Computational design of protein catalysts with enhanced stabilities for use in research and enzyme technologies is a challenging task. Using force-field calculations and phylogenetic analysis, we previously designed the haloalkane dehalogenase DhaA115 which contains 11 mutations that confer upon it outstanding thermostability (T m = 73.5 °C; ΔT m > 23 °C). An understanding of the structural basis of this hyperstabilization is required in order to develop computer algorithms and predictive tools. Here, we report X-ray structures of DhaA115 at 1.55 Å and 1.6 Å resolutions and their molecular dynamics trajectories, which unravel the intricate network of interactions that reinforce the αβα-sandwich architecture. Unexpectedly, mutations toward bulky aromatic amino acids at the protein surface triggered long-distance (∼27 Å) backbone changes due to cooperative effects. These cooperative interactions produced an unprecedented double-lock system that: (i) induced backbone changes, (ii) closed the molecular gates to the active site, (iii) reduced the volumes of the main and slot access tunnels, and (iv) occluded the active site. Despite these spatial restrictions, experimental tracing of the access tunnels using krypton derivative crystals demonstrates that transport of ligands is still effective. Our findings highlight key thermostabilization effects and provide a structural basis for designing new thermostable protein catalysts.

• Publikační typ
• časopisecké články MeSH

Haloalkane dehalogenases catalyze the hydrolysis of halogen-carbon bonds in organic halogenated compounds and as such are of great utility as biocatalysts. The crystal structures of the haloalkane dehalogenase DhlA from the bacterium from Xanthobacter autotrophicus GJ10, specifically adapted for the conversion of the small 1,2-dichloroethane (DCE) molecule, display the smallest catalytic site (110 Å3) within this enzyme family. However, during a substrate-specificity screening, we noted that DhlA can catalyze the conversion of far bulkier substrates, such as the 4-(bromomethyl)-6,7-dimethoxy-coumarin (220 Å3). This large substrate cannot bind to DhlA without conformational alterations. These conformational changes have been previously inferred from kinetic analysis, but their structural basis has not been understood. Using molecular dynamic simulations, we demonstrate here the intrinsic flexibility of part of the cap domain that allows DhlA to accommodate bulky substrates. The simulations displayed two routes for transport of substrates to the active site, one of which requires the conformational change and is likely the route for bulky substrates. These results provide insights into the structure-dynamics function relationships in enzymes with deeply buried active sites. Moreover, understanding the structural basis for the molecular adaptation of DhlA to 1,2-dichloroethane introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature.

• Klíčová slova
• active site, conformational change, dichloroethane degradation, enzyme catalysis, enzyme kinetics, enzyme mechanism, ethylene dichloride, haloalkane dehalogenase, molecular dynamics, molecular evolution, organic halogen, organohalogen, protein conformation,
• MeSH
• ethylendichloridy metabolismus   MeSH
• halogenace MeSH
• hydrolasy chemie   metabolismus   MeSH
• katalytická doména MeSH
• kinetika MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• kumariny chemie   metabolismus   MeSH
• metylace MeSH
• simulace molekulární dynamiky MeSH
• simulace molekulového dockingu MeSH
• substrátová specifita MeSH
• Xanthobacter chemie   enzymologie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ethylendichloridy MeSH
• ethylene dichloride MeSH Prohlížeč
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• kumariny MeSH

ChannelsDB (http://ncbr.muni.cz/ChannelsDB) is a database providing information about the positions, geometry and physicochemical properties of channels (pores and tunnels) found within biomacromolecular structures deposited in the Protein Data Bank. Channels were deposited from two sources; from literature using manual deposition and from a software tool automatically detecting tunnels leading to the enzymatic active sites and selected cofactors, and transmembrane pores. The database stores information about geometrical features (e.g. length and radius profile along a channel) and physicochemical properties involving polarity, hydrophobicity, hydropathy, charge and mutability. The stored data are interlinked with available UniProt annotation data mapping known mutation effects to channel-lining residues. All structures with channels are displayed in a clear interactive manner, further facilitating data manipulation and interpretation. As such, ChannelsDB provides an invaluable resource for research related to deciphering the biological function of biomacromolecular channels.

