75 research outputs found
Structures of EccB\u3csub\u3e1\u3c/sub\u3e and EccD\u3csub\u3e1\u3c/sub\u3e from the Core Complex of the Mycobacterial ESX-1 Type VII Secretion System
Background: The ESX-1 type VII secretion system is an important determinant of virulence in pathogenic mycobacteria, including Mycobacterium tuberculosis. This complicated molecular machine secretes folded proteins through the mycobacterial cell envelope to subvert the host immune response. Despite its important role in disease very little is known about the molecular architecture of the ESX-1 secretion system. Results: This study characterizes the structures of the soluble domains of two conserved core ESX-1 components – EccB1 and EccD1. The periplasmic domain of EccB1 consists of 4 repeat domains and a central domain, which together form a quasi 2-fold symmetrical structure. The repeat domains of EccB1 are structurally similar to a known peptidoglycan binding protein suggesting a role in anchoring the ESX-1 system within the periplasmic space. The cytoplasmic domain of EccD1has a ubiquitin-like fold and forms a dimer with a negatively charged groove. Conclusions: These structures represent a major step towards resolving the molecular architecture of the entire ESX-1 assembly and may contribute to ESX-1 targeted tuberculosis intervention strategies
Enhanced Antifibrinolytic Efficacy of a Plasmin-Specific Kunitz-Inhibitor (60-Residue Y11T/L17R with C-Terminal IEK) of Human Tissue Factor Pathway Inhibitor Type-2 Domain1
Current antifibrinolytic agents reduce blood loss by inhibiting plasmin active sites (e.g., aprotinin) or by preventing plasminogen/tissue plasminogen activator (tPA) binding to fibrin clots (e.g., ε-aminocaproic acid and tranexamic acid); however, they have adverse side effects. Here, we expressed 60-residue (NH2NAE . . . IEKCOOH) Kunitz domain1 (KD1) mutants of human tissue factor pathway inhibitor type-2 that inhibit plasmin as well as plasminogen activation. A single (KD1-L17R-KCOOH) and a double mutant (KD1-Y11T/L17R- KCOOH) were expressed in Escherichia coli as His-tagged constructs, each with enterokinase cleavage sites. KD1-Y11T/L17R-KCOOH was also expressed in Pichia pastoris. KD1-Y11T/L17R-KCOOH inhibited plasmin comparably to aprotinin and bound to the kringle domains of plasminogen/plasmin and tPA with Kd of ~50 nM and ~35 nM, respectively. Importantly, compared to aprotinin, KD1-L17R-KCOOH and KD1-Y11T/L17R-KCOOH did not inhibit kallikrein. Moreover, the antifibrinolytic potential of KD1-Y11T/L17R-KCOOH was better than that of KD1-L17R-KCOOH and similar to that of aprotinin in plasma clot-lysis assays. In thromboelastography experiments, KD1-Y11T/L17R-KCOOH was shown to inhibit fibrinolysis in a dose dependent manner and was comparable to aprotinin at a higher concentration. Further, KD1-Y11T/L17R-KCOOH did not induce cytotoxicity in primary human endothelial cells or fibroblasts. We conclude that KD1-Y11T/L17R-KCOOH is comparable to aprotinin, the most potent known inhibitor of plasmin and can be produced in large amounts using Pichia
Electrophysiological properties of porin, the major outer membrane protein of Haemophilus influenzae
Haemophilus influenzae (Hi) is a Gram-negative bacteria that is the causative agent of bacterial meningitis. The outer membrane (OM) of Gram-negative bacteria functions as a selectively permeable barrier. The exchange of small hydrophilic solutes between the external environment and the periplasm is mediated by large water-filled channels, porins. Charged residues of the pore determine the functional properties of the protein, which include: ion conductance, ionic selectivity, and voltage gating.To study the properties of the porin (341 amino acids; Mr 37,782) of Haemophilus influenzae type b (Hib), purified porin was subjected to chemical modification. The covalent modification of lysine residues with succinic anhydride (SA; Mr 100.08) results in charge reversal. The addition of up to 12 succinate groups per porin molecule was identified using electrospray ionization mass spectrometry (MS). Tryptic digestion of the modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight MS identified the sites of succinylation. The majority of modified lysines were positioned in surface-located loops, numbers 1 and 4 to 7. When the electrophysiological properties of SA-modified porin were analyzed in planar lipid bilayers (PLBs) and compared to Hib porin it was found that the single channel conductance was increased, while the threshold for voltage gating was decreased. The