Assessment on the binding affinity between ritonavir with model transport protein: a combined multi-spectroscopic approaches with computer simulation

Expression of the α-amylase gene (amyE) ofBacillus subtilisis subject to CcpA (catabolite control protein A)-mediated catabolite repression, a global regulatory mechanism inBacillusand other Gram-positive bacteria. To determine effectors of CcpA, we tested the ability of glycolytic metabolites, nucleotides, and cofactors to affect CcpA binding to theamyEoperator,amyO. Those that stimulated the DNA-binding affinity of CcpA were tested for their effect on transcription. HPr-P (Ser-46), proposed as an effector of CcpA, also was tested. In DNase I footprint assays, the affinity of CcpA foramyOwas stimulated 2-fold by fructose-1,6-diphosphate (FDP), 1.5-fold by oxidized or reduced forms of NADP, and 10-fold by HPr-P (Ser-46). However, the triple combinations, CcpA/NADP/HPr-P (Ser-46) and CcpA/FDP/HPr-P (Ser-46) synergistically stimulated DNA-binding affinity by 120- and 300-fold, respectively. NADP added to CcpA specifically stimulated transcription inhibition of theamyEpromoter by 120-fold. CcpA combined with HPr (Ser-46) inhibited transcription from theamyEpromoter, but it also inhibited several control promoters. FDP did not stimulate transcription inhibition by CcpA nor did the triple combinations. The finding that NADP had little effect on CcpA DNA binding but increased the ability of CcpA to inhibit transcription suggests that catabolite repression is not simply caused by CcpA bindingamyObut rather a result of interactions with the transcription machinery enhanced by NADP.

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Identification and characterization of two drug-like fragments that bind to the same cryptic binding pocket of Burkholderia pseudomallei DsbA

10.1101/2021.03.25.436878 ◽

2021 ◽

Author(s):

Guillaume A. Petit ◽

Biswarajan Mohanty ◽

Róisín M. McMahon ◽

Stefan Nebl ◽

David H. Hilko ◽

...

Keyword(s):

Crystal Structures ◽

Binding Affinity ◽

Disulfide Bond ◽

Burkholderia Pseudomallei ◽

Protein A ◽

Binding Pocket ◽

New Drugs ◽

Nmr Studies ◽

Small Surface ◽

Starting Point

AbstractDiSulfide Bond forming proteins (DSB) play a crucial role in the pathogenicity of many Gram-negative bacteria. Disulfide bond protein A (DsbA) catalyzes the formation of disulfide bonds necessary for the activity and stability of multiple substrate proteins, including many virulence factors. Hence, DsbA is an attractive target for the development of new drugs to combat bacterial infections. Here, we identified two fragments - 1 (bromophenoxy propanamide) and 2 (4-methoxy-N-phenylbenzenesulfonamide), that bind to the DsbA from the pathogenic bacterium Burkholderia pseudomallei, the causative agent of melioidosis. Crystal structures of the oxidized B. pseudomallei DsbA (termed BpsDsbA) co-crystallized with 1 or 2 suggests that both fragments bind to a hydrophobic pocket that is formed by a change in the side chain orientation of tyrosine 110. This conformational change opens a “cryptic” pocket that is not evident in the apo-protein structure. This binding location was supported by 2D-NMR studies which identified a chemical shift perturbation of the tyrosine 110 backbone amide resonance of more than 0.05 ppm upon addition of 2 mM of fragment 1 and over 0.04 ppm upon addition of 1 mM of fragment 2. Although binding was detected by both X-ray crystallography and NMR, the binding affinity (KD) for both fragments was low (above 2 mM), suggesting weak interactions with BpsDsbA. This conclusion is also supported by the modelled crystal structures which ascribe partial occupancy to the ligands in the cryptic binding pocket. Small fragments such as 1 and 2 are not expected to have high binding affinity due to their size and the relatively small surface area that can be involved in intermolecular interactions. However, their simplicity makes them ideal for functionalization and optimization. Identification of the binding sites of 1 and 2 to BpsDsbA could provide a starting point for the development of more potent novel antimicrobial compounds that target DsbA and bacterial virulence.SynopsisDescribes the binding properties of two drug-like fragments to a conformationally dynamic site in the disulfide-bond forming protein A from Burkholderia pseudomallei.

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Exposure of African Catfish (Clarias gariepinus) to Lead and Zinc Modulates Membrane-Bound Transport Protein: A Plausible Effect on Na+/K+-ATPase Activity

Biological Trace Element Research ◽

10.1007/s12011-021-03005-5 ◽

2021 ◽

Author(s):

Augustine Apiamu ◽

Sophia U. Osawaru ◽

Samuel O. Asagba ◽

Uduenevwo F. Evuen ◽

Fidelis I. Achuba

Keyword(s):

Atpase Activity ◽

Clarias Gariepinus ◽

Protein A ◽

Transport Protein ◽

African Catfish ◽

Lead And Zinc ◽

Membrane Bound

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Phosphorylation of a PDZ Domain Extension Modulates Binding Affinity and Interdomain Interactions in Postsynaptic Density-95 (PSD-95) Protein, a Membrane-associated Guanylate Kinase (MAGUK)

Journal of Biological Chemistry ◽

10.1074/jbc.m111.272583 ◽

2011 ◽

Vol 286 (48) ◽

pp. 41776-41785 ◽

Cited By ~ 39

Author(s):

Jun Zhang ◽

Chad M. Petit ◽

David S. King ◽

Andrew L. Lee

Keyword(s):

Binding Affinity ◽

Pdz Domain ◽

Protein A ◽

Postsynaptic Density ◽

Guanylate Kinase ◽

Domain Extension ◽

Interdomain Interactions

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Gold Nanoparticle Based Immunostrip Assay Method for Detection of Protein-A

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.229-231.260 ◽

2012 ◽

Vol 229-231 ◽

pp. 260-266 ◽

Cited By ~ 1

Author(s):

Ajit Zambre ◽

Nripen Chanda ◽

Sudhirdas Prayaga ◽

Rosana Almudhafar ◽

Raghuraman Kannan ◽

...

Keyword(s):

Gold Nanoparticles ◽

Gold Nanoparticle ◽

Binding Affinity ◽

Polyclonal Antibody ◽

Protein A ◽

Assay Method ◽

Rabbit Polyclonal Antibody

We have successfully developed gold nanoparticle based immunostrip assay to detect protein-A (PA). Rabbit polyclonal antibody IGg (αPA) that has affinity to PA was conjugated to gold nanoparticles (GNPs) and the gold nanoconjugate (αPA-GNP) was used to detect protein-A by simple immunostrip assay method. ELISA experiments were used to confirm the retention of binding affinity of antibody towards protein-A after conjugation with gold nanoparticles.

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