RESUMEN
P450nor is a heme-containing enzyme that catalyzes the conversion of nitric oxide (NO) to nitrous oxide (N2O). Its catalytic mechanism has attracted attention in chemistry, biology, and environmental engineering. The catalytic cycle of P450nor is proposed to consist of three major steps. The reaction mechanism for the last step, N2O generation, remains unknown. In this study, the reaction pathway of the N2O generation from the intermediate I was explored with the B3LYP calculations using an active center model after the examination of the validity of the model. In the validation, we compared the heme distortions between P450nor and other oxidoreductases, suggesting a small effect of protein environment on the N2O generation reaction in P450nor. We then evaluated the electrostatic environment effect of P450nor on the hydride affinity to the active site with quantum mechanics/molecular mechanics (QM/MM) calculations, confirming that the affinity was unchanged with or without the protein environment. The active center model for P450nor showed that the N2O generation process in the enzymatic reaction undergoes a reasonable barrier height without protein environment. Consequently, our findings strongly suggest that the N2O generation reaction from the intermediate I depends sorely on the intrinsic reactivity of the heme cofactor bound on cysteine residue.
Asunto(s)
Óxido Nítrico , Oxidorreductasas , Oxidorreductasas/metabolismo , Óxido Nítrico/metabolismo , Óxido Nitroso/metabolismo , Simulación de Dinámica Molecular , HemoRESUMEN
Heme proteins play diverse and important biological roles, from electron transfer and chemical catalysis to oxygen transport and/or storage. Although the distortion of heme porphyrin correlates with the physical properties of heme, such as the redox potential and oxygen affinity, the relationship between heme distortion and the heme protein environment is unclear. Here, we tested the hypothesis that the protein environment of the heme-binding pocket determines heme distortion (conformation). We analyzed the correlations between the amino acid composition of the heme-binding pocket and the magnitude of heme distortion along 12 vibrational modes using machine learning. A correlation was detected in the three lowest vibrational modes. Analysis of heme distortions in nearly the same environments of the heme-binding pocket supported this notion. Our analyses indicate that the heme-binding pocket environment is a major factor impacting the distortion of heme porphyrin along the three lowest vibrational modes. In addition, statistical analysis of the distortion of heme porphyrin revealed that the peaks of distributions of the ruffling and breathing distortions are shifted from 0 (the equilibrium structure). Both the ruffling and breathing distortions are correlated with the redox potential of heme, so that heme molecules with these distortions have a lower redox potential than planar molecules. These findings explain the structure-function relationship of heme.
Asunto(s)
Hemo , Hemoproteínas , Transporte de Electrón , Hemo/metabolismo , Hemoproteínas/química , Hemoproteínas/metabolismo , Conformación Molecular , VibraciónRESUMEN
Hydrogen-bond (H-bond) energies in 310-helices of short alanine peptides were systematically examined by precise DFT calculations with the negative fragmentation approach (NFA), a modified method based on the molecular tailoring approach. The contribution of each H-bond was evaluated in detail from the 310-helical conformation of total energies (whole helical model, WH3-10 model), and the results were compared with the property of H-bond in α-helix from our previous study. The H-bond energies of the WH3-10 model exhibited tendencies different from those exhibited by the α-helix in that they depended on the helical position of the relevant H-bond pair. H-bond pairs adjacent to the terminal H-bond pairs were observed to be strongly destabilized. The analysis of electronic structures indicated that structural characteristics cause the destabilization of the H-bond in 310-helices. We also found that the longer the helix length, the more stable the H-bond in the terminal pairs of the WH3-10 model, suggesting the action of H-bond cooperativity.
Asunto(s)
Péptidos , Teoría Funcional de la Densidad , Enlace de Hidrógeno , Modelos Moleculares , Péptidos/química , Conformación Proteica en Hélice alfaRESUMEN
"Plant-type" ferredoxins (Fds) in the thylakoid membranes of plants, algae, and cyanobacteria possess a single [2Fe-2S] cluster in active sites and mediate light-induced electron transfer from Photosystem I reaction centers to various Fd-dependent enzymes. Structural knowledge of plant-type Fds is relatively limited to static structures, and the detailed behavior of oxidized and reduced Fds has not been fully elucidated. It is important that the investigations of the effects of active-center reduction on the structures and dynamics for elucidating electron-transfer mechanisms. In this study, model systems of oxidized and reduced Fds were constructed from the high-resolution crystal structure of Chlamydomonas reinhardtii Fd1, and three 200 ns molecular dynamics simulations were performed for each system. The force field parameters of the oxidized and reduced active centers were independently obtained using quantum chemical calculations. There were no substantial differences in the global conformations of the oxidized and reduced forms. In contrast, active-center reduction affected the hydrogen-bond network and compactness of the surrounding residues, leading to the increased flexibility of the side chain of Phe61, which is essential for the interaction between Fd and the target protein. These computational results will provide insight into the electron-transfer mechanisms in the Fds.