addition of extra negative charges increase the single channel conductance of Hib porin and function as voltage sensors.Selected lysine residues that were found to be modified with SA were substituted with glutamic acid using site-directed mutagenesis. Single point mutations were made in a residue assigned to the barrel lumen and to three residues in each of loops 4 and 6. The mutant Hib porins had increased single channel conductances relative to wild-type Hib porin. Voltage gating of mutant Hib porins was altered by the introduction of negative charges into loops 4 and 6 and in the barrel lumen. Previous experiments had implicated surface-exposed loop 4 in voltage gating. This study ascribes a role for residues in loop 6 and a residue within the barrel lumen in the changes that accompany pore closure.Hi strains causing infection in cystic fibrosis patients are capable of persistent infection despite prolonged antibiotic treatment with beta-lactam antibiotics. During the course of infection porin properties may be altered due to the changes in porin sequences that are attributed to antigenic drift. The electrophysiological properties of four porins from CF patient-derived Hi strains were characterized to examine changes in porin properties arising from persistent infection of the CF lung. The clinical Hi porins displayed altered channel properties that included increased voltage sensitivity and single channel conductances that were either greater or smaller than that of Hib porin. The decreased single channel conductance of one of the porins was associated with an increase in the minimal inhibitory concentration of the antibiotics novobiocin and streptomycin. These results demonstrate a porin-mediated decrease in OM permeability as an antibiotic resistance mechanism for Hi
Expanding the use of ethanol as a feedstock for cell-free synthetic biochemistry by implementing acetyl-CoA and ATP generating pathways.
Ethanol is a widely available carbon compound that can be increasingly produced with a net negative carbon balance. Carbon-negative ethanol might therefore provide a feedstock for building a wider range of sustainable chemicals. Here we show how ethanol can be converted with a cell free system into acetyl-CoA, a central precursor for myriad biochemicals, and how we can use the energy stored in ethanol to generate ATP, another key molecule important for powering biochemical pathways. The ATP generator produces acetone as a value-added side product. Our ATP generator reached titers of 27 ± 6 mM ATP and 59 ± 15 mM acetone with maximum ATP synthesis rate of 2.8 ± 0.6 mM/h and acetone of 7.8 ± 0.8 mM/h. We illustrated how the ATP generating module can power cell-free biochemical pathways by converting mevalonate into isoprenol at a titer of 12.5 ± 0.8 mM and a maximum productivity of 1.0 ± 0.05 mM/h. These proof-of-principle demonstrations may ultimately find their way to the manufacture of diverse chemicals from ethanol and other simple carbon compounds
Mutagenesis Identifies Amino Acid Residues in Extracellular Loops and within the Barrel Lumen That Determine Voltage Gating of Porin from Haemophilus influenzae
Crystal structure of the toxin Msmeg_6760, the structural homolog of Mycobacterium tuberculosis Rv2035, a novel type II toxin involved in the hypoxic response
The structure of Msmeg_6760, a protein of unknown function, has been determined. Biochemical and bioinformatics analyses determined that Msmeg_6760 interacts with a protein encoded in the same operon, Msmeg_6762, and predicted that the operon is a toxin-antitoxin (TA) system. Structural comparison of Msmeg_6760 with proteins of known function suggests that Msmeg_6760 binds a hydrophobic ligand in a buried cavity lined by large hydrophobic residues. Access to this cavity could be controlled by a gate-latch mechanism. The function of the Msmeg_6760 toxin is unknown, but structure-based predictions revealed that Msmeg_6760 and Msmeg_6762 are homologous to Rv2034 and Rv2035, a predicted novel TA system involved in Mycobacterium tuberculosis latency during macrophage infection. The Msmeg_6760 toxin fold has not been previously described for bacterial toxins and its unique structural features suggest that toxin activation is likely to be mediated by a novel mechanism
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Design and Construction of a Designed Ankyrin Repeat Protein (DARPin) Display Library
