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The coiled-coil domains of the putative yeast cargo receptors Emp46p and Emp47p (Emp46pcc and Emp47pcc) assemble into heterocomplexes at neutral pH. Upon lowering the pH, the complex dissociates and reassembles into homo-oligomers. A glutamate residue (E303) located on the hydrophobic surface of Emp46pcc serves as the pH-sensing switch for assembly and segregation, and we have suggested that its side chains are protonated in the heterocomplex, even at neutral pH. To examine this hypothesis, we constructed two structural models in which the side chains of E303 were negatively charged or protonated and analyzed the effects of these charged states on the structure of the heterocomplex using molecular dynamics (MD) simulations. The calculated structures suggested the side chains of E303 to be protonated in the heterocomplex, even at neutral pH. Based on these computational results, the pH dependence of Emp47pcc homo-oligomer assembly was experimentally modified by a glutamate mutation on its hydrophobic surface. The Q306E mutant of Emp47pcc underwent a structural transition at physiological pH. Our results suggest a method for modifying pH-dependent protein-protein interactions.

Androgen receptor (AR) has a key role in the development and progression of prostate cancer, and AR antagonists are used for its remedy. Recently, carborane derivatives, which are carbon-containing boron clusters have attracted attention as new AR ligands. Here we determined the force field parameters of 10-vertex and 12-vertex p-carborane to facilitate in silico drug design of boron clusters. Then, molecular dynamics (MD) simulations of complexes of AR-carborane derivatives were performed to evaluate the parameters and investigate the influences of carborane derivatives on the three-dimensional structure of AR. Energy profiles were obtained using quantum chemical calculations, and the force-field parameters were determined by curve fitting of the energy profiles. The results of MD simulations indicated that binding of the antagonist-BA341 changed some hydrogen-bond formations involved in the structure and location of helix 12. Those results were consistent with previously reported data. The determined parameters are also useful for refining the structure of the carborane-receptor complex obtained by docking simulations and development of new ligands with carborane cages not only for AR but also for various receptors.

Proteins of modern terrestrial organisms are composed of nearly 20 amino acids; however, the amino acid sets of primitive organisms may have contained fewer than 20 amino acids. Furthermore, the full set of 20 amino acids is not required by some proteins to encode their function. Indeed, simplified variants of Escherichia coli (E. coli) orotate phosphoribosyltransferase (OPRTase) constructed by Akanuma et al. and composed of a limited amino acid set exhibit significant catalytic activity for the growth of E. coli. However, its structural details are currently unclear. Here, we predict the structures of simplified variants of OPRTase using molecular dynamics (MD) simulations and evaluate the accuracy of the MD simulations for simplified proteins. The three-dimensional structure of the wild-type was largely maintained in the simplified variants, but differences in the catalyst loop and C-terminal helix were observed. These results are considered sufficient to elucidate the differences in catalytic activity between the wild-type and simplified OPRTase variants. Thus, using MD simulations to make structural predictions appears to be a useful strategy when investigating non-wild-type proteins composed of reduced amino acid sets.

The stereoinversion of amino acid residues in proteins is considered to trigger various age-related diseases. Serine (Ser) residues are relatively prone to stereoinversion. It is assumed that threonine (Thr) residues also undergo stereoinversion, which results in the formation of the d

The deamidation of glutamine (Gln) residues, which occurs non-enzymatically under physiological conditions, triggers protein denaturation and aggregation. Gln residues are deamidated via the cyclic glutarimide intermediates to l-α-, d-α-, l-β-, and d-β-glutamate residues. The production of these biologically uncommon amino acid residues is implicated in the pathogenesis of autoimmune diseases. The reaction rate of Gln deamidation is influenced by the C-terminal adjacent (N +1) residue and is highest in the Gln-glycine (Gly) sequence. Here, we investigated the effect of the (N + 1) Gly on the mechanism of Gln deamidation and the activation barrier using quantum chemical calculations. Energy-minima and transition-state geometries were optimized by the B3LYP density functional theory, and MP2 calculations were used to obtain the single-point energy. The calculated activation barrier (85.4 kJ mol−1) was sufficiently low for the reactions occurring under physiological conditions. Furthermore, the hydrogen bond formation between the catalytic ion and the main chain of Gly on the C-terminal side was suggested to accelerate Gln deamidation by stabilizing the transition state.

