Michal Kolinski

Coarse-Grained Protein Models and Their Applications.

The traditional computational modeling of protein structure, dynamics, and interactions remains difficult for many protein systems. It is mostly due to the size of protein conformational spaces and required simulation time scales that are still too large to be studied in atomistic detail. Lowering the level of protein representation from all-atom to coarse-grained opens up new possibilities for studying protein systems. In this review we provide an overview of coarse-grained models focusing on their design, including choices of representation, models of energy functions, sampling of conformational space, and applications in the modeling of protein structure, dynamics, and interactions. A more detailed description is given for applications of coarse-grained models suitable for efficient combinations with all-atom simulations in multiscale modeling strategies.

Structural investigation of the C-terminal catalytic fragment of presenilin 1

The γ-secretase complex has a decisive role in the development of Alzheimer’s disease, in that it cleaves a precursor to create the amyloid β peptide whose aggregates form the senile plaques encountered in the brains of patients. Γ-secretase is a member of the intramembrane-cleaving proteases which process their transmembrane substrates within the bilayer. Many of the mutations encountered in early onset familial Alzheimer’s disease are linked to presenilin 1, the catalytic component of γ-secretase, whose active form requires its endoproteolytic cleavage into N-terminal and C-terminal fragments. Although there is general agreement regarding the topology of the N-terminal fragment, studies of the C-terminal fragment have yielded ambiguous and contradictory results that may be difficult to reconcile in the absence of structural information. Here we present the first structure of the C-terminal fragment of human presenilin 1, as obtained from NMR studies in SDS micelles. The structure reveals a topology where the membrane is likely traversed three times in accordance with the more generally accepted nine transmembrane domain model of presenilin 1, but contains unique structural features adapted to accommodate the unusual intramembrane catalysis. These include a putative half-membrane-spanning helix N-terminally harboring the catalytic aspartate, a severely kinked helical structure toward the C terminus as well as a soluble helix in the assumed-to-be unstructured N-terminal loop.

Stabilizing Effect of Zn2+ in Native Bovine Rhodopsin

Single-molecule force spectroscopy (SMFS) is a powerful tool to dissect molecular interactions that govern the stability and function of proteins. We applied SMFS to understand the effect of Zn2+ on the molecular interactions underlying the structure of rhodopsin. Force-distance curves obtained from SMFS assays revealed the strength and location of molecular interactions that stabilize structural segments within this receptor. The inclusion of ZnCl2 in SMFS assay buffer increased the stability of most structural segments. This effect was not mimicked by CaCl2, CdCl2, or CoCl2 Thus, Zn2+. stabilizes the structure of rhodopsin in a specific manner.

The Mechanism of Ligand-Induced Activation or Inhibition of μ- and κ-Opioid Receptors †

G-protein-coupled receptors (GPCRs) are important targets for treating severe diseases. However why certain molecules act as activators whereas others, with similar structures, block GPCR activation, is poorly understood since the same molecule can activate one receptor subtype while blocking another closely related receptor. To shed light on these central questions, we used all-atom, long-time-scale molecular dynamics simulations on the κ-opioid and μ-opioid receptors (κOR and μOR). We found that water molecules penetrating into the receptor interior mediate the activating versus blocking effects of a particular ligand-receptor interaction. Both the size and the flexibility of the bound ligand regulated water influx into the receptor. The solvent-accessible inner surface area was found to be a parameter that can help predict the function of the bound ligand.

Interactions of Benzo[a]pyrene and Diesel Exhaust Particulate Matter with the Lung Surfactant System

One of the reasons for cellular changes in the lung tissue exposed to the diesel exhaust composed of soot particles with adsorbed volatile organic molecules is the reduction of the clearance rate in the pulmonary region of the respiratory system. The interaction of the fractal-like particles and organic substances with a surfactant monolayer limits its dynamic activity. The surface properties of Survanta, a purified extract of bovine lung surfactant (LS), which interacted with carbon particles (200 nm aggregates) and benzo[a]pyrene (BaP), molecules were measured with the oscillating bubble technique. The results showed a significant lowering of the dynamics of the surfactant monolayer compared to the control case (no exposure). Additional measurements of surface pressure during the monotonic compression of the air-water interface containing the major LS phospholipid, dipalmitoylphosphatidylcholine (DPPC), showed that the presence of BaP molecules in the system influenced its stability. The experimental results were supplemented with a theoretical molecular dynamics model of the interaction between BaP and DPPC molecules. The simulation results indicated the insertion of BaP molecules into the lipid layer, which explained the measured effects.

Molecular interactions between fenoterol stereoisomers and derivatives and the β2-adrenergic receptor binding site studied by docking and molecular dynamics simulations

The β2 adrenergic receptor (β2-AR) has become a model system for studying the ligand recognition process and mechanism of the G protein coupled receptors activation. In the present study stereoisomers of fenoterol and some of its derivatives (N = 94 molecules) were used as molecular probes to identify differences in stereo-recognition interactions between β2-AR and structurally similar agonists. The present study aimed at determining the 3D molecular models of the fenoterol derivative-β2-AR complexes. Molecular models of β2-AR have been developed by using the crystal structure of the human β2-AR T4 lysozyme fusion protein with bound (S)-carazolol (PDB ID: 2RH1) and more recently reported structure of a nanobody-stabilized active state of the β2-AR with the bound full agonist BI-167107 (PDB ID: 3P0G). The docking procedure allowed us to study the similarities and differences in the recognition binding site(s) for tested ligands. The agonist molecules occupied the same binding region, between TM III, TM V, TM VI and TM VII. The residues identified by us during docking procedure (Ser203, Ser207, Asp113, Lys305, Asn312, Tyr308, Asp192) were experimentally indicated in functional and biophysical studies as being very important for the agonist-receptor interactions. Moreover, the additional space, an extension of the orthosteric pocket, was identified and described. Furthermore, the molecular dynamics simulations were used to study the molecular mechanism of interaction between ligands ((R,R’)- and (S,S’)-fenoterol) and β2-AR. Our research offers new insights into the ligand stereoselective interaction with one of the most important GPCR member. This study may also facilitate the design of improved selective medications, which can be used to treat, prevent and control heart failure symptoms.