• MeSH
• aminokyseliny chemie   metabolismus   MeSH
• cytochrom P-450 CYP2D6 chemie   genetika   metabolismus   MeSH
• databáze proteinů * MeSH
• eukaryotické buňky cytologie   enzymologie   MeSH
• exprese genu MeSH
• hydrofobní a hydrofilní interakce MeSH
• iontové kanály chemie   genetika   metabolismus   MeSH
• jaderný pór chemie   genetika   metabolismus   MeSH
• katalytická doména MeSH
• koenzymy chemie   metabolismus   MeSH
• lidé MeSH
• mutace MeSH
• prokaryotické buňky cytologie   enzymologie   MeSH
• software * MeSH
• statická elektřina MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• cytochrom P-450 CYP2D6 MeSH
• iontové kanály MeSH
• koenzymy MeSH

Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs.

• MeSH
• buněčná membrána chemie   metabolismus   MeSH
• cholesterol chemie   metabolismus   MeSH
• kompetitivní vazba MeSH
• krysa rodu Rattus MeSH
• nádorové buněčné linie MeSH
• proteinové domény * MeSH
• receptor adenosinový A2A chemie   metabolismus   MeSH
• receptory spřažené s G-proteiny chemie   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol MeSH
• receptor adenosinový A2A MeSH
• receptory spřažené s G-proteiny MeSH

This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to "helicopter" above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function.

• MeSH
• buněčná membrána metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• interakční proteinové domény a motivy fyziologie   MeSH
• jaterní mikrozomy metabolismus   MeSH
• lidé MeSH
• sekundární struktura proteinů MeSH
• systém (enzymů) cytochromů P-450 chemie   fyziologie   MeSH
• výzkumná zpráva * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• systém (enzymů) cytochromů P-450 MeSH

BACKGROUND: Channels and pores in biomacromolecules (proteins, nucleic acids and their complexes) play significant biological roles, e.g., in molecular recognition and enzyme substrate specificity. RESULTS: We present an advanced software tool entitled MOLE 2.0, which has been designed to analyze molecular channels and pores. Benchmark tests against other available software tools showed that MOLE 2.0 is by comparison quicker, more robust and more versatile. As a new feature, MOLE 2.0 estimates physicochemical properties of the identified channels, i.e., hydropathy, hydrophobicity, polarity, charge, and mutability. We also assessed the variability in physicochemical properties of eighty X-ray structures of two members of the cytochrome P450 superfamily. CONCLUSION: Estimated physicochemical properties of the identified channels in the selected biomacromolecules corresponded well with the known functions of the respective channels. Thus, the predicted physicochemical properties may provide useful information about the potential functions of identified channels. The MOLE 2.0 software is available at http://mole.chemi.muni.cz.

• Publikační typ
• časopisecké články MeSH

Tunnels and channels facilitate the transport of small molecules, ions and water solvent in a large variety of proteins. Characteristics of individual transport pathways, including their geometry, physico-chemical properties and dynamics are instrumental for understanding of structure-function relationships of these proteins, for the design of new inhibitors and construction of improved biocatalysts. CAVER is a software tool widely used for the identification and characterization of transport pathways in static macromolecular structures. Herein we present a new version of CAVER enabling automatic analysis of tunnels and channels in large ensembles of protein conformations. CAVER 3.0 implements new algorithms for the calculation and clustering of pathways. A trajectory from a molecular dynamics simulation serves as the typical input, while detailed characteristics and summary statistics of the time evolution of individual pathways are provided in the outputs. To illustrate the capabilities of CAVER 3.0, the tool was applied for the analysis of molecular dynamics simulation of the microbial enzyme haloalkane dehalogenase DhaA. CAVER 3.0 safely identified and reliably estimated the importance of all previously published DhaA tunnels, including the tunnels closed in DhaA crystal structures. Obtained results clearly demonstrate that analysis of molecular dynamics simulation is essential for the estimation of pathway characteristics and elucidation of the structural basis of the tunnel gating. CAVER 3.0 paves the way for the study of important biochemical phenomena in the area of molecular transport, molecular recognition and enzymatic catalysis. The software is freely available as a multiplatform command-line application at http://www.caver.cz.