Dihydropyrimidinase (DHP) is an enzyme that catabolizes the degradation of pyrimidine and fluoropyrimidine drugs such as 5-fluorouracil. DHP deficiency triggers various clinical symptoms and increases the risk of fluoropyrimidine drug toxicity. Various pathogenic variants of DHP cause DHP deficiency, and their catalytic activities have been well studied. However, the three-dimensional structures of DHP variants have not been clarified. In this study, we investigated the effects of mutations on DHP structures using the molecular dynamics simulations. Simulations of the wild type and 10 variants were performed and compared. In the T68R, D81G, G278D, and L337P variants, the flexibilities of structures related to the interaction for oligomer formation increased in comparison with those of the wild type. W117R, T343A, and R412 M mutations affected the structures of stereochemistry gate loops or the substrate-binding pocket. The three-dimensional structures of W360R and G435R variants were suggested to collapse. On the other hand, only slight structural changes were observed in the R181W variant, whose experimentally observed activity was similar to that of the wild type. The computational results are expected to clarify the relationship between clinical mutations and structural effects of drug-metabolizing enzymes.

Recently, inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) have been proposed as potential therapeutic agents for COVID-19. Studying effects of amino acid mutations in the conformation of drug targets is necessary for anticipating drug resistance. In this study, with the structure of the SARS-CoV-2 Mpro complexed with a non-covalent inhibitor, we performed molecular dynamics (MD) simulations to determine the conformation of the complex when single amino acid residue in the active site is mutated. As a model of amino acid mutation, we constructed mutant proteins with one residue in the active site mutated to alanine. This method is called virtual alanine scan. The results of the MD simulations showed that the conformation and configuration of the ligand was changed for mutants H163A and E166A, although the structure of the whole protein and of the catalytic dyad did not change significantly, suggesting that mutations in His163 and Glu166 may be linked to drug resistance.

Isomerized aspartic acid (Asp) residues have previously been identified in various aging tissues, and are suspected to contribute to age-related diseases. Asp-residue isomerization occurs nonenzymatically under physiological conditions, resulting in the formation of three types of isomerized Asp (i.e., L-isoAsp, D-Asp, and D-isoAsp) residues. Asp-residue isomerization often accelerates protein aggregation and insolubilization, making structural biology analyses difficult. Recently, Sakaue et al. reported the synthesis of a ribonuclease A (RNase A) in which Asp121 was artificially replaced with different isomerized Asp residues, and experimentally demonstrated that the enzymatic activities of these artificial mutants were completely lost. However, their structural features have not yet been elucidated. In the present study, the three-dimensional (3D) structures of these artificial-mutant RNases A were predicted using molecular dynamics (MD) simulations. The 3D structures of wild-type and artificial-mutant RNases A were converged by 3000-ns MD simulations. Our computational data show that the structures of the active site and the formation frequencies of the appropriate catalytic dyad structures in the artificial-mutant RNases A were quite different from wild-type RNase A. These computational findings may provide an explanation for the experimental data which show that artificial-mutant RNases A lack enzymatic activity. Herein, MD simulations have been used to evaluate the influences of isomerized Asp residues on the 3D structures of proteins.

Aspartic acid (Asp) residues are prone to nonenzymatic isomerization via a succinimide (Suc) intermediate. The formation of isomerized Asp residues is considered to be associated with various age-related diseases, such as cataracts and Alzheimer&rsquo, s disease. In the present paper, we describe the reaction pathway of Suc residue formation from Asp residues catalyzed by two water molecules using the B3LYP/6-31+G(d,p) level of theory. Single-point energies were calculated using the MP2/6-311+G(d,p) level of theory. For these calculations, we used a model compound in which an Asp residue was capped with acetyl and methylamino groups on the N- and C-termini, respectively. In the aqueous phase, Suc residue formation from an Asp residue was roughly divided into three steps, namely, iminolization, cyclization, and dehydration, with the activation energy estimated to be 109 kJ mol&minus, 1. Some optimized geometries and reaction modes in the aqueous phase were observed that differed from those in the gas phase.

