanine nucleotide exchange factor (GEF) CaCdc25, triggering hyphal growth-related signaling pathways through its conserved GTP-binding (G)-domain. An important function in hyphal growth has also been proposed for the long hypervariable region downstream the G-domain, whose unusual content of polyglutamine stretches and Q/N repeats make CaRas1 unique within Ras proteins. Despite its biological importance, both the structure of CaRas1 and the molecular basis of its activation by CaCdc25 remain unexplored. Here, we show that CaRas1 has an elongated shape and limited conformational flexibility and that its hypervariable region contains helical structural elements, likely forming an intramolecular coiled-coil. Functional assays disclosed that CaRas1-activation by CaCdc25 is highly efficient, with activities up to 2,000-fold higher than reported for human GEFs. The crystal structure of the CaCdc25 catalytic region revealed an active conformation for the α-helical hairpin, critical for CaRas1-activation, unveiling a specific region exclusive to CTG-clade species. Structural studies on CaRas1/CaCdc25 complexes also revealed an interaction surface clearly distinct from that of homologous human complexes. Furthermore, we identified an inhibitory synthetic peptide, prompting the proposal of a key regulatory mechanism for CaCdc25. To our knowledge, this is the first report of specific inhibition of the CaRas1-activation via targeting its GEF. This, together with their unique pathogen-structural features, disclose a set of novel strategies to specifically block this important virulence-related mechanism. IMPORTANCE Candida albicans is the main causative agent of candidiasis, the commonest fungal infection in humans. The eukaryotic nature of C. albicans and the rapid emergence of antifungal resistance raise the challenge of identifying novel drug targets to battle this prevalent and life-threatening disease. CaRas1 and CaCdc25 are key players in the activation of signaling pathways triggering multiple virulence traits, including the yeast-to-hypha interconversion. The structural similarity of the conserved G-domain of CaRas1 to those of human homologs and the lack of structural information on CaCdc25 has impeded progress in targeting these proteins. The unique structural and functional features for CaRas1 and CaCdc25 presented here, together with the identification of a synthetic peptide capable of specifically inhibiting the GEF activity of CaCdc25, open new possibilities to uncover new antifungal drug targets against C. albicans virulence.

Bacteria are challenged to adapt to environmental variations in order to survive. Under nutritional stress, several bacteria are able to slow down their metabolism into a nonreplicating state and wait for favourable conditions. It is almost universal that bacteria accumulate carbon stores to survive during this nonreplicating state and to fuel rapid proliferation when the growth-limiting stress disappears. Mycobacteria are exceedingly successful in their ability to become dormant under harsh circumstances and to be able to resume growth when conditions are favourable. Rapidly growing mycobacteria accumulate glucosylglycerate under nitrogen-limiting conditions and quickly mobilize it when nitrogen availability is restored. The depletion of intracellular glucosylglycerate levels in Mycolicibacterium hassiacum (basonym Mycobacterium hassiacum) was associated with the up-regulation of the gene coding for glucosylglycerate hydrolase (GgH), an enzyme that is able to hydrolyse glucosylglycerate to glycerate and glucose, a source of readily available energy. Highly conserved among unrelated phyla, GgH is likely to be involved in bacterial reactivation following nitrogen starvation, which in addition to other factors driving mycobacterial recovery may also provide an opportunity for therapeutic intervention, especially in the serious infections caused by some emerging opportunistic pathogens of this group, such as Mycobacteroides abscessus (basonym Mycobacterium abscessus). Using a combination of biochemical methods and hybrid structural approaches, the oligomeric organization of M. hassiacum GgH was determined and molecular determinants of its substrate binding and specificity were unveiled.

Peptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, generating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or join its components.

Reports on phase separation and amyloid formation for multiple proteins and aggregation-prone peptides are recurrently used to explore the molecular mechanisms associated with several human diseases. The information conveyed by these reports can be used directly in translational investigation, e.g., for the design of better drug screening strategies, or be compiled in databases for benchmarking novel aggregation-predicting algorithms. Given that minute protocol variations determine different outcomes of protein aggregation assays, there is a strong urge for standardized descriptions of the different types of aggregates and the detailed methods used in their production. In an attempt to address this need, we assembled the Minimum Information Required for Reproducible Aggregation Experiments (MIRRAGGE) guidelines, considering first-principles and the established literature on protein self-assembly and aggregation. This consensus information aims to cover the major and subtle determinants of experimental reproducibility while avoiding excessive technical details that are of limited practical interest for non-specialized users. The MIRRAGGE table (template available in Supplementary Information) is useful as a guide for the design of new studies and as a checklist during submission of experimental reports for publication. Full disclosure of relevant information also enables other researchers to reproduce results correctly and facilitates systematic data deposition into curated databases.

