Crystal structure of Pseudomonas aeruginosa apo-azurin at 1.85 Å resolution

AbstractThe 3D structure of apo-azurin from Pseudomonas aeruginosa has been determined at 1.85 Å resolution. The crystal structure is composed of two different molecular forms of apo-azurin arranged as hetero-dimers in the tetramer of the asymmetric unit. Form 1 closely resembles the holo-protein lacking copper. Form 2 shows differences in the metal binding site region induced by the incorporation of a solvent molecule into this site. The positions of the copper ligands His46 and His117 are shifted by 0.6 Å and 1.6 Å. The His117 side chain adopts a position at the surface of the protein, thereby facilitating access to the copper site. The presence of two different molecular forms of apo-azurin in the crystal lattice may reflect an equilibrium between the two forms in solution. 1H-NMR spectra or apo-azurin recorded as a function or pH show that at high pH the line broadening of His35, His46 and His117 resonances is consistent with an interconversion between forms 1 and 2. At low pH, no broadening is observed. This may indicate that here the interconversion is fast on the NMR timescale

Protons taken hostage: Dynamic H-bond networks of the pH-sensing GPR68

Proton-sensing G Protein Coupled Receptors (GPCRs) sense changes in the extracellular pH to effect cell signaling for cellular homeostasis. They tend to be overexpressed in solid tumors associated with acidic extracellular pH, and are of direct interest as drug targets. How proton-sensing GPCRs sense extracellular acidification and activate upon protonation change is important to understand, because it may guide the design of therapeutics. Lack of publicly available experimental structures make it challenging to discriminate between conflicting mechanisms proposed for proton-binding, as main roles have been assigned to either an extracellular histidine cluster or to an internal carboxylic triad. Here we present a protocol to derive and evaluate structural models of the proton-sensing GPR68. This approach integrates state-of-the-art homology modeling with microsecond-timescale atomistic simulations, and with a detailed assessment of the compatibility of the structural models with known structural features of class A GPCRs. To decipher structural elements of potential interest for protonation-coupled conformational changes of GPR68, we used the best-compatible model as a starting point for independent atomistic simulations of GPR68 with different protonation states, and graph computations to characterize the response of GPR68 to changes in protonation. We found that GPR68 hosts an extended hydrogen-bond network that inter-connects the extracellular histidine cluster to the internal carboxylic triad, and which can even reach groups at the cytoplasmic G-protein binding site. Taken together, results suggest that GPR68 relies on dynamic, hydrogen-bond networks to inter-connect extracellular and internal proton-binding sites, and to elicit conformational changes at the cytoplasmic G-protein binding site

Biophysics in drug discovery : impact, challenges and opportunities

Over the past 25 years, biophysical technologies such as X-ray crystallography, nuclear magnetic resonance spectroscopy, surface plasmon resonance spectroscopy and isothermal titration calorimetry have become key components of drug discovery platforms in many pharmaceutical companies and academic laboratories. There have been great improvements in the speed, sensitivity and range of possible measurements, providing high-resolution mechanistic, kinetic, thermodynamic and structural information on compound-target interactions. This Review provides a framework to understand this evolution by describing the key biophysical methods, the information they can provide and the ways in which they can be applied at different stages of the drug discovery process. We also discuss the challenges for current technologies and future opportunities to use biophysical methods to solve drug discovery problems

Structural Basis for Inhibition Promiscuity of Dual Specific Thrombin and Factor Xa Blood Coagulation Inhibitors

AbstractBackground: A major current focus of pharmaceutical research is the development of selective inhibitors of the blood coagulation enzymes thrombin or factor Xa to be used as orally bioavailable anticoagulant drugs in thromboembolic disorders and in the prevention of venous and arterial thrombosis. Simultaneous direct inhibition of thrombin and factor Xa by synthetic proteinase inhibitors as a novel approach to antithrombotic therapy could result in potent anticoagulants with improved pharmacological properties.Results: The binding mode of such dual specific inhibitors of thrombin and factor Xa was determined for the first time by comparative crystallography using human α-thrombin, human des-Gla (1–44) factor Xa and bovine trypsin as the ligand receptors. The benzamidine-based inhibitors utilize two different conformations for the interaction with thrombin and factor Xa/trypsin, which are evoked by the steric requirements of the topologically different S2 subsites of the enzymes. Compared to the unliganded forms of the proteinases, ligand binding induces conformational adjustments of thrombin and factor Xa active site residues indicative of a pronounced induced fit mechanism.Conclusion: The structural data reveal the molecular basis for a desired unselective inhibition of the two key components of the blood coagulation cascade. The 4-(1-methyl-benzimidazole-2-yl)-methylamino-benzamidine moieties of the inhibitors are able to fill both the small solvent accessible as well as the larger hydrophobic S2 pockets of factor Xa and thrombin, respectively. Distal fragments of the inhibitors are identified which fit into both the cation hole/aromatic box of factor Xa and the hydrophobic aryl binding site of thrombin. Thus, binding constants in the medium-to-low nanomolar range are obtained against both enzymes

Protons taken hostage: Dynamic H-bond networks of the pH-sensing GPR68

Proton-sensing G Protein Coupled Receptors (GPCRs) sense changes in the extracellular pH to effect cell signaling for cellular homeostasis. They tend to be overexpressed in solid tumors associated with acidic extracellular pH, and are of direct interest as drug targets. How proton-sensing GPCRs sense extracellular acidification and activate upon protonation change is important to understand, because it may guide the design of therapeutics. Lack of publicly available experimental structures make it challenging to discriminate between conflicting mechanisms proposed for proton-binding, as main roles have been assigned to either an extracellular histidine cluster or to an internal carboxylic triad. Here we present a protocol to derive and evaluate structural models of the proton-sensing GPR68. This approach integrates state-of-the-art homology modeling with microsecond-timescale atomistic simulations, and with a detailed assessment of the compatibility of the structural models with known structural features of class A GPCRs. To decipher structural elements of potential interest for protonation-coupled conformational changes of GPR68, we used the best-compatible model as a starting point for independent atomistic simulations of GPR68 with different protonation states, and graph computations to characterize the response of GPR68 to changes in protonation. We found that GPR68 hosts an extended hydrogen-bond network that inter-connects the extracellular histidine cluster to the internal carboxylic triad, and which can even reach groups at the cytoplasmic G-protein binding site. Taken together, results suggest that GPR68 relies on dynamic, hydrogen-bond networks to inter-connect extracellular and internal proton-binding sites, and to elicit conformational changes at the cytoplasmic G-protein binding site

Comparative Analysis of Binding Kinetics and Thermodynamics of Dipeptidyl Peptidase‑4 Inhibitors and Their Relationship to Structure

The binding kinetics and thermodynamics of dipeptidyl peptidase (DPP)-4 inhibitors (gliptins) were investigated using surface plasmon resonance and isothermal titration calorimetry. Binding of gliptins to DPP-4 is a rapid electrostatically driven process. Off-rates were generally slow partly because of reversible covalent bond formation by some gliptins, and partly because of strong and extensive interactions. Binding of all gliptins is enthalpy-dominated due to strong ionic interactions and strong solvent-shielded hydrogen bonds. Using a congeneric series of molecules which represented the intermediates in the lead optimization program of linagliptin, the onset of slow binding kinetics and development of the thermodynamic repertoire were analyzed in the context of incremental changes of the chemical structures. All compounds rapidly associated, and therefore the optimization of affinity and residence time is highly correlated. The major contributor to the increasing free energy of binding was a strong increase of binding enthalpy, whereas entropic contributions remained low and constant despite significant addition of lipophilicity