ABSTRACT
The type VI secretion system (T6SS) is a complex molecular nanomachine used by Gram-negative bacteria to deliver diverse effectors into adjacent cells. A membrane complex (MC) anchors this transport system to the bacterial cell wall. One of the proteins forming the MC is TssL, a cytoplasmic protein bound to the inner membrane through a single transmembrane helix. Here, we report the structure of the cytoplasmic N-terminal region of TssL from Acinetobacter baumannii, a bacterium encoding in a single locus a secretion system that is a special case among other T6SSs. The protein structure, consisting of two antiparallel alpha-helical bundles connected by a short loop, reveals several interesting particularities compared with homologous proteins from other organisms. In addition, we demonstrate the structural significance of residues Asp98 and Glu99, which are strongly conserved among T6SS-encoding Gram-negative bacteria. Mutations in these two residues strongly impact protein dynamics, expression, and functionality. Our results improve our understanding of the T6SS of A. baumannii, which remains largely understudied compared with that of other pathogens.IMPORTANCE Several Acinetobacter species carry one functional type VI secretion system (T6SS). The T6SS is encoded in a single locus containing 16 conserved genes, most of which code for proteins essential to T6SS activity. One of these key components is TssL, a cytoplasmic protein bound to the inner membrane. Despite its importance and its particular characteristics, the structure of T6SS in A. baumannii remains understudied. Here, we present structural, in silico, and in vivo studies of TssL, highlighting the importance of two well-conserved residues and improving our understanding of this secretion system in this bacterium.
Subject(s)
Acinetobacter baumannii/metabolism , Bacterial Proteins/metabolism , Type VI Secretion Systems/metabolism , Acinetobacter baumannii/genetics , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Molecular Dynamics Simulation , MutationABSTRACT
Acinetobacter baumannii is an extracellular opportunistic human pathogen that is becoming increasingly problematic in hospitals. In the present study, we demonstrate that the A. baumannii Omp 33- to 36-kDa protein (Omp33-36) is a porin that acts as a channel for the passage of water. The protein is found on the cell surface and is released along with other porins in the outer membrane vesicles (OMVs). In immune and connective cell tissue, this protein induced apoptosis by activation of caspases and modulation of autophagy, with the consequent accumulation of p62/SQSTM1 (sequestosome 1) and LC3B-II (confirmed by use of autophagy inhibitors). Blockage of autophagy enables the bacterium to persist intracellularly (inside autophagosomes), with the subsequent development of cytotoxicity. Finally, we used macrophages and a mouse model of systemic infection to confirm that Omp33-36 is a virulence factor in A. baumannii. Overall, the study findings show that Omp33-36 plays an important role in the pathogenesis of A. baumannii infections.
Subject(s)
Acinetobacter baumannii , Apoptosis/drug effects , Autophagy/drug effects , Bacterial Proteins/metabolism , Porins/metabolism , Virulence Factors/metabolism , Acinetobacter Infections/microbiology , Animals , Bacterial Proteins/genetics , Cell Line , DNA Fragmentation , Humans , Macrophages/microbiology , Male , Mice , Mice, Inbred BALB C , Porins/genetics , Protein Conformation , Virulence Factors/geneticsABSTRACT
BACKGROUND: Manganese peroxidases (MnPs) are, together with lignin peroxidases and versatile peroxidases, key elements of the enzymatic machineries secreted by white-rot fungi to degrade lignin, thus providing access to cellulose and hemicellulose in plant cell walls. A recent genomic analysis of 52 Agaricomycetes species revealed the existence of novel MnP subfamilies differing in the amino-acid residues that constitute the manganese oxidation site. Following this in silico analysis, a comprehensive structure-function study is needed to understand how these enzymes work and contribute to transform the lignin macromolecule. RESULTS: Two MnPs belonging to the subfamilies recently classified as MnP-DGD and MnP-ESD-referred to as Ape-MnP1 and Cst-MnP1, respectively-were identified as the primary peroxidases secreted by the Agaricales species Agrocybe pediades and Cyathus striatus when growing on lignocellulosic substrates. Following heterologous expression and in vitro activation, their biochemical characterization confirmed that these enzymes are active MnPs. However, crystal structure and mutagenesis studies revealed manganese coordination spheres different from those expected after their initial classification. Specifically, a glutamine residue (Gln333) in the C-terminal tail of Ape-MnP1 was found to be involved in manganese binding, along with Asp35 and Asp177, while Cst-MnP1 counts only two amino acids (Glu36 and Asp176), instead of three, to function as a MnP. These findings led to the renaming of these subfamilies as MnP-DDQ and MnP-ED and to re-evaluate their evolutionary origin. Both enzymes were also able to directly oxidize lignin-derived phenolic compounds, as seen for other short MnPs. Importantly, size-exclusion chromatography analyses showed that both enzymes cause changes in polymeric lignin in the presence of manganese, suggesting their relevance in lignocellulose transformation. CONCLUSIONS: Understanding the mechanisms used by basidiomycetes to degrade lignin is of particular relevance to comprehend carbon cycle in nature and to design biotechnological tools for the industrial use of plant biomass. Here, we provide the first structure-function characterization of two novel MnP subfamilies present in Agaricales mushrooms, elucidating the main residues involved in catalysis and demonstrating their ability to modify the lignin macromolecule.