Structure Prediction of the Second Extracellular Loop in G-Protein-Coupled Receptors

G-protein-coupled receptors (GPCRs) play key roles in living organisms. Therefore, it is important to determine their functional structures. The second extracellular loop (ECL2) is a functionally important region of GPCRs, which poses significant challenge for computational structure prediction methods. In this work, we evaluated CABS, a well-established protein modeling tool for predicting ECL2 structure in 13 GPCRs. The ECL2s (with between 13 and 34 residues) are predicted in an environment of other extracellular loops being fully flexible and the transmembrane domain fixed in its x-ray conformation. The modeling procedure used theoretical predictions of ECL2 secondary structure and experimental constraints on disulfide bridges. Our approach yielded ensembles of low-energy conformers and the most populated conformers that contained models close to the available x-ray structures. The level of similarity between the predicted models and x-ray structures is comparable to that of other state-of-the-art computational methods. Our results extend other studies by including newly crystallized GPCRs.

Structural elucidation of specific noncovalent association of folic acid with native cyclodextrins using an ion mobility mass spectrometry and theoretical approach.

The combination of ion mobility mass spectrometry studies and theoretical calculations including docking studies permitted a detailed structural description of noncovalent complexes of folic acid (FA) and native cyclodextrins (α-CD, β-CD, and γ-CD). The mode of noncovalent association depended on the cavity size of the cyclodextrin. The structure of FA/α-CD represented the exclusion complex in which the aminobenzoic moiety and the aromatic pteridine ring of folic acid remain outside the cyclodextrin cavity, while the glutamate residue is anchored in the interior of the α-cyclodextrin. A rotaxane-type structure was proposed for the FA/β-CD complex with the aminobenzoic part of FA being trapped in the central cavity of β-CD. The glutamate residue and the aromatic pteridine ring interact with the primary and secondary rim hydroxyl residues, respectively, enhancing complex stability. Two possible structures of FA/γ-CD were suggested, the first one being analogous to the FA/β-CD complex and the second one being more stable-in which the aromatic pteridine ring penetrates into the CD cavity while the glutamate residue with the aminobenzoic part of FA is exposed to the cone exterior of CD at its wider edge. Further insight into the association behavior of the folic acid toward cyclodextrins evaluated by thermodynamic calculations indicates that the process is highly exothermic. The complex stability increased in the order FA/α-CD < FA/β-CD < FA/γ-CD. This order is consistent with the previously determined relative gas-phase stability established based on the dissociation efficiency curves of the FA/CD complexes.

Modeling of ligand binding to G protein coupled receptors: cannabinoid CB1, CB2 and adrenergic β2AR

AbstractCannabinoid and adrenergic receptors belong to the class A (similar to rhodopsin) G protein coupled receptors. Docking of agonists and antagonists to CB1 and CB2 cannabinoid receptors revealed the importance of a centrally located rotamer toggle switch and its possible participation in the mechanism of agonist/antagonist recognition. The switch is composed of two residues, F3.36 and W6.48, located on opposite transmembrane helices TM3 and TM6 in the central part of the membranous domain of cannabinoid receptors. The CB1 and CB2 receptor models were constructed based on the adenosine A2A receptor template. The two best scored conformations of each receptor were used for the docking procedure. In all poses (ligand-receptor conformations) characterized by the lowest ligand-receptor intermolecular energy and free energy of binding the ligand type matched the state of the rotamer toggle switch: antagonists maintained an inactive state of the switch, whereas agonists changed it. In case of agonists of β2AR, the (R,R) and (S,S) stereoisomers of fenoterol, the molecular dynamics simulations provided evidence of different binding modes while preserving the same average position of ligands in the binding site. The (S,S) isomer was much more labile in the binding site and only one stable hydrogen bond was created. Such dynamical binding modes may also be valid for ligands of cannabinoid receptors because of the hydrophobic nature of their ligand-receptor interactions. However, only very long molecular dynamics simulations could verify the validity of such binding modes and how they affect the process of activation. FigureThe rotamer toggle switch in cannabinoid receptors is comprised of two residues, F3.36 and W6.48, which are located on transmembrane helices TM3 and TM6. Docking of agonists and antagonists to CB1 and CB2 cannabinoid receptors revealed the importance of this centrally located switch and its possible participation in the mechanism of agonist/antagonist sensing. The best scored poses (ligand-receptor conformations) were obtained for the ligands matching the switch state: antagonists maintained the state of the rotamer toggle switch, whereas agonists changed it

Determination and modelling of stereoselective interactions of ligands with drug transporters: a key dimension in the understanding of drug disposition.

1. Stereochemistry is an important dimension in pharmacology and plays a role in every aspect of the pharmacological fate of chiral xenobiotics. This includes small molecule–drug transporter binding. 2. This paper reviews the reported stereoselectivities of substrate and inhibitor interactions with P-glycoprotein and the organic cation transporter obtained using standard functional and binding studies, as well as data obtained from online cellular membrane affinity chromatography studies. 3. The use of stereochemical data in quantitative structure–activity relationship (QSAR) and pharmacophore modelling is also addressed as is the effect of ignoring the fact that small molecule–drug transporter interactions take place in three-dimensional and asymmetric space.