Asunto(s)
Cianobacterias , Ferredoxinas , Ferredoxinas/metabolismo , Simulación de Dinámica Molecular , Transporte de Electrón , Cianobacterias/metabolismo , Plantas/metabolismo , Oxidación-ReducciónRESUMEN
Hydrogen-bond (H-bond) interaction energies in α-helices of short alanine peptides were systematically examined by precise density functional theory calculations, followed by a molecular tailoring approach. The contribution of each H-bond interaction in α-helices was estimated in detail from the entire conformation energies, and the results were compared with those in the minimal H-bond models, in which only H-bond donors and acceptors exist with the capping methyl groups. The former interaction energies were always significantly weaker than the latter energies, when the same geometries of the H-bond donors and acceptors were applied. The chemical origin of this phenomenon was investigated by analyzing the differences among the electronic structures of the local peptide backbones of the α-helices and those of the minimal H-bond models. Consequently, we found that the reduced H-bond energy originated from the depolarizations of both the H-bond donor and acceptor groups, due to the repulsive interactions with the neighboring polar peptide groups in the α-helix backbone. The classical force fields provide similar H-bond energies to those in the minimal H-bond models, which ignore the current depolarization effect, and thus they overestimate the actual H-bond energies in α-helices. © 2019 The Authors. Journal of Computational Chemistry published by Wiley Periodicals, Inc.
Asunto(s)
Alanina/química , Péptidos/química , Teoría Funcional de la Densidad , Enlace de Hidrógeno , Modelos Moleculares , Conformación Proteica en Hélice alfaRESUMEN
BACKGROUND: Maleness in mammals is genetically determined by the Y chromosome. On the Y chromosome SRY is known as the mammalian male-determining gene. Both placental mammals (Eutheria) and marsupial mammals (Metatheria) have SRY genes. However, only eutherian SRY genes have been empirically examined by functional analyses, and the involvement of marsupial SRY in male gonad development remains speculative. RESULTS: In order to demonstrate that the marsupial SRY gene is similar to the eutherian SRY gene in function, we first examined the sequence differences between marsupial and eutherian SRY genes. Then, using a parsimony method, we identify 7 marsupial-specific ancestral substitutions, 13 eutherian-specific ancestral substitutions, and 4 substitutions that occurred at the stem lineage of therian SRY genes. A literature search and molecular dynamics computational simulations support that the lineage-specific ancestral substitutions might be involved with the functional differentiation between marsupial and eutherian SRY genes. To address the function of the marsupial SRY gene in male determination, we performed luciferase assays on the testis enhancer of Sox9 core (TESCO) using the marsupial SRY. The functional assay shows that marsupial SRY gene can weakly up-regulate the luciferase expression via TESCO. CONCLUSIONS: Despite the sequence differences between the marsupial and eutherian SRY genes, our functional assay indicates that the marsupial SRY gene regulates SOX9 as a transcription factor in a similar way to the eutherian SRY gene. Our results suggest that SRY genes obtained the function of male determination in the common ancestor of Theria (placental mammals and marsupials). This suggests that the marsupial SRY gene has a function in male determination, but additional experiments are needed to be conclusive.