ABSTRACT
The coronavirus disease 2019 (COVID-19) pandemic continues to represent a global public health issue. The viral main protease (Mpro) represents one of the most attractive targets for the development of antiviral drugs. Herein we report peptidyl nitroalkenes exhibiting enzyme inhibitory activity against Mpro (Ki: 1-10 µM) good anti-SARS-CoV-2 infection activity in the low micromolar range (EC50: 1-12 µM) without significant toxicity. Additional kinetic studies of compounds FGA145, FGA146 and FGA147 show that all three compounds inhibit cathepsin L, denoting a possible multitarget effect of these compounds in the antiviral activity. Structural analysis shows the binding mode of FGA146 and FGA147 to the active site of the protein. Furthermore, our results illustrate that peptidyl nitroalkenes are effective covalent reversible inhibitors of the Mpro and cathepsin L, and that inhibitors FGA145, FGA146 and FGA147 prevent infection against SARS-CoV-2.
ABSTRACT
In vitro mitogenesis assays have shown that sulfated glycosaminoglycans (GAGs; heparin and heparan sulfate) cause an enhancement of the mitogenic activity of fibroblast growth factors (FGFs). Herein, we report that the simultaneous presence of FGF and the GAG is not an essential requisite for this event to take place. Indeed, preincubation with heparin (just before FGF addition) of cells lacking heparan sulfate produced an enhancing effect equivalent to that observed when the GAG and the protein are simultaneously added. A first structural characterization of this effect by analytical ultracentrifugation of a soluble preparation of the heparin-binding domain of fibroblast growth factor receptor 2 (FGFR2) and a low molecular weight (3 kDa) heparin showed that the GAG induces dimerization of FGFR2. To derive a high resolution structural picture of this molecular recognition process, the interactions of a soluble heparin-binding domain of FGFR2 with two different homogeneous, synthetic, and mitogenically active sulfated GAGs were analyzed by NMR spectroscopy. These studies, assisted by docking protocols and molecular dynamics simulations, have demonstrated that the interactions of these GAGs with the soluble heparin-binding domain of FGFR induces formation of an FGFR dimer; its architecture is equivalent to that in one of the two distinct crystallographic structures of FGFR in complex with both heparin and FGF1. This preformation of the FGFR dimer (with similar topology to that of the signaling complex) should favor incorporation of the FGF component to form the final assemblage of the signaling complex, without major entropy penalty. This cascade of events is probably at the heart of the observed activating effect of heparin in FGF-driven mitogenesis.
Subject(s)
Fibroblast Growth Factors/metabolism , Heparin/metabolism , Nuclear Magnetic Resonance, Biomolecular , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Binding Sites , Cell Line , Dimerization , Fibroblast Growth Factors/chemistry , Fibroblast Growth Factors/genetics , Heparitin Sulfate/metabolism , Humans , Molecular Dynamics Simulation , Protein Structure, Tertiary , Receptor, Fibroblast Growth Factor, Type 2/chemistry , Receptor, Fibroblast Growth Factor, Type 2/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , UltracentrifugationABSTRACT
Unspecific peroxygenases (UPOs) are extracellular fungal enzymes of biotechnological interest as self-sufficient (and more stable) counterparts of cytochrome P450 monooxygenases, the latter being present in most living cells. Expression hosts and structural information are crucial for exploiting UPO diversity (over eight thousand UPO-type genes were identified in sequenced genomes) in target reactions of industrial interest. However, while many thousands of entries in the Protein Data Bank include molecular coordinates of P450 enzymes, only 19 entries correspond to UPO enzymes, and UPO structures from only two species (Agrocybe aegerita and Hypoxylon sp.) have been published to date. In the present study, two UPOs from the basidiomycete Marasmius rotula (rMroUPO) and the ascomycete Collariella virescens (rCviUPO) were crystallized after sequence optimization and Escherichia coli expression as active soluble enzymes. Crystals of rMroUPO and rCviUPO were obtained at sufficiently high resolution (1.45 and 1.95 Å, respectively) and the corresponding structures were solved by molecular replacement. The crystal structures of the two enzymes (and two mutated variants) showed dimeric proteins. Complementary biophysical and molecular biology studies unveiled the diverse structural bases of the dimeric nature of the two enzymes. Intermolecular disulfide bridge and parallel association between two α-helices, among other interactions, were identified at the dimer interfaces. Interestingly, one of the rCviUPO variants incorporated the ability to produce fatty acid diepoxides-reactive compounds with valuable cross-linking capabilities-due to removal of the enzyme C-terminal tail located near the entrance of the heme access channel. In conclusion, different dimeric arrangements could be described in (short) UPO crystal structures.