Deamidation of asparagine (Asn) residues is a nonenzymatic post-translational modification of proteins. Asn deamidation is associated with pathogenesis of age-related diseases and hypofunction of monoclonal antibodies. Deamidation rate is known to be affected by the residue following Asn on the carboxyl side and by secondary structure. Information about main-chain conformation of Asn residues is necessary to accurately predict deamidation rate. In this study, the effect of main-chain conformation of Asn residues on deamidation rate was computationally investigated using molecular dynamics (MD) simulations and quantum chemical calculations. The results of MD simulations for &gamma, S-crystallin suggested that frequently deamidated Asn residues have common main-chain conformations on the N-terminal side. Based on the simulated structure, initial structures for the quantum chemical calculations were constructed and optimized geometries were obtained using the B3LYP density functional method. Structures that were frequently deamidated had a lower activation energy barrier than that of the little deamidated structure. We also showed that dihydrogen phosphate and bicarbonate ions are important catalysts for deamidation of Asn residues.

In contrast with the common belief that all the amino acid residues in higher organisms are l-forms, d-amino acid residues have been recently detected in various aging tissues. Aspartic acid (Asp) residues are known to be the most prone to stereoinvert via cyclic imide intermediate. Although the glutamic acid (Glu) is similar in chemical structure to Asp, little has been reported to detect d-Glu residues in human proteins. In this study, we investigated the mechanism of the Glu-residue stereoinversion catalyzed by water molecules using B3LYP/6-31+G(d,p) density functional theory calculations. We propose that the Glu-residue stereoinversion proceeds via a cyclic imide intermediate, i.e., glutarimide (GI). All calculations were performed by using a model compound in which a Glu residue was capped with acetyl and methylamino groups on the N- and C-termini, respectively. We found that two water molecules catalyze the three steps involved in the GI formation: iminolization, cyclization, and dehydration. The activation energy required for the Glu residue to form a GI intermediate was estimated to be 32.3 kcal mol-1 , which was higher than that of the experimental Asp-residue stereoinversion. This calculation result suggests that the Glu-residue stereoinversion is not favored under the physiological condition.

Recently, stereoinversions and isomerizations of amino acid residues in the proteins of living beings have been observed. Because isomerized amino acids cause structural changes and denaturation of proteins, isomerizations of amino acid residues are suspected to cause age-related diseases. In this study, AMBER molecular force field parameters were tested by using computationally generated nonapeptides and tripeptides including stereoinverted and/or isomerized amino acid residues. Energy calculations by using density functional theory were also performed for comparison. Although the force field parameters were developed by parameter fitting for l-α-amino acids, the accuracy of the computational results for d-amino acids and β-amino acids was comparable to those for l-α-amino acids. The conformational energies for tripeptides calculated by using density functional theory were reproduced more accurately than those for nonapeptides calculated by using the molecular mechanical force field. The evaluations were performed for the ff99SB, ff03, ff12SB, and the latest ff14SB force field parameters.

The coiled-coil domains of the putative yeast cargo receptors Emp46p and Emp47p are responsible for their complex-formation in the Endoplasmic Reticulum. In vitro experiments using coiled-coil domains (Emp46pcc/47pcc) have indicated that formation of the hetero-complex is pH-dependent and that amino acid Glu303 of Emp46pcc is a key residue in this process. In this study, we investigated the effects of various mutations on complex formation and discovered the mechanism for its pH-dependency, which is that dissociation of the complex at low pH arises mainly from stabilization of Emp46pcc itself. Moreover, destabilization by the introduction of a histidine residue in Emp46pcc to repel a lysine residue in Emp47pcc, caused an upward shift in the pH profile of complex formation. Another mutation in Emp46pcc, a proline to an alanine (P291A), increased the stability of the helical structure, especially at low pH and shifted the transition pH upward. Combination of these pH-shifting mutations had an additive effect on the pH profile of complex formation. Thus, we successfully constructed coiled-coils that can react to a wide range of pH, encompassing more appropriate values for use in sensing physiological pH changes in the cell.

