scholarly journals Structural characterization of the virulence factor nuclease A fromStreptococcus agalactiae

2014 ◽  
Vol 70 (11) ◽  
pp. 2937-2949 ◽  
Author(s):  
Andrea F. Moon ◽  
Philippe Gaudu ◽  
Lars C. Pedersen
Keyword(s):  
Virulence Factor ◽  
Substrate Binding ◽  
Nuclease Activity ◽  
Structure Based Drug Design ◽  
X Ray Crystallography ◽  
High Resolution Structure ◽  
Group B ◽  
Increased Activity ◽  
Dna Substrate

The group B pathogenStreptococcus agalactiaecommonly populates the human gut and urogenital tract, and is a major cause of infection-based mortality in neonatal infants and in elderly or immunocompromised adults. Nuclease A (GBS_NucA), a secreted DNA/RNA nuclease, serves as a virulence factor forS. agalactiae, facilitating bacterial evasion of the human innate immune response. GBS_NucA efficiently degrades the DNA matrix component of neutrophil extracellular traps (NETs), which attempt to kill and clear invading bacteria during the early stages of infection. In order to better understand the mechanisms of DNA substrate binding and catalysis of GBS_NucA, the high-resolution structure of a catalytically inactive mutant (H148G) was solved by X-ray crystallography. Several mutants on the surface of GBS_NucA which might influence DNA substrate binding and catalysis were generated and evaluated using an imidazole chemical rescue technique. While several of these mutants severely inhibited nuclease activity, two mutants (K146R and Q183A) exhibited significantly increased activity. These structural and biochemical studies have greatly increased our understanding of the mechanism of action of GBS_NucA in bacterial virulence and may serve as a foundation for the structure-based drug design of antibacterial compounds targeted toS. agalactiae.

scholarly journals Applications of Cryo-EM in small molecule and biologics drug design

2021 ◽  
Author(s):  
Joshua A. Lees ◽  
Joao M. Dias ◽  
Seungil Han
Keyword(s):  
Drug Design ◽  
Structural Characterization ◽  
Viral Vectors ◽  
Biological Macromolecules ◽  
Structure Based Drug Design ◽  
X Ray Crystallography ◽  
Technological Advances ◽  
High Resolution Analysis ◽  
Resolution Analysis

Electron cryo-microscopy (cryo-EM) is a powerful technique for the structural characterization of biological macromolecules, enabling high-resolution analysis of targets once inaccessible to structural interrogation. In recent years, pharmaceutical companies have begun to utilize cryo-EM for structure-based drug design. Structural analysis of integral membrane proteins, which comprise a large proportion of druggable targets and pose particular challenges for X-ray crystallography, by cryo-EM has enabled insights into important drug target families such as G protein-coupled receptors (GPCRs), ion channels, and solute carrier (SLCs) proteins. Structural characterization of biologics, such as vaccines, viral vectors, and gene therapy agents, has also become significantly more tractable. As a result, cryo-EM has begun to make major impacts in bringing critical therapeutics to market. In this review, we discuss recent instructive examples of impacts from cryo-EM in therapeutics design, focusing largely on its implementation at Pfizer. We also discuss the opportunities afforded by emerging technological advances in cryo-EM, and the prospects for future development of the technique.

scholarly journals "Structural characterization of aminoglycoside modifying enzyme ANT(2"")-Ia"

2014 ◽  
Vol 70 (a1) ◽  
pp. C702-C702
Author(s):  
Angelia Bassenden ◽  
Dmitry Rodionov ◽  
Nilu Sabet-Kassouf ◽  
Tahereh Haji ◽  
Kun Shi ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Specific Activity ◽  
Ribosomal Subunit ◽  
Bactericidal Effect ◽  
Nmr Studies ◽  
Structure Based Drug Design ◽  
X Ray Crystallography ◽  
16S Rna

Aminoglycosides are a class of broad-spectrum antibiotics used in the treatment of serious Gram-negative bacterial infections, they target the 16S RNA subunit and upon binding cause errors in translation, eventually inducing a bactericidal effect [1]. Aminoglycoside nucleotidyltransferase (2")-Ia (ANT(2")-Ia) is an aminoglycoside modifying enzyme that prevents aminoglycosides from binding to the ribosomal subunit, making this enzyme a principle candidate structure-based drug design [2]. Characterization of ANT(2")-Ia has been proven to be difficult due to the low stability and solubility of overexpressed protein, where 95% of the protein being expressed is in the form of inclusion bodies [3]. We describe a protocol that has lead to successful expression and purification of ANT(2")-Ia. A successful enzymatic assay has also been adapted and the protein is active and stable under these conditions with a specific activity of 0.14 U/mg. Furthermore, nuclear magnetic resonance (NMR) studies have allowed for the assignment of 144 of the 176 non-proline backbone residues. Substrate binding NMR experiments have shown unique global chemical shift perturbations upon binding ATP and tobramycin, suggesting unique binding sites for each substrate. Structural determination of ANT(2")-Ia using NMR in conjunction with x-ray crystallography can be utilized in order to develop small molecules that will act as more effective aminoglycosides in order to inhibit ANT(2")-Ia from binding and modifying these antibiotics.