Asunto(s)
Evolución Molecular , Genes sry , Marsupiales/genética , Procesos de Determinación del Sexo/genética , Secuencia de Aminoácidos , Animales , ADN/metabolismo , Genes Reporteros , Luciferasas/metabolismo , Masculino , Filogenia , Unión Proteica , TermodinámicaRESUMEN
The replica exchange method (REM) is a powerful tool for the conformational sampling of biomolecules. In this study, we propose an enhanced exchange algorithm for REM not meeting the detailed balance condition (DBC), but satisfying the balance condition in all considered exchanges between two replicas. Breaking the DBC can minimize the rejection rate and make an exchange process rejection-free as the number of replicas increases. To enhance the efficiency of REM, all possible pairs--not only the nearest neighbor--were considered in the exchange process. The test simulations of the alanine dipeptide confirmed the correctness of our method. The average traveling distance of each replica in the temperature distribution was also increased in proportion to an increase in the exchange rate. Furthermore, we applied our algorithm to the conformational sampling of the 10-residue miniprotein, chignolin, with an implicit solvent model. The results showed a faster convergence in the calculation of its free energy landscape, compared to that achieved using the normal exchange method of adjacent pairs. This algorithm can also be applied to the conventional near neighbor method and is expected to reduce the required number of replicas.
Asunto(s)
Alanina/química , Algoritmos , Dipéptidos/química , Oligopéptidos/químicaRESUMEN
Structure-function relationships in proteins have been one of the crucial scientific topics in recent research. Heme proteins have diverse and pivotal biological functions. Therefore, clarifying their structure-function correlation is significant to understand their functional mechanism and is informative for various fields of science. In this study, we constructed convolutional neural network models for predicting protein functions from the tertiary structures of heme-binding sites (active sites) of heme proteins to examine the structure-function correlation. As a result, we succeeded in the classification of oxygen-binding protein (OB), oxidoreductase (OR), proteins with both functions (OB-OR), and electron transport protein (ET) with high accuracy. Although the misclassification rate for OR and ET was high, the rates between OB and ET and between OB and OR were almost zero, indicating that the prediction model works well between protein groups with quite different functions. However, predicting the function of proteins modified with amino acid mutation(s) remains a challenge. Our findings indicate a structure-function correlation in the active site of heme proteins. This study is expected to be applied to the prediction of more detailed protein functions such as catalytic reactions.
Asunto(s)
Hemoproteínas , Hemoproteínas/genética , Dominio Catalítico , Redes Neurales de la Computación , Sitios de Unión , AminoácidosRESUMEN
Heme is located in the active site of proteins and has diverse and important biological functions, such as electron transfer and oxygen transport and/or storage. The distortion of heme porphyrin is considered an important factor for the diverse functions of heme because it correlates with the physical properties of heme, such as oxygen affinity and redox potential. Therefore, clarification of the relationship between heme distortion and the protein environment is crucial in protein science. Here, we analyzed the fluctuation in heme distortion in the protein environment for hemoglobin and myoglobin using molecular dynamics (MD) simulations and quantum mechanical (QM) calculations as well as statistical analysis of the protein structures of hemoglobin and myoglobin stored in Protein Data Bank. Our computation and statistical analysis showed that the protein environment for hemoglobin and myoglobin prominently affects the doming distortion of heme porphyrin, which correlates with its oxygen affinity, and that the magnitude of distortion is different between hemoglobin and myoglobin. These results suggest that heme distortion is affected by its protein environment and fluctuates around its fitted conformation, leading to physical properties that are appropriate for protein functions.
RESUMEN
We present a brief review of our recent computational studies of hydrogen bonds (H-bonds) in helical secondary structures of proteins, α-helix and 310-helix, using a Negative Fragmentation Approach with density functional theory. We found that the depolarized electronic structures of the carbonyl oxygen of the ith residue and the amide hydrogen of the (i + 4)th residue cause weaker H-bond in an α-helix than in an isolated H-bond. Our calculations showed that the H-bond energies in the 310-helix were also weaker than those of the isolated H-bonds. In the 310-helices, the adjacent N-H group at the (i + 1)th residue was closer to the C=O group of the H-bond pair than the adjacent C=O group in the 310-helices, whereas the adjacent C=O group at the (i + 1)th residue was close to the H-bond acceptor in α-helices. Therefore, the destabilization of the H-bond is attributed to the depolarization caused by the adjacent residue of the helical backbone connecting the H-bond donor and acceptor. The differences in the change in electron density revealed that such depolarizations were caused by the local electronic interactions in their neighborhood inside the helical structure and redistributed the electron density. We also present the improvements in the force field of classical molecular simulation, based on our findings. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-022-01034-5.