Computational docking programs generally predict many candidates for ligand poses, and only one or few poses should be selected for later drug design. In this study, we performed molecular dynamics (MD) simulations on 81 test sets of protein–ligand complexes and determined how well MD simulations evaluated docking poses. Our results suggest that appropriate candidate poses can be chosen from the many docking poses by using MD simulations with explicit solvents. To distinguish the appropriate pose from other candidates, 5-ns and 10-ns MD simulations were effective.

Cytochrome P450 (CYP) 2A6 is a monooxygenase involved in the metabolism of various endogenous and exogenous chemicals, such as nicotine and therapeutic drugs. The genetic polymorphisms in CYP2A6 are a cause of individual variation in smoking behavior and drug toxicities. The enzymatic activities of the allelic variants of CYP2A6 were analyzed in previous studies. However, the three-dimensional structures of the mutants were not investigated, and the mechanisms underlying activity reduction remain unknown. In this study, to investigate the structural changes involved in the reduction in enzymatic activities, we performed molecular dynamics simulations for ten allelic mutants of CYP2A6. For the calculated wild type structure, no significant structural changes were observed in comparison with the experimental structure. On the other hand, the mutations affected the interaction with heme, substrates, and the redox partner. In CYP2A6.44, a structural change in the substrate access channel was also observed. Those structural effects could explain the alteration of enzymatic activity caused by the mutations. The results of simulations provide useful information regarding the relationship between genotype and phenotype.

The molecular force field plays an important role in molecular design. The force field parameters of small molecules were obtained by applying quantum chemical calculations on a limited number of compounds. However, force field parameters for atypical compounds such as organic radicals have not been obtained yet. In this study, the energy profiles covering conformational changes of nitronyl nitroxide derivatives are calculated using quantum chemical calculations, and the force field parameters are obtained by curve fitting of the energy profiles. For calculating energy curves, we applied the hybrid density functional theory; the atomic charges of the test compounds were calculated based on the restricted electrostatic potential method. As the nitronyl nitroxide derivatives are considered to have great potential applications in biological sciences, the determined parameters are expected to be useful for the molecular design of organic radicals.

The 26S proteasome is a large protein complex, responsible for degradation of ubiquinated proteins in eukaryotic cells. Eukaryotic proteasome formation is a highly ordered process that is assisted by several assembly chaperones. The assembly of its catalytic 20S core particle depends on at least five proteasome-specific chaperones, i.e., proteasome-assembling chaperons 1-4 (PAC1-4) and proteasome maturation protein (POMP). The orthologues of yeast assembly chaperones have been structurally characterized, whereas most mammalian assembly chaperones are not. In the present study, we determined a crystal structure of human PAC4 at 1.90-A resolution. Our crystallographic data identify a hydrophobic surface that is surrounded by charged residues. The hydrophobic surface is complementary to that of its binding partner, PAC3. The surface also exhibits charge complementarity with the proteasomal α4-5 subunits. This will provide insights into human proteasome-assembling chaperones as potential anticancer drug targets.

Spontaneous cyclisation of glutamic acid (Glu) residues located at N-termini in peptides and proteins is called ‘pyroglutamylation’ and is assumed to be involved in several neurodegenerative diseases. Although it has long been believed that N-terminal Glu residues undergo pyroglutamylation enzymatically, it has recently been experimentally confirmed that nonenzymatic pyroglutamylation can proceed in some types of aqueous buffer. However, the detailed mechanism has not been proposed or investigated, and even whether some small-molecule catalysts are required for pyroglutamylation has not been clarified. Therefore, we investigated three types of pyroglutamylation mechanism of N-terminal Glu residues using quantum chemical calculations: in the absence of any catalysts, catalysed by one water molecule, and catalysed by two water molecules. All calculations were performed using N-terminal Glu residues capped with a methylamino group on the C-terminal as a model compound. Optimised energy minima and transition state geometries were obtained using the B3LYP density functional method. The pyroglutamylation mechanism is roughly divided into two steps: cyclisation and dehydration, and the calculated activation barrier was 108 and 107 kJ mol−1 in the two- and three-water-assisted pathways, respectively. The results of computational analysis suggest that water molecules can act as catalysts for pyroglutamylation. The calculated activation barrier of two-water-assisted pyroglutamylation was 108 kJ mol−1, and the results of computational analysis indicate that water molecules can act as catalysts for pyroglutamylation.