ABSTRACT
Class D ß-lactamases represent a growing and diverse class of penicillin-inactivating enzymes that are usually resistant to commercial ß-lactamase inhibitors. As many such enzymes are found in multi-drug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa, novel ß-lactamase inhibitors are urgently needed. Five unique 6-alkylidene-2'-substituted penicillanic acid sulfones (1-5) were synthesized and tested against OXA-24, a clinically important ß-lactamase that inactivates carbapenems and is found in A. baumannii. Based upon the roles Tyr112 and Met223 play in the OXA-24 ß-lactamase, we also engineered two variants (Tyr112Ala and Tyr112Ala,Met223Ala) to test the hypothesis that the hydrophobic tunnel formed by these residues influences inhibitor recognition. IC(50) values against OXA-24 and two OXA-24 ß-lactamase variants ranged from 10 ± 1 (4 vs WT) to 338 ± 20 nM (5 vs Tyr112Ala, Met223Ala). Compound 4 possessed the lowest K(i) (500 ± 80 nM vs WT), and 1 possessed the highest inactivation efficiency (k(inact)/K(i) = 0.21 ± 0.02 µM(-1) s(-1)). Electrospray ionization mass spectrometry revealed a single covalent adduct, suggesting the formation of an acyl-enzyme intermediate. X-ray structures of OXA-24 complexed to four inhibitors (2.0-2.6 Å) reveal the formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanion hole. These data provide the first structural evidence that 6-alkylidene-2'-substituted penicillin sulfones are effective mechanism-based inactivators of class D ß-lactamases. Their unique chemistry makes them developmental candidates. Mechanisms for class D hydrolysis and inhibition are discussed, and a pathway for the evolution of the BlaR1 sensor of Staphylococcus aureus to the class D ß-lactamases is proposed.
Subject(s)
Acinetobacter baumannii/enzymology , Bacterial Proteins/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Penicillanic Acid/chemistry , Sulfones/chemistry , beta-Lactamase Inhibitors , Acinetobacter baumannii/drug effects , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Microbial Sensitivity Tests , Molecular Structure , Spectrometry, Mass, Electrospray Ionization , Sulfones/chemical synthesis , Sulfones/pharmacology , beta-LactamasesABSTRACT
Lignin biodegradation, a key step in carbon recycling in land ecosystems, is carried out by white-rot fungi through an H(2)O(2)-dependent process defined as enzymatic combustion. Pleurotus eryngii is a selective lignin-degrading fungus that produces H(2)O(2) during redox cycling of p-anisylic compounds involving the secreted flavoenzyme aryl-alcohol oxidase (AAO). Here, the 2.4 A resolution X-ray crystal structure of this oxidoreductase, which catalyzes dehydrogenation reactions on various primary polyunsaturated alcohols, yielding the corresponding aldehydes, is reported. The AAO crystal structure was solved by single-wavelength anomalous diffraction of a selenomethionine derivative obtained by Escherichia coli expression and in vitro folding. This monomeric enzyme is composed of two domains, the overall folding of which places it into the GMC (glucose-methanol-choline oxidase) oxidoreductase family, and a noncovalently bound FAD cofactor. However, two additional structural elements exist in the surroundings of its active site that modulate the access of substrates; these are absent in the structure of the model GMC oxidoreductase glucose oxidase. The folding of these novel elements gives rise to a funnel-like hydrophobic channel that connects the solvent region to the buried active-site cavity of AAO. This putative active-site cavity is located in front of the re side of the FAD isoalloxazine ring and near two histidines (His502 and His546) that could contribute to alcohol activation as catalytic bases. Moreover, three aromatic side chains from two phenylalanines (Phe397 and Phe502) and one tyrosine (Tyr92) at the inner region of the channel form an aromatic gate that may regulate the access of the enzyme substrates to the active site as well as contribute to the recognition of the alcohols that can effectively be oxidized by AAO.