RESUMEN
Heme proteins serve diverse and pivotal biological functions. Therefore, clarifying the mechanisms of these diverse functions of heme is a crucial scientific topic. Distortion of heme porphyrin is one of the key factors regulating the chemical properties of heme. Here, we constructed convolutional neural network models for predicting heme distortion from the tertiary structure of the heme-binding pocket to examine their correlation. For saddling, ruffling, doming, and waving distortions, the experimental structure and predicted values were closely correlated. Furthermore, we assessed the correlation between the cavity shape and molecular structure of heme and demonstrated that hemes in protein pockets with similar structures exhibit near-identical structures, indicating the regulation of heme distortion through the protein environment. These findings indicate that the tertiary structure of the heme-binding pocket is one of the factors regulating the distortion of heme porphyrin, thereby controlling the chemical properties of heme relevant to the protein function; this implies a structure-function correlation in heme proteins.
Asunto(s)
Hemoproteínas , Porfirinas , Hemo/metabolismo , Estructura Molecular , Redes Neurales de la Computación , Porfirinas/químicaRESUMEN
Heme participates in a wide range of biological functions such as oxygen transport, electron transport, oxygen reduction, transcriptional regulation and so on. While the mechanism of each function has been investigated for many heme proteins, the origin of the diversity of the heme functions is still unclear and a crucial scientific issue. We have constructed a database of heme proteins, named Python-based database and analyzer for DIStortion of Heme porphyrin (PyDISH), which also contains some analysis tools. The aim of PyDISH is to integrate the information on the structures of hemes and heme proteins and the functions of heme proteins. This database will provide the structure-function relationships focusing on heme porphyrin distortion and lead to the elucidation of the origin of the functional diversity of heme proteins. In addition, the insights obtained from the database can be used for the design of protein function. PyDISH contains the structural data of more than 13 000 hemes extracted from the Protein Data Bank, including heme porphyrin distortion, axial ligands coordinating to the heme and the orientation of the propionate sidechains of heme. PyDISH also has information about the protein domains, including Uniprot ID, protein fold by CATH ID, organism, coordination distance and so on. The analytical tools implemented in PyDISH allow users to not only browse and download the data but also analyze the structures of heme porphyrin by using the analytical tools implemented in PyDISH. PyDISH users will be able to utilize the obtained results for the design of protein function. Database URL: http://pydish.bio.info.hiroshima-cu.ac.jp/.
RESUMEN
PG16 is a broadly neutralizing antibody to the human immunodeficiency virus (HIV). A crystal structure of PG16 revealed that the unusually long 28-residue complementarity determining region (CDR) H3 forms a unique subdomain, referred to as a "hammerhead", that directly contacts the antigen. The hammerhead apparently governs the function of PG16 while a previous experimental assay showed that the mutation of TyrH100Q to Ala, which does not directly contact the antigen, decreased the neutralization ability of PG16. However, the molecular mechanism by which a remote mutation from the hammerhead or contact paratope affects the neutralization potency has remained unclear. Here, we performed molecular dynamics simulations of the wild-type and variants (TyrH100Q to Ala, and TyrH100Q to Phe) of PG16, to clarify the effects of these mutations on the dynamics of CDR-H3. Our simulations revealed that the structural rigidity of the CDR-H3 in PG16 is attributable to the hydrogen bond interaction between TyrH100Q and ProH99, as well as the steric support by TyrH100Q. The loss of both interactions increases the intrinsic fluctuations of the CDR-H3 in PG16, leading to a conformational transition of CDR-H3 toward an inactive state.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Antígenos/inmunología , Regiones Determinantes de Complementariedad/inmunología , Anticuerpos Anti-VIH/inmunología , Simulación de Dinámica Molecular , Mutación , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/metabolismo , Antígenos/genética , Antígenos/metabolismo , Sitios de Unión de Anticuerpos/genética , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/metabolismo , Cristalografía por Rayos X , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/metabolismo , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/genética , VIH-1/inmunología , VIH-1/fisiología , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Conformación ProteicaRESUMEN
Voltage-gated potassium channels play crucial roles in regulating membrane potential. They are activated by membrane depolarization, allowing the selective permeation of K+ ions across the plasma membrane, and enter a nonconducting state after lasting depolarization, a process known as inactivation. Inactivation in voltage-activated potassium channels occurs through two distinct mechanisms, N-type and C-type inactivation. C-type inactivation is caused by conformational changes in the extracellular mouth of the channel, whereas N-type inactivation is elicited by changes in the cytoplasmic mouth of the protein. The W434F-mutated Shaker channel is known as a nonconducting mutant and is in a C-type inactivation state at a depolarizing membrane potential. To clarify the structural properties of C-type inactivated protein, we performed molecular dynamics simulations of the wild-type and W366F (corresponding to W434F in Shaker) mutant of the Kv1.2-2.1 chimera channel. The W366F mutant was in a nearly nonconducting state with a depolarizing voltage and recovered from inactivation with a reverse voltage. Our simulations and three-dimensional reference interaction site model analysis suggested that structural changes in the selectivity filter upon membrane depolarization trap K+ ions around the inner mouth of the selectivity filter and prevent ion permeation. This pore restriction is involved in the molecular mechanism of C-type inactivation.
