The endoplasmic reticulum is organized into ordered regions enriched in cholesterol and sphingomyelin, and disordered microdomains characterized by more fluidity. Rabbit CYP1A1 and CYP1A2 localize into disordered and ordered microdomains, respectively. Previously, a CYP1A2 chimera containing the first 109 amino acids of CYP1A1 showed altered microdomain localization. The goal of this study was to identify specific residues responsible for CYP1A microdomain localization. Thus, CYP1A2 chimeras containing substitutions from homologous regions of CYP1A1 were expressed in HEK 293T/17 cells, and the localization was examined after solubilization with Brij 98. A CYP1A2 mutant with the three amino acids from CYP1A1 (VAG) at positions 27 to 29 of CYP1A2 was generated that showed a distribution pattern similar to those of CYP1A1/1A2 chimeras containing both the first 109 amino acids and the first 31 amino acids of CYP1A1 followed by remaining amino acids of CYP1A2. Similarly, the reciprocal substitution of three amino acids from CYP1A2 (AVR) into CYP1A1 resulted in a partial redistribution of the chimera into ordered microdomains. Molecular dynamic simulations indicate that the positive charges of the CYP1A1 and CYP1A2 linker regions between the N termini and catalytic domains resulted in different depths of immersion of the N termini in the membrane. The overlap of the distribution of positively charged residues in CYP1A2 (AVR) and negatively charged phospholipids was higher in the ordered than in the disordered microdomain. These findings identify three residues in the CYP1AN terminus as a novel microdomain-targeting motif of the P450s and provide a mechanistic explanation for the differential microdomain localization of CYP1A.

• Keywords
• CYP1A1, CYP1A2, cytochrome P450, membrane charge depth, membrane protein, microdomain localization, microdomain-targeting motif, protein chimera, protein-lipid interaction, structure-function,
• MeSH
• Cytochrome P-450 CYP1A1 * genetics   metabolism   chemistry   MeSH
• Cytochrome P-450 CYP1A2 * metabolism   genetics   chemistry   MeSH
• Endoplasmic Reticulum metabolism   MeSH
• HEK293 Cells MeSH
• Rabbits MeSH
• Humans MeSH
• Membrane Microdomains metabolism   genetics   MeSH
• Protein Domains MeSH
• Amino Acid Sequence MeSH
• Molecular Dynamics Simulation MeSH
• Amino Acid Substitution MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cytochrome P-450 CYP1A1 * MeSH
• Cytochrome P-450 CYP1A2 * MeSH

Protein structural families are groups of homologous proteins defined by the organization of secondary structure elements (SSEs). Nowadays, many families contain vast numbers of structures, and the SSEs can help to orient within them. Communities around specific protein families have even developed specialized SSE annotations, always assigning the same name to the equivalent SSEs in homologous proteins. A detailed analysis of the groups of equivalent SSEs provides an overview of the studied family and enriches the analysis of any particular protein at hand. We developed a workflow for the analysis of the secondary structure anatomy of a protein family. We applied this analysis to the model family of cytochromes P450 (CYPs)-a family of important biotransformation enzymes with a community-wide used SSE annotation. We report the occurrence, typical length and amino acid sequence for the equivalent SSE groups, the conservation/variability of these properties and relationship to the substrate recognition sites. We also suggest a generic residue numbering scheme for the CYP family. Comparing the bacterial and eukaryotic part of the family highlights the significant differences and reveals a well-known anomalous group of bacterial CYPs with some typically eukaryotic features. Our workflow for SSE annotation for CYP and other families can be freely used at address https://sestra.ncbr.muni.cz .

• MeSH
• Humans MeSH
• Sequence Analysis, Protein methods   MeSH
• Molecular Dynamics Simulation MeSH
• Software * MeSH
• Cytochrome P-450 Enzyme System chemistry   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytochrome P-450 Enzyme System 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
• Amino Acids chemistry   metabolism   MeSH
• Cytochrome P-450 CYP2D6 chemistry   genetics   metabolism   MeSH
• Databases, Protein * MeSH
• Eukaryotic Cells cytology   enzymology   MeSH
• Gene Expression MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Ion Channels chemistry   genetics   metabolism   MeSH
• Nuclear Pore chemistry   genetics   metabolism   MeSH
• Catalytic Domain MeSH
• Coenzymes chemistry   metabolism   MeSH
• Humans MeSH
• Mutation MeSH
• Prokaryotic Cells cytology   enzymology   MeSH
• Software * MeSH
• Static Electricity MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amino Acids MeSH
• Cytochrome P-450 CYP2D6 MeSH
• Ion Channels MeSH
• Coenzymes 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
• Cell Membrane metabolism   MeSH
• Endoplasmic Reticulum metabolism   MeSH
• Protein Interaction Domains and Motifs physiology   MeSH
• Microsomes, Liver metabolism   MeSH
• Humans MeSH
• Protein Structure, Secondary MeSH
• Cytochrome P-450 Enzyme System chemistry   physiology   MeSH
• Research Report * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cytochrome P-450 Enzyme System 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.

• Publication type
• Journal Article 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
• Algorithms * MeSH
• Hydrolases chemistry   metabolism   MeSH
• Protein Conformation * MeSH
• Crystallography MeSH
• Proteins chemistry   metabolism   MeSH
• Cluster Analysis MeSH
• Molecular Dynamics Simulation MeSH
• Software * MeSH
• Computational Biology methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• haloalkane dehalogenase MeSH Browser
• Hydrolases MeSH
• Proteins 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
• Cytochrome P-450 CYP3A chemistry   MeSH
• Enzymes chemistry   MeSH
• Internet MeSH
• Ion Channels chemistry   MeSH
• Protein Conformation MeSH
• Models, Molecular MeSH
• Computer Graphics MeSH
• Ribosomes chemistry   MeSH
• Software * MeSH
• User-Computer Interface MeSH
• Ribosome Subunits, Large, Archaeal chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytochrome P-450 CYP3A MeSH
• Enzymes MeSH
• Ion Channels 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
• Aryl Hydrocarbon Hydroxylases chemistry   metabolism   MeSH
• Cytochrome P-450 CYP2C9 MeSH
• Phosphatidylcholines chemistry   metabolism   MeSH
• Ibuprofen chemistry   metabolism   MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Membranes, Artificial * MeSH
• Models, Molecular MeSH
• Surface Properties MeSH
• Molecular Dynamics Simulation MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1,2-oleoylphosphatidylcholine MeSH Browser
• Aryl Hydrocarbon Hydroxylases MeSH
• CYP2C9 protein, human MeSH Browser
• Cytochrome P-450 CYP2C9 MeSH
• Phosphatidylcholines MeSH
• Ibuprofen MeSH
• Membranes, Artificial * MeSH