Abstract
Virstatin is a small molecule that inhibits Vibrio cholerae virulence regulation, the causative agent for cholera. Here we report the interaction of virstatin with human serum albumin (HSA) using various biophysical methods. The drug binding was monitored using different isomeric forms of HSA (N form ∼pH 7.2, B form ∼pH 9.0 and F form ∼pH 3.5) by absorption and fluorescence spectroscopy. There is a considerable quenching of the intrinsic fluorescence of HSA on binding the drug. The distance (r) between donor (Trp214 in HSA) and acceptor (virstatin), obtained from Forster-type fluorescence resonance energy transfer (FRET), was found to be 3.05 nm. The ITC data revealed that the binding was an enthalpy-driven process and the binding constants K a for N and B isomers were found to be 6.09×105 M−1 and 4.47×105 M−1, respectively. The conformational changes of HSA due to the interaction with the drug were investigated from circular dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. For 1∶1 molar ratio of the protein and the drug the far-UV CD spectra showed an increase in α- helicity for all the conformers of HSA, and the protein is stabilized against urea and thermal unfolding. Molecular docking studies revealed possible residues involved in the protein-drug interaction and indicated that virstatin binds to Site I (subdomain IIA), also known as the warfarin binding site.
Highlights
Chemical genetics is an emerging field of research which employs small molecules to dissect complex biological processes and for studying microbial pathogenesis [1,2]
In order to dissect the pathogenesis cholera, which still poses a threat to many parts of the world, high throughput screen of 50,000 compounds in small molecule library from Chembridge Research Laboratories was carried out to identify inhibitors of Vibrio cholerae virulence factor expression [3,4,5,6]
4-[N-(1,8-naphthalimide)]-nbutyric acid (Figure 1), is such a small molecule that attenuates the intestinal colonization of Vibrio cholerae by preventing the dimerization of the transcriptional activator ToxT [7,8]
Summary
Chemical genetics is an emerging field of research which employs small molecules to dissect complex biological processes and for studying microbial pathogenesis [1,2]. The most important physiological role of HSA is to bind a large variety of ligands (fatty acids, hormones, amino acids, drugs, etc.), and deliver them to the target organs. The X-ray crystallographic studies reveal that the heart shaped HSA consists of three structurally similar domains (I, II and III), each of which contains two subdomains (A and B) [11,12]. These subdomains are predominantly helical and extensively cross-linked through several disulfide bridges, with one tryptophan residue (Trp214) in subdomain IIA [13,14]. Since HSA is known to undergo different pH-dependent conformational transitions it is an ideal candidate for studying protein-drug interaction [17]. The probable binding site of virstatin to HSA is predicted from molecular docking studies
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