ABSTRACT Actinobacteria have long been the main source of antibiotics, secondary metabolites with tightly controlled biosynthesis by environmental and physiological factors. Phosphorylation of exogenous glucosamine has been suggested as a mechanism for incorporation of this extracellular material into secondary metabolite biosynthesis, but experimental evidence of specific glucosamine kinases in Actinobacteria is lacking. Here, we present the molecular fingerprints for the identification of a unique family of actinobacterial glucosamine kinases. Structural and biochemical studies on a distinctive kinase from the soil bacterium Streptacidiphilus jiangxiensis unveiled its preference for glucosamine and provided structural evidence of a phosphoryl transfer to this substrate. Conservation of glucosamine-contacting residues across a large number of uncharacterized actinobacterial proteins unveiled a specific glucosamine binding sequence motif. This family of kinases and their genetic context may represent the missing link for the incorporation of environmental glucosamine into the antibiotic biosynthesis pathways in Actinobacteria and can be explored to enhance antibiotic production. IMPORTANCE The discovery of novel enzymes involved in antibiotic biosynthesis pathways is currently a topic of utmost importance. The high levels of antibiotic resistance detected worldwide threaten our ability to combat infections and other 20th-century medical achievements, namely, organ transplantation or cancer chemotherapy. We have identified and characterized a unique family of enzymes capable of phosphorylating glucosamine to glucosamine-6-phosphate, a crucial molecule directly involved in the activation of antibiotic production pathways in Actinobacteria, nature’s main source of antimicrobials. The consensus sequence identified for these glucosamine kinases will help establish a molecular fingerprint to reveal yet-uncharacterized sequences in antibiotic producers, which should have an important impact in biotechnological and biomedical applications, including the enhancement and optimization of antibiotic production.

The validation and analysis of X-ray crystallographic data is essential for reproducibility and the development of crystallographic methods. Here, the authors describe a repository for crystallographic datasets and demonstrate some of the ways it could serve the crystallographic community.

Candida albicans is a polymorphic opportunistic fungal pathogen normally residing as commensal on mucosal surfaces, skin and gastrointestinal and genitourinary tracts. However, in immunocompromised patients C. albicans can cause superficial mucosal infections or life-threatening disseminated candidemia. A change in physiological conditions triggers a cascade of molecular events leading to morphogenetic alterations and increased resistance to damage induced by host defenses. The complex biology of this human pathogen is reflected in its morphological plasticity and reinforced by the ability to ambiguously translate the universal leucine CUG codon predominantly as serine, but also as leucine. Mistranslation affects more than half of C. albicans proteome and it is widespread across many biological processes. A previous analysis of CTG-codon containing gene products in C. albicans suggested that codon ambiguity subtly shapes protein function and might have a pivotal role in signalling cascades associated with morphological changes and pathogenesis. In this review we further explore this hypothesis by highlighting the role of ambiguous decoding in macromolecular recognition of key effector proteins associated with the regulation of signal transduction cascades and the cell cycle, which are critical processes for C. albicans morphogenic plasticity under a variety of environmental conditions.

The cysteine-less peptidic anticoagulants madanin-1 and madanin-2 from the bush tick Haemaphysalis longicornis are the founding members of the MEROPS inhibitor family I53. It has been previously suggested that madanins exert their functional activity by competing with physiological substrates for binding to the positively charged exosite I (fibrinogen-binding exosite) of α-thrombin. We hereby demonstrate that competitive inhibition of α-thrombin by madanin-1 or madanin-2 involves binding to the enzyme's active site. Moreover, the blood coagulation factors IIa and Xa are shown to hydrolyze both inhibitors at different, although partially overlapping cleavage sites. Finally, the three-dimensional structure of the complex formed between human α-thrombin and a proteolytic fragment of madanin-1, determined by X-ray crystallography, elucidates the molecular details of madanin-1 recognition and processing by the proteinase. Taken together, the current findings establish the mechanism of action of madanins, natural anticoagulants that behave as cleavable competitive inhibitors of thrombin.

Tuberculosis constitutes today a serious threat to human health worldwide, aggravated by the increasing number of identified multi-resistant strains of Mycobacterium tuberculosis, its causative agent, as well as by the lack of development of novel mycobactericidal compounds for the last few decades. The increased resilience of this pathogen is due, to a great extent, to its complex, polysaccharide-rich, and unusually impermeable cell wall. The synthesis of this essential structure is still poorly understood despite the fact that enzymes involved in glycosidic bond synthesis represent more than 1% of all M. tuberculosis ORFs identified to date. One of them is GpgS, a retaining glycosyltransferase (GT) with low sequence homology to any other GTs of known structure, which has been identified in two species of mycobacteria and shown to be essential for the survival of M. tuberculosis. To further understand the biochemical properties of M. tuberculosis GpgS, we determined the three-dimensional structure of the apo enzyme, as well as of its ternary complex with UDP and 3-phosphoglycerate, by X-ray crystallography, to a resolution of 2.5 and 2.7 A, respectively. GpgS, the first enzyme from the newly established GT-81 family to be structurally characterized, displays a dimeric architecture with an overall fold similar to that of other GT-A-type glycosyltransferases. These three-dimensional structures provide a molecular explanation for the enzyme's preference for UDP-containing donor substrates, as well as for its glucose versus mannose discrimination, and uncover the structural determinants for acceptor substrate selectivity. Glycosyltransferases constitute a growing family of enzymes for which structural and mechanistic data urges. The three-dimensional structures of M. tuberculosis GpgS now determined provide such data for a novel enzyme family, clearly establishing the molecular determinants for substrate recognition and catalysis, while providing an experimental scaffold for the structure-based rational design of specific inhibitors, which lay the foundation for the development of novel anti-tuberculosis therapies.

Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine alpha-thrombin.boophilin complex, refined at 2.35 A resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S(1) pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9 degrees and is displaced by 6 A, while the C-terminal domain rotates almost 6 degrees accompanied by a 3 A displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P(1) residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin.boophilin.trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.