Influence of ligand binding on structure and thermostability of human α₁-acid glycoprotein

Abstract

Ligand binding of neutral progesterone, basic propranolol, and acidic warfarin to human α1-acid glycoprotein (AGP) was investigated by Raman spectroscopy. The binding itself is characterized by a uniform conformational shift in which a tryptophan residue is involved. Slight differences corresponding to different contacts of the individual ligands inside the β-barrel are described. Results are compared with in silico ligand docking into the available crystal structure of deglycosylated AGP using quantum/molecular mechanics. Calculated binding energies are -18.2, -14.5, and -11.5 kcal/mol for warfarin, propranolol, and progesterone, respectively. These calculations are consistent with Raman difference spectroscopy; nevertheless, minor discrepancies in the precise positions of the ligands point to structural differences between deglycosylated and native AGP. Thermal dynamics of AGP with/without bounded warfarin was followed by Raman spectroscopy in a temperature range of 10-95 °C and analyzed by principal component analysis. With increasing temperature, a slight decrease of α-helical content is observed that coincides with an increase in β-sheet content. Above 45 °C, also β-strands tend to unfold, and the observed decrease in β-sheet coincides with an increase of β-turns accompanied by a conformational shift of the nearby disulfide bridge from high-energy trans-gauche-trans to more relaxed gauche-gauche-trans. This major rearrangement in the vicinity of the bridge is not only characterized by unfolding of the β-sheet but also by subsequent ligand release. Hereby, ligand binding alters the protein dynamics, and the more rigid protein-ligand complex shows an improved thermal stability, a finding that contributes to the reported chaperone-like function of AGP.

Identification of Natural Product Sulfuretin Derivatives as Inhibitors for the Endoplasmic Reticulum Redox Protein ERO1α

“…We utilized the cellular thermal shift assay (CETSA) to determine target engagement of T151742 in vitro using the non-small cell lung cancer (NSCLC) cell line PC-9. The EGFR-driven PC-9 cell line was chosen as new treatment strategies are needed in EGFR-driven NSCLC as most patients relapse and become resistant to current targeted therapies with the development of further mutations such as T790M and C797S, as well as the emergence of EGFR-independent tumors. , CETSA utilizes thermodynamic principles that have been well described and upon binding of ligands to a protein, the ligand-bound protein becomes more thermostable with respect to the temperature required to denature the protein or expose hydrophobic regions of the protein leading to aggregation and insolubility of the protein . All proteins have an aggregation temperature, which is inherent to their intracellular thermostability.…”

“…42,43 CETSA utilizes thermodynamic principles that have been well described and upon binding of ligands to a protein, the ligand-bound protein becomes more thermostable with respect to the temperature required to denature the protein or expose hydrophobic regions of the protein leading to aggregation and insolubility of the protein. 44 All proteins have an aggregation temperature, which is inherent to their intracellular thermostability. Upon the addition of a ligand binding, the thermostability increases.…”

Characterization of the mechanism of interaction between α1‐acid glycoprotein and lipid membranes by vacuum‐ultraviolet circular‐dichroism spectroscopy

“…Knowledge of both the secondary and tertiary structures of proteins is useful for understanding their structure‐function relationships. The secondary structure of AGP in the N state has been investigated using circular dichroism (CD), Raman, and Fourier‐transform infrared spectroscopy to reveal the binding sites of drugs and the structure‐function relationships of AGP . CD spectroscopy has been used to analyze the structure of AGP in lipid membranes because it is very sensitive to local peptide structures and applicable to any size of protein at a low concentration under various experimental conditions, such as in the presence of a membrane .…”

Quorum sensing‐associated bacterial phenazines are potential ligands of human α1‐acid glycoprotein

“…6,7 This crevice can accommodate and transport several hundred, mostly basic and neutral small molecules of both endoand exogenous origin. 1,8 The binding promiscuity of AGP is reflected in the diversity of molecular shapes and dimensions of its ligands that are varied from complex, large macrocycles (macrolide antibiotics, 9 porphyrin pigments, 10,11 steroids, 12 organogold complexes 13 ) to much smaller molecules such as lidocaine, 14 atenolol, or disopyramide. 6 Besides serum albumin, AGP is the second most important drug carrier protein in the circulatory system.…”