Asunto(s)
Canal de Potasio Kv.1.2/metabolismo , Animales , Canal de Potasio Kv.1.2/química , Canal de Potasio Kv.1.2/genética , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Mutación , Fosfatidiletanolaminas/química , Potasio/química , Potasio/metabolismo , Unión Proteica , Conformación Proteica , Ratas , Agua/químicaRESUMEN
The voltage-gated proton channel (Hv1/VSOP) is inhibited by Zn2+, of which the binding site is located in the extracellular region. We utilized attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to examine the coordination structure by monitoring protein structural changes induced by Zn2+-binding. The Zn2+-induced difference ATR-FTIR spectra of Hv1 showed IR features that can be assigned to the histidine C5-N1 and carboxylate-COO- stretches as well as amide I changes likely in α-helical peptide bonds. Analysis of vibrational frequencies indicated that the Zn2+ is coordinated by the anionic carboxylate with monodentate mode and by the histidine at N1 (Nτ) position of the neutral imidazole form. Combined with quantum chemical calculations, the most probable coordination structure was proposed as a tetrahedral geometry with ligands of carboxylate and imidazole groups in addition to a water molecule.
RESUMEN
By clustering various ion channels and transporters, ankyrin-G (AnkG) configures the membrane-excitation platforms in neurons and cardiomyocytes. AnkG itself localizes to specific areas on the plasma membrane via s-palmitoylation of Cys. However, the structural mechanism by which AnkG anchors to the membrane is not understood. In this study, we solved the crystal structures of the reduced and oxidized forms of the AnkG s-palmitoylation domain and used multiple long-term coarse-grained molecular dynamics simulations to analyze their membrane association. Here we report that the membrane anchoring of AnkG was facilitated by s-palmitoylation, defining a stable binding interface on the lipid membrane, and that AnkG without s-palmitoylation also preferred to stay near the membrane but did not have a unique binding interface. This suggests that AnkG in the juxtamembrane region is primed to accept lipid modification at Cys, and once that happens AnkG constitutes a rigid structural base upon which a membrane-excitation platform can be assembled.
Asunto(s)
Ancirinas/química , Lipoilación/fisiología , Animales , Axones/metabolismo , Membrana Celular/química , Cromatografía en Gel , Cristalografía por Rayos X , Cisteína/química , Humanos , Lípidos/química , Simulación de Dinámica Molecular , Neuronas/metabolismo , Oxidación-Reducción , Oxígeno/química , Dominios Proteicos , Multimerización de Proteína , Ratas , Espectrina/metabolismoRESUMEN
The conformation and functions of proteins are closely linked, and many proteins undergo conformational changes upon ligand binding. The X-ray crystallographic studies have revealed conformational differences in proteins between the liganded and unliganded states. Currently, the conformational transitions that originate in the ligand binding are explained on the basis of two representative models, the induced-fit and preexisting equilibrium dynamics models. However, the actual dynamics of the proteins remain ambiguous. Though these two models are the extreme ones, it is important to understand the difference between these two, particularly in structural biology and medicinal chemistry studies. Here, we clarified the difference in the mechanisms responsible for the conformational changes induced in two proteins upon ligand binding by examining computationally determined free-energy profiles of the apo- and holoproteins. The lysine/arginine/ornithine-binding protein and maltose/maltodextrin-binding protein were chosen as the target proteins, and the energy profiles were generated by a molecular simulation approach. Our results revealed that fluctuations in the apo state and protein-ligand interactions both play important roles in conformational transition, and the mechanism is highly influenced by the fluctuations in the apo state, which are unique to a particular structure.