Interaction of meropenem with ‘N’ and ‘B’ isoforms of human serum albumin: a spectroscopic and molecular docking study

<p>Carbapenems are used to control the outbreak of β-lactamases expressing bacteria. The effectiveness of drugs is influenced by its interaction with human serum albumin (HSA). Strong binding of carbapenems to HSA may lead to decreased bioavailability of the drug. The non-optimal drug dosage will provide a positive selection pressure on bacteria to develop resistance. Here, we investigated the interaction between meropenem and HSA at physiological pH 7.5 (N-isoform HSA) and non-physiological pH 9.2 (B-isoform HSA). Results showed that meropenem quenches the fluorescence of both ‘N’ and ‘B’ isoforms of HSA (Δ<i>G</i> < 0 and binding constant ~10⁴ M⁻¹). Electrostatic interactions and van der Waal interactions along with H-bonds stabilized the complex of meropenem with ‘N’ and ‘B’ isoforms of HSA, respectively. Molecular docking results revealed that meropenem binds to HSA near Sudlow’s site II (subdomain IIIA) close to Trp-214 with a contribution of a few residues of subdomain IIA. CD spectroscopy showed a change in the conformation of both the isoforms of HSA upon meropenem binding. The catalytic efficiency of HSA (only N-isoform) on p-nitrophenyl acetate was increased primarily due to a decrease in <i>K</i>_m and an increase in <i>k</i>_cat values. This study provides an insight into the molecular basis of interaction between meropenem and HSA.</p

Michelis-Menten curves and catalytic efficiencies of HPR and its variants on poly(A).ploy(U).

<p>The ribonuclease activity of HPR and its variants was analysed on the double stranded RNA substrate, poly(A).poly(U) as described. A. Michelis-Menten curves; B. Catalytic efficiencies (<i>k</i>_cat/<i>K</i>_m).</p

Effect of HPR and its variants on thermal transition profile of double stranded DNA poly (dA−dT).poly(dA−dT).

<p>The thermal transition profiles of DNA alone or with protein were studied spectrophotometrically at 260 nm in 10 mM MOPS buffer containing 50 mM NaCl (pH 7.5). Melted fraction of DNA (F_t) was plotted against temperature. <i>Panel A</i>: thermal transition profile of DNA in the presence of HPR, Q28A, Q28L, G38D, R39A and RNase A. <i>Panel B</i>: thermal transition profile of DNA in the presence of HPR, Q28A/G38D, Q28A/R39A, Q28L/R39A, R39A/G38D, Q28A/G38D/R39A and RNase A.</p

Transition temperatures (<i>T</i>_m) for the melting of DNA in the presence of HPR and variants.

<p>The transition temperatures were derived from the thermal denaturation curves of DNA in the absence and presence of HPR and its variants. Each experiment was done three times and the standard errors are given.</p

Transition temperatures of HPR and variants.

<p>The transition temperatures were derived from the thermal denaturation curves of HPR and its variants. Each experiment was done three times and the standard errors are given.</p

Steady state kinetics of HPR and variants on poly(A).poly(U).

<p>The kinetic parameters were obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017159#s4" target="_blank">Materials and Methods</a>. Each experiment was done three times and the standard errors are given.</p

<i>In silico</i> analysis of HPR variants.

<p>The structures were drawn in PyMOL software using the coordinates of 1DZA (19). All important residues are shown in ball and stick model. The three panels show effect of respective mutations on various interactions in HPR variants. A. Gln28; B. Gly38; and C. Arg39. The hydrogen bond and van der Waal interactions are shown in blue and red dotted lines, respectively.</p

CD spectra of HPR and its variants.

<p>CD spectra were recorded in the far-UV region (200–250 nm) at pH 7.4 and 25°C. The spectra are presented as mean residue ellipticity, expressed in degrees.cm².dmol⁻¹. <i>Panel A</i>: CD spectra of HPR, Q28A, Q28L, G38D and R39A. <i>Panel B</i>: CD spectra of HPR, Q28A/G38D, Q28A/R39A, Q28L/R39A, R39A/G38D and Q28A/G38D/R39A. <i>Panel C</i>: [<i>θ</i>]₂₂₂ of HPR and its variants.</p

Thermal denaturation profiles of HPR and its variants.

<p>Heat induced unfolding curves of HPR and its variants are shown as plots of <i>f</i>_D values vs temperature. <i>f</i>_D is the fraction of the protein in denatured state as defined in the text. <i>Panel A</i>: denaturation profiles of HPR, Q28A, Q28L, G38D and R39A. <i>Panel B</i>: denaturation profile of HPR, Q28A/G38D, Q28A/R39A, Q28L/R39A, R39A/G38D and Q28A/G38D/R39A.</p

Cartoon model of HPR (golden) superimposed over RNase A (light green).

<p>The structure was drawn by taking atomic coordinates from Protein Data Bank in PyMol. PDB IDs of HPR and RNase A are 1DZA and 3JW1, respectively. All residues are shown in ball and stick model. The residues under investigation, Gln28, Gly38 and Arg39 are shown in yellow. Asp38 of RNase A is shown in red, active site residues His12 and His119 in light pink and the ligand, uridine-5'-monophosphate in grey.</p