Glutamine (Gln) residues located at N-termini undergo spontaneous intramolecular cyclization, causing the formation of pyroglutamic acid (pGlu) residues. pGlu residues have been detected at the N-termini in various peptides and proteins. The formation of pGlu residues during the fermentation and purification processes of antibody drugs is one of the concerns in the design and formulation of these drugs and has been reported to proceed rapidly in a phosphate buffer. In this study, we have examined the phosphate-catalyzed mechanisms of the pGlu residue formation from N-terminal Gln residues via quantum chemical calculations using B3LYP density functional methods. Single-point energies were calculated using the second-order Moller-Plesset perturbation theory. We performed the calculations for the model compound in which an uncharged N-terminal Gln residue is capped with a methyl amino group on the C-terminal. The activation energy of the formation of pGlu residues was calculated as 83.8 kJ mol-1, which was lower than that of the typical nonenzymatic reaction of amino acid residues. In addition, the computational results indicate that the flexibility of the main and side chains in N-terminal Gln residues was necessary for the formation of pGlu residues to proceed. In the obtained pathway, inorganic phosphate species act as the catalyst by mediating the proton transfer.

The 26S proteasome is critical for the selective degradation of proteins in eukaryotic cells. This enzyme complex is composed of approximately 70 subunits, including the structurally homologous proteins &alpha, 1&ndash, &alpha, 7, which combine to form heptameric rings. The correct arrangement of these &alpha, subunits is essential for the function of the proteasome, but their assembly does not occur autonomously. Assembly of the &alpha, subunit is assisted by several chaperones, including the PAC3-PAC4 heterodimer. In this study we showed that the PAC3-PAC4 heterodimer functions as a molecular matchmaker, stabilizing the &alpha, 4-&alpha, 5-&alpha, 6 subcomplex during the assembly of the &alpha, ring. We solved a 0.96-&Aring, atomic resolution crystal structure for a PAC3 homodimer which, in conjunction with nuclear magnetic resonance (NMR) data, highlighted the mobility of the loop comprised of residues 51 to 61. Based on these structural and dynamic data, we created a three-dimensional model of the PAC3-4/&alpha, 4/&alpha, 5/&alpha, 6 quintet complex, and used this model to investigate the molecular and structural basis of the mechanism of proteasome &alpha, subunit assembly, as mediated by the PAC3-PAC4 heterodimeric chaperone. Our results provide a potential basis for the development of selective inhibitors against proteasome biogenesis.

Aspartic acid (Asp) residues are prone to non-enzymatic stereoinversion, and Asp-residue stereoinversion is believed to be mediated via a succinimide (SI) intermediate. The stereoinverted Asp residues are believed to cause several age-related diseases. However, in peptides and proteins, few studies have reported the stereoinversion of glutamic acid (Glu) residues whose structures are similar to that of Asp. We previously presumed that Glu-residue stereoinversion proceeds via a glutarimide (GI) intermediate and showed that the calculated activation barriers of SI- and GI-intermediate stereoinversion are almost equivalent in the gas phase. In this study, we investigated the stereoinversion pathways of the l-GI intermediate in the aqueous phase using B3LYP density functional methods. The calculated activation barrier of l-GI-intermediate stereoinversion in the aqueous phase was approximately 36 kcal&middot, mol&minus, 1, which was much higher than that in the gas phase. Additionally, as this activation barrier exceeded that of Asp-residue stereoinversion, it is presumed that Glu-residue stereoinversion has a lower probability of proceeding under physiological conditions than Asp-residue stereoinversion.

Uncommon crosslinked amino acids have been identified in several aging tissues and are suspected to trigger various age-related diseases. Several uncommon residues are formed when the dehydroalanine (Dha) residue undergoes a nucleophilic attack by surrounding residues. Dha residues are considered to be formed by posttranslational modification of serine (Ser) and cysteine residues. In the present study, we investigated the Dha residue formation mechanism catalyzed by dihydrogen phosphate ion (H2PO4-) using quantum chemical calculations. We obtained optimized geometries using the B3LYP density functional method and carried out single-point energy calculations using the second-order Moller-Plesset perturbation method. All calculations were performed using Ace-Ser-Nme (Ace = acetyl, Nme = methylamino) as a model compound. Results of the computational analysis suggest that the mechanism underlying the Dha residue formation from Ser consists of two steps: enolization and 1,3-elimination. The H2PO4- catalyzed both reactions as a proton-relay mediator. The calculated activation barrier for Dha residue formation was estimated as 30.4 kcal mol-1. In this pathway, the catalytic H2PO4- interacts with the Ser residue α-proton, carbonyl oxygen of Ser, and C-terminal side adjacent residues, and the calculated activation energy produced was the same as the experimentally reported value for nonenzymatic modifications of amino acid residues. Therefore, our calculation suggests that H2PO4--catalyzed Ser residue dehydration can proceed nonenzymatically.

Eukaryotic proteasomes harbor heteroheptameric &alpha, rings, each composed of seven different but homologous subunits &alpha, 1&ndash, &alpha, 7, which are correctly assembled via interactions with assembly chaperones. The human proteasome &alpha, 7 subunit is reportedly spontaneously assembled into a homotetradecameric double ring, which can be disassembled into single rings via interaction with monomeric &alpha, 6. We comprehensively characterized the oligomeric state of human proteasome &alpha, subunits and demonstrated that only the &alpha, 7 subunit exhibits this unique, self-assembling property and that not only &alpha, 6 but also &alpha, 4 can disrupt the &alpha, 7 double ring. We also demonstrated that mutationally monomerized &alpha, 7 subunits can interact with the intrinsically monomeric &alpha, 4 and &alpha, 6 subunits, thereby forming heterotetradecameric complexes with a double-ring structure. The results of this study provide additional insights into the mechanisms underlying the assembly and disassembly of proteasomal subunits, thereby offering clues for the design and creation of circularly assembled hetero-oligomers based on homo-oligomeric structural frameworks.

The isomerization rate of aspartic acid (Asp) residue is known to be affected by the three-dimensional structures of peptides and proteins. Although the isomerized Asp residues were experimentally observed, structural features which affect the isomerization cannot be elucidated sufficiently because of protein denaturation and aggregation. In this study, molecular dynamics (MD) simulations were conducted on three αA-crystallin peptides (T6, T10, and T18), each containing a single Asp residue with different isomerization rate (T18 > T6 > T10) to clarify the structural factors of Asp isomerization tendency. For MD trajectories, distances between side-chain carboxyl carbon of Asp and main-chain amide nitrogen of (n + 1) residue (Cγ–N distances), root mean square fluctuations (RMSFs), and polar surface areas for main-chain amide nitrogen of (n + 1) residues (PSAN) were calculated, because these structural features are considered to relate to the formations of cyclic imide intermediates. RMSFs and PSAN are indexes of peptide backbone flexibilities and solvent exposure of the amide nitrogen, respectively. The average Cγ–N distances of T10 was longer than those of the other two peptides. In addition, the peptide containing Asp residue with a higher isomerization rate showed higher flexibility of the peptide backbone around the Asp residue. PSAN for amide nitrogen in T18 were much larger than those of other two peptides. The computational results suggest that Asp-residue isomerization rates are affected by these factors.

In peptides and proteins, nonenzymatic deamidation of asparagine (Asn) residues can trigger some age-related diseases by disrupting the conformation of the biological proteins. In addition, Asn-residue deamidation, which has been observed in various protein preparations, is an important determinant of the quality of protein preparations. In the present study, we investigated the molecular mechanisms of Asn-residue deamidation catalysed by acetic acid, which is frequently used as a buffer in protein preparations. The calculations were conducted using an Asn residue capped with acetyl and methylamino groups on the N- and C-termini, respectively. Energy minima and transition-state geometries were optimised using B3LYP density functional theory (DFT) calculations. The relative energies of all optimised geometries obtained by the MP2 single-point energy calculations were corrected for zero-point energies calculated using the B3LYP DFT method. Asn-residue deamidation was divided roughly into two processes (cyclisation and deammoniation). Computational results indicate that cyclisation is rate-determining. A catalytic acetic acid molecule acted as a proton-transfer mediator in both processes. These results provide useful information for improving formulations of protein preparation.

In the biological proteins, aspartic acid (Asp) residues are prone to nonenzymatic isomerization via a succinimide (Suc) intermediate. Asp-residue isomerization causes the aggregation and the insolubilization of proteins, and is considered to be involved in various age-related diseases. Although Suc intermediate was considered to be formed by nucleophilic attack of the main-chain amide nitrogen of N-terminal side adjacent residue to the side-chain carboxyl carbon of Asp residue, previous studies have shown that the nucleophilic attack is more likely to proceed via iminol tautomer when the water molecules act as catalysts. However, the full pathway to Suc-intermediate formation has not been investigated, and the experimental analyses for the Asp-residue isomerization mechanism at atomic and molecular levels, such as the analysis of the transition state geometry, are difficult. In the present study, we computationally explored the full pathways for Suc-intermediate formation from Asp residues. The calculations were performed two types of reactant complexes, and all energy minima and TS geometries were optimized using B3LYP density functional methods. As a result, the SI-intermediate formation was divided into three processes, i.e., iminolization, cyclization, and dehydration processes, and the activation energies were calculated to be 26.1 or 28.4 kcal mol−1. These values reproduce the experimental data. The computational results show that abundant water molecules in living organisms are effective catalysts for the Asp-residue isomerization.

The l-α-Asp residues in peptides or proteins are prone to undergo nonenzymatic reactions to form l-β-Asp, d-α-Asp, and d-β-Asp residues via a succinimide five-membered ring intermediate. From these three types of isomerized aspartic acid residues, particularly d-β-Asp has been widely detected in aging tissue. In this study, we computationally investigated the cyclization of α- and β-Asp residues to form succinimide with dihydrogen phosphate ion as a catalyst (H2PO4-). We performed the study using B3LYP/6-31+G(d,p) density functional theory calculations. The comparison of the activation barriers of both residues is discussed. All the calculations were performed using model compounds in which an α/β-Asp-Gly sequence is capped with acetyl and methylamino groups on the N- and C-termini, respectively. Moreover, H2PO4- catalyzes all the steps of the succinimide formation (cyclization-dehydration) acting as a proton-relay mediator. The calculated activation energy barriers for succinimide formation of α- and β-Asp residues are 26.9 and 26.0kcalmol-1, respectively. Although it was experimentally confirmed that β-Asp has higher stability than α-Asp, there was no clear difference between the activation barriers. Therefore, the higher stability of β-Asp residue than α-Asp residue may be caused by an entropic effect associated with the succinimide formation.

Although various higher-order protein structure prediction methods have been developed, almost all of them were developed based on the three-dimensional (3D) structure information of known proteins. Here we predicted the short protein structures by molecular dynamics (MD) simulations in which only Newton’s equations of motion were used and 3D structural information of known proteins was not required. To evaluate the ability of MD simulationto predict protein structures, we calculated seven short test protein (10–46 residues) in the denatured state and compared their predicted and experimental structures. The predicted structure for Trp-cage (20 residues) was close to the experimental structure by 200-ns MD simulation. For proteins shorter or longer than Trp-cage, root-mean square deviation values were larger than those for Trp-cage. However, secondary structures could be reproduced by MD simulations for proteins with 10–34 residues. Simulations by replica exchange MD were performed, but the results were similar to those from normal MD simulations. These results suggest that normal MD simulations can roughly predict short protein structures and 200-ns simulations are frequently sufficient for estimating the secondary structures of protein (approximately 20 residues). Structural prediction method using only fundamental physical laws are useful for investigating non-natural proteins, such as primitive proteins and artificial proteins for peptide-based drug delivery systems.