Protein display systems are powerful techniques used to identify protein molecules that bind with high affinity to target proteins of interest. The initial challenge in implementing a display system is the construction of a high-diversity naïve library. Here, we describe the methods to generate a designed ankyrin repeat protein (DARPin) display library using degenerate oligonucleotides. Specifically described is the construction of a single DARPin repeat module by overlap extension PCR, concatenation of the module by restriction enzyme digestion and ligation, and incorporation of the concatenated modules into a full-length DARPin sequence in a bacterial cloning or display vector containing the hydrophilic N- and C-terminal capping domains. Protocols for PCR amplification of DARPin sequences to estimate diversity of naïve and enriched libraries via next-generation sequencing are included, as is a simple Linux-based program for analysis of naïve and enriched sequences. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of a single DARPin repeat by overlap extension PCR Basic Protocol 2: Concatenation of DARPin repeats Basic Protocol 3: Ligation of internal repeats into cloning/display vector containing N- and C-terminal capping repeats Basic Protocol 4: Estimation of library size and diversity by next-generation sequencing (NGS) Basic Protocol 5: NGS analysis of naïve and enriched libraries
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X-ray crystal structure of a designed rigidified imaging scaffold in the ligand-free conformation.
Imaging scaffolds composed of designed protein cages fused to designed ankyrin repeat proteins (DARPins) have enabled the structure determination of small proteins by cryogenic electron microscopy (cryo-EM). One particularly well characterized scaffold type is a symmetric tetrahedral assembly composed of 24 subunits, 12 A and 12 B, which has three cargo-binding DARPins positioned on each vertex. Here, the X-ray crystal structure of a representative tetrahedral scaffold in the apo state is reported at 3.8 Å resolution. The X-ray crystal structure complements recent cryo-EM findings on a closely related scaffold, while also suggesting potential utility for crystallographic investigations. As observed in this crystal structure, one of the three DARPins, which serve as modular adaptors for binding diverse `cargo proteins, present on each of the vertices is oriented towards a large solvent channel. The crystal lattice is unusually porous, suggesting that it may be possible to soak crystals of the scaffold with small (≤30 kDa) protein cargo ligands and subsequently determine cage-cargo structures via X-ray crystallography. The results suggest the possibility that cryo-EM scaffolds may be repurposed for structure determination by X-ray crystallography, thus extending the utility of electron-microscopy scaffold designs for alternative structural biology applications
Escherichia coli RNase R Has Dual Activities, Helicase and RNase â–¿ â€
In Escherichia coli, the cold shock response occurs when there is a temperature downshift from 37°C to 15°C, and this response is characterized by induction of several cold shock proteins, including the DEAD-box helicase CsdA, during the acclimation phase. CsdA is involved in a variety of cellular processes. Our previous studies showed that the helicase activity of CsdA is critical for its function in cold shock acclimation of cells and that the only proteins that were able to complement its function were another helicase, RhlE, an RNA chaperone, CspA, and a cold-inducible exoribonuclease, RNase R. Interestingly, other major 3′-to-5′ processing exoribonucleases of E. coli, such as polynucleotide phosphorylase and RNase II, cannot complement the cold shock function of CsdA. Here we carried out a domain analysis of RNase R and showed that this protein has two distinct activities, RNase and helicase, which are independent of each other and are due to different domains. Mutant RNase R proteins that lack the RNase activity but exhibit the helicase activity were able to complement the cold shock function of CsdA, suggesting that only the helicase activity of RNase R is essential for complementation of the cold shock function of CsdA. We also observed that in vivo deletion of the two cold shock domains resulted in a loss of the ability of RNase R to complement the cold shock function of CsdA. We further demonstrated that RNase R exhibits helicase activity in vitro independent of its RNase activity. Our results shed light on the unique properties of RNase R and how it is distinct from other exoribonucleases in E. coli
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