scholarly journals Structural Characterization of Heterodinuclear ZnII-LnIII Complexes (Ln = Pr, Nd) with a Ring-Contracted H2valdien-Derived Schiff Base Ligand

2021 ◽  
Author(s):  
Shabana Noor ◽  
Richard Goddard ◽  
Fehmeeda Khatoon ◽  
Sarvendra Kumar ◽  
Rüdiger W. Seidel
Keyword(s):  
Molecular Structure ◽  
Schiff Base ◽  
Structural Characterization ◽  
Schiff Base Ligand ◽  
Crystal And Molecular Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Imidazoline Ring

AbstractSynthesis and structural characterization of two heterodinuclear ZnII-LnIII complexes with the formula [ZnLn(HL)(µ-OAc)(NO3)2(H2O)x(MeOH)1-x]NO3 · n H2O · n MeOH [Ln = Pr (1), Nd (2)] and the crystal and molecular structure of [ZnNd(HL)(µ-OAc)(NO3)2(H2O)] [ZnNd(HL)(OAc)(NO3)2(H2O)](NO3)2 · n H2O · n MeOH (3) are reported. The asymmetrical compartmental ligand (E)-2-(1-(2-((2-hydroxy-3-methoxybenzylidene)amino)-ethyl)imidazolidin-2-yl)-6-methoxyphenol (H2L) is formed from N1,N3-bis(3-methoxysalicylidene)diethylenetriamine (H2valdien) through intramolecular aminal formation, resulting in a peripheral imidazoline ring. The structures of 1–3 were revealed by X-ray crystallography. The smaller ZnII ion occupies the inner N2O2 compartment of the ligand, whereas the larger and more oxophilic LnIII ions are found in the outer O2O2’ site. Graphic Abstract Synthesis and structural characterization of two heterodinuclear ZnII-LnIII complexes (Ln = Pr, Nd) bearing an asymmetrical compartmental ligand formed in situ from N1,N3-bis(3-methoxysalicylidene)diethylenetriamine (H2valdien) through intramolecular aminal formation are reported.

scholarly journals Molecular and Functional Characterization of a ToxR-Regulated Lipoprotein from a Clinical Isolate of Aeromonas hydrophila

2006 ◽  
Vol 74 (7) ◽  
pp. 3742-3755 ◽  
Author(s):  
Lakshmi Pillai ◽  
Jian Sha ◽  
Tatiana E. Erova ◽  
Amin A. Fadl ◽  
Bijay K. Khajanchi ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Aeromonas Hydrophila ◽  
Functional Characterization ◽  
Affinity Column ◽  
Serum Resistance ◽  
Classical Pathway ◽  
Dependent Manner ◽  
T7 Promoter

ABSTRACT Human diseases caused by species of Aeromonas have been classified into two major groups: septicemia and gastroenteritis. In this study, we reported the molecular and functional characterization of a new virulence factor, ToxR-regulated lipoprotein, or TagA, from a diarrheal isolate, SSU, of Aeromonas hydrophila. The tagA gene of A. hydrophila exhibited 60% identity with that of a recently identified stcE gene from Escherichia coli O157:H7, which encoded a protein (StcE) that provided serum resistance to the bacterium and prevented erythrocyte lysis by controlling classical pathway of complement activation by cleaving the complement C1-esterase inhibitor (C1-INH). We purified A. hydrophila TagA as a histidine-tagged fusion protein (rTagA) from E. coli DE3 strain using a T7 promoter-based pET30 expression vector and nickel affinity column chromatography. rTagA cleaved C1-INH in a time-dependent manner. The tagA isogenic mutant of A. hydrophila, unlike its corresponding wild-type (WT) or the complemented strain, was unable to cleave C1-INH, which is required to potentiate the C1-INH-mediated lysis of host and bacterial cells. We indeed demonstrated colocalization of C1-INH and TagA on the bacterial surface by confocal fluorescence microscopy, which ultimately resulted in increased serum resistance of the WT bacterium. Likewise, we delineated the role of TagA in contributing to the enhanced ability of C1-INH to inhibit the classical complement-mediated lysis of erythrocytes. Importantly, we provided evidence that the tagA mutant was significantly less virulent in a mouse model of infection (60%) than the WT bacterium at two 50% lethal doses, which resulted in 100% mortality within 48 h. Taken together, our data provided new information on the role of TagA as a virulence factor in bacterial pathogenesis. This is the first report of TagA characterization from any species of Aeromonas.

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