Subject(s)
Alcohol Oxidoreductases/chemistry , Pleurotus/enzymology , Alcohol Oxidoreductases/metabolism , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Protein Folding , Protein Structure, Tertiary , Sequence Alignment , Sequence Homology, Amino Acid , Structural Homology, Protein , Substrate SpecificityABSTRACT
The type VI secretion system (T6SS) is a mechanism that is commonly used by pathogenic bacteria to infect host cells and for survival in competitive environments. This system assembles on a core baseplate and elongates like a phage puncturing device; it is thought to penetrate the target membrane and deliver effectors into the host or competing bacteria. Valine-glycine repeat protein G1 (VgrG1) forms the spike at the tip of the elongating tube formed by haemolysin co-regulated protein 1 (Hcp1); it is structurally similar to the T4 phage (gp27)3-(gp5)3 puncturing complex. Here, the crystal structure of full-length VgrG1 from Pseudomonas aeruginosa is reported at a resolution of 2.0 Å, which through a trimeric arrangement generates a needle-like shape composed of two main parts, the head and the spike, connected via a small neck region. The structure reveals several remarkable structural features pointing to the possible roles of the two main segments of VgrG1: the head as a scaffold cargo domain and the ß-roll spike with implications in the cell-membrane puncturing process and as a carrier of cognate toxins.
Subject(s)
Bacterial Proteins/chemistry , Pseudomonas aeruginosa/chemistry , Type VI Secretion Systems/chemistry , Crystallography, X-Ray , Protein Conformation , Protein MultimerizationABSTRACT
The type VI secretion system (T6SS) is a bacterial macromolecular machine widely distributed in Gram-negative bacteria, which transports effector proteins into eukaryotic host cells or other bacteria. Membrane complexes and a central tubular structure, which resembles the tail of contractile bacteriophages, compose the T6SS. One of the proteins forming this tube is the hemolysin co-regulated protein (Hcp), which acts as virulence factor, as transporter of effectors and as a chaperone. In this study, we present the structure of Hcp from Acinetobacter baumannii, together with functional and oligomerization studies. The structure of this protein exhibits a tight ß barrel formed by two ß sheets and flanked at one side by a short α-helix. Six Hcp molecules associate to form a donut-shaped hexamer, as observed in both the crystal structure and solution. These results emphasize the importance of this oligomerization state in this family of proteins, despite the low similarity of sequence among them. The structure presented in this study is the first one for a protein forming part of a functional T6SS from A. baumannii. These results will help us to understand the mechanism and function of this secretion system in this opportunistic nosocomial pathogen.
Subject(s)
Acinetobacter baumannii , Bacterial Proteins/chemistry , Type VI Secretion Systems/chemistry , Bacterial Proteins/metabolism , Crystallography, X-Ray , Models, Molecular , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, SecondaryABSTRACT
Combating bacterial resistance to beta-lactams, the most widely used antibiotics, is an emergent and clinically important challenge. OXA-24 is a class D beta-lactamase isolated from a multiresistant epidemic clinical strain of Acinetobacter baumannii. We have investigated how OXA-24 specifically hydrolyzes the last resort carbapenem antibiotic, and we have determined the crystal structure of OXA-24 at a resolution of 2.5 A. The structure shows that the carbapenem's substrate specificity is determined by a hydrophobic barrier that is established through the specific arrangement of the Tyr-112 and Met-223 side chains, which define a tunnel-like entrance to the active site. The importance of these residues was further confirmed by mutagenesis studies. Biochemical and microbiological analyses of specific point mutants selected on the basis of structural criteria significantly reduced the catalytic efficiency (k(cat)/K(m)) against carbapenems, whereas the specificity for oxacillin was noticeably increased. This is the previously unrecognized crystal structure that has been obtained for a class D carbapenemase enzyme. Accordingly, this information may help to improve the development of effective new drugs to combat beta-lactam resistance. More specifically, it may help to overcome carbapenem resistance in A. baumannii, probably one of the most worrying infectious threats in hospitals worldwide.