A major form of proteasome activator PA28 is a heteroheptamer composed of interferon-γ-inducible α and β subunits, which share approximately 50% amino acid identity and possess distinct insert loops. This activator forms a complex with the 20S proteasome and thereby stimulates proteasomal degradation of peptides in an ATP-independent manner, giving rise to smaller antigenic peptides presented by major histocompatibility complex class I molecules. In this study, we performed biophysical and biochemical characterization of the structure and function of the PA28 hetero-oligomer. Deuteration-assisted small-angle neutron scattering demonstrated three α and four β subunits are alternately arranged in the heptameric ring. In this arrangement, PA28 loops surround the central pore of the heptameric ring (site for peptide entry). Activating the 20S proteasome with a PA28 mutant that lacked the α subunit loops cleaved model substrates longer than a nonapeptide with better efficiency when compared to wild-type PA28. Based on these data, we hypothesize that the flexible PA28 loops act as gatekeepers, which function to select the length of peptide substrates to be transported between the proteolytic chamber and the extra-proteasomal medium.

We developed a novel, to our knowledge, technique for real-time monitoring of subunit exchange in homooligomeric proteins, using deuteration-assisted small-angle neutron scattering (SANS), and applied it to the tetradecamer of the proteasome α7 subunit. Isotopically normal and deuterated tetradecamers exhibited identical SANS profiles in 81% D2O solution. After mixing these solutions, the isotope sensitive SANS intensity in the low-q region gradually decreased, indicating subunit exchange, whereas the small-angle x-ray scattering profile remained unchanged confirming the structural integrity of the tetradecamer particles during the exchange. Kinetic analysis of zero-angle scattering intensity indicated that 1), only two of the 14 subunits were exchanged in each tetradecamer and 2), the exchange process involves at least two steps. This study underscores the usefulness of deuteration-assisted SANS, which can provide quantitative information not only on the molecular sizes and shapes of homooligomeric proteins, but also on their kinetic properties.

Transcription of the human immunodeficiency virus type 1 (HIV-1) requires the interaction of the cyclin T1 (CycT1) subunit of a host cellular factor, positive transcription elongation factor b, with the viral Tat protein at the transactivation response (TAR) element of nascent viral transcripts. The involvement of the interaction between Tat and CycT1 is known to be through the Tat–TAR recognition motif (TRM) on CycT1. Here, we have further characterized this molecular interaction and clarified the role of the CycT1 N-terminal region in Tat action. We found crucial and distinctive roles of Q46, Q50 and F176 of human CycT1 protein in Tat-mediated transcription by creating various Ala substitution mutants of CycT1 based on its three-dimensional structure. We confirmed the involvement of these amino acid residues in binding to Tat with Q46 and Q50, and to a lesser extent with F176, by in vitro pull-down assay. Relative transactivation activities of wild-type CycT1 chimeras and mutant derivatives on the HIV-1 long terminal repeat were determined by luciferase reporter assays. Whereas CycT1 Q46A alone had impaired transcriptional activity, the CycT1(Q46A)–Tat chimeric protein retained almost full activity of the wild-type CycT1. However, CycT1 mutants (C261Y, Q50A or F176A) or their chimeric counterparts had lost the transactivation capacity. Moreover, a triple-mutant chimera containing Q46A, Q50A and F176A mutations completely abolished the transcriptional activity, indicating that these amino acid residues are involved through distinct mechanisms. These findings provide new insights for the development of anti-HIV drugs.

Protein disulfide isomerase (PDI) acts as folding catalyst and molecular chaperone for disulfide-containing proteins through the formation, breakage, and rearrangement of disulfide bonds. PDI has a modular structure comprising four thioredoxin domains, a, b, b', and a', followed by a short segment, c. The a and a' domains have an active site cysteine pair for the thiol-disulfide exchange reaction, which alters PