title: Insight into the binding mechanism of imipenem to human serum albumin by spectroscopic and computational approaches.
abstract: The mechanism of interaction between imipenem and HSA was investigated by various techniques like fluorescence, UV.vis absorbance, FRET, circular dichroism, urea denaturation, enzyme kinetics, ITC, and molecular docking. We found that imipenem binds to HSA at a high affinity site located in subdomain IIIA (Sudlow's site I) and a low affinity site located in subdomain IIA.IIB. Electrostatic interactions played a vital role along with hydrogen bonding and hydrophobic interactions in stabilizing the imipenem.HSA complex at subdomain IIIA, while only electrostatic and hydrophobic interactions were present at subdomain IIA.IIB. The binding and thermodynamic parameters obtained by ITC showed that the binding of imipenem to HSA was a spontaneous process (ΔGD⁰(D)= -32.31 kJ mol(-1) for high affinity site and ΔGD⁰(D) = -23.02 kJ mol(-1) for low affinity site) with binding constants in the range of 10(4)-10(5) M(-1). Spectroscopic investigation revealed only one binding site of imipenem on HSA (Ka∼10(4) M(-1)). FRET analysis showed that the binding distance between imipenem and HSA (Trp-214) was optimal (r = 4.32 nm) for quenching to occur. Decrease in esterase-like activity of HSA in the presence of imipenem showed that Arg-410 and Tyr-411 of subdomain IIIA (Sudlow's site II) were directly involved in the binding process. CD spectral analysis showed altered conformation of HSA upon imipenem binding. Moreover, the binding of imipenem to subdomain IIIA (Sudlow's site II) of HSA also affected its folding pathway as clear from urea-induced denaturation studies.
authors.affiliation: Medical Microbiology and Molecular Biology Laboratory, Interdisciplinary Biotechnology Unit, Aligarh Muslim University , Aligarh, Uttar Pradesh, 202 002, India.
authors.forename: Md Tabish
authors.initials: MT
authors.lastname: Rehman
authors.forename: Hira
authors.initials: H
authors.lastname: Shamsi
authors.forename: Asad U
authors.initials: AU
authors.lastname: Khan
mesh_terms: D001665:Binding Sites / Q000502:physiology; D002942:Circular Dichroism / Q000379:methods; D019295:Computational Biology / Q000379:methods; D006801:Humans; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D015378:Imipenem / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D012709:Serum Albumin / Q000378:metabolism*; D013057:Spectrum Analysis / Q000379:methods; D013816:Thermodynamics
chemical_list: D012709:Serum Albumin; D015378:Imipenem