• MeSH
• algoritmy * MeSH
• hydrolasy chemie   metabolismus   MeSH
• konformace proteinů * MeSH
• krystalografie MeSH
• proteiny chemie   metabolismus   MeSH
• shluková analýza MeSH
• simulace molekulární dynamiky MeSH
• software * MeSH
• výpočetní biologie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• proteiny MeSH

Biomolecular channels play important roles in many biological systems, e.g. enzymes, ribosomes and ion channels. This article introduces a web-based interactive MOLEonline 2.0 application for the analysis of access/egress paths to interior molecular voids. MOLEonline 2.0 enables platform-independent, easy-to-use and interactive analyses of (bio)macromolecular channels, tunnels and pores. Results are presented in a clear manner, making their interpretation easy. For each channel, MOLEonline displays a 3D graphical representation of the channel, its profile accompanied by a list of lining residues and also its basic physicochemical properties. The users can tune advanced parameters when performing a channel search to direct the search according to their needs. The MOLEonline 2.0 application is freely available via the Internet at http://ncbr.muni.cz/mole or http://mole.upol.cz.

• MeSH
• cytochrom P-450 CYP3A chemie   MeSH
• enzymy chemie   MeSH
• internet MeSH
• iontové kanály chemie   MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• počítačová grafika MeSH
• ribozomy chemie   MeSH
• software * MeSH
• uživatelské rozhraní počítače MeSH
• velké podjednotky ribozomu archebakteriální chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytochrom P-450 CYP3A MeSH
• enzymy MeSH
• iontové kanály MeSH

Cytochrome P450 2C9 (CYP2C9) is a membrane-anchored human microsomal protein involved in the drug metabolism in liver. CYP2C9 consists of an N-terminal transmembrane anchor and a catalytic cytoplasmic domain. While the structure of the catalytic domain is well-known from X-ray experiments, the complete structure and its incorporation into the membrane remains unsolved. We constructed an atomistic model of complete CYP2C9 in a dioleoylphosphatidylcholine membrane and evolved it by molecular dynamics simulations in explicit water on a 100+ ns time-scale. The model agrees well with known experimental data about membrane positioning of cytochromes P450. The entry to the substrate access channel is proposed to be facing the membrane interior while the exit of the product egress channel is situated above the interface pointing toward the water phase. The positions of openings of the substrate access and product egress channels correspond to free energy minima of CYP2C9 substrate ibuprofen and its metabolite in the membrane, respectively.

• MeSH
• aromatické hydroxylasy chemie   metabolismus   MeSH
• cytochrom P450 CYP2C9 MeSH
• fosfatidylcholiny chemie   metabolismus   MeSH
• ibuprofen chemie   metabolismus   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• membrány umělé * MeSH
• molekulární modely MeSH
• povrchové vlastnosti MeSH
• simulace molekulární dynamiky MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,2-oleoylphosphatidylcholine MeSH Prohlížeč
• aromatické hydroxylasy MeSH
• CYP2C9 protein, human MeSH Prohlížeč
• cytochrom P450 CYP2C9 MeSH
• fosfatidylcholiny MeSH
• ibuprofen MeSH
• membrány umělé * MeSH

Engineering enzymes to degrade anthropogenic compounds efficiently is challenging. We obtained Rhodococcus rhodochrous haloalkane dehalogenase mutants with up to 32-fold higher activity than wild type toward the toxic, recalcitrant anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. We identified key residues in access tunnels connecting the buried active site with bulk solvent by rational design and randomized them by directed evolution. The most active mutant has large aromatic residues at two out of three randomized positions and two positions modified by site-directed mutagenesis. These changes apparently enhance activity with TCP by decreasing accessibility of the active site for water molecules, thereby promoting activated complex formation. Kinetic analyses confirmed that the mutations improved carbon-halogen bond cleavage and shifted the rate-limiting step to the release of products. Engineering access tunnels by combining computer-assisted protein design with directed evolution may be a valuable strategy for refining catalytic properties of enzymes with buried active sites.

• MeSH
• biodegradace MeSH
• cirkulární dichroismus MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• látky znečišťující životní prostředí chemie   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• počítačová simulace MeSH
• propan analogy a deriváty   chemie   MeSH
• proteinové inženýrství * MeSH
• Rhodococcus enzymologie   genetika   růst a vývoj   MeSH
• řízená evoluce molekul MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,2,3-trichloropropane MeSH Prohlížeč
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• látky znečišťující životní prostředí MeSH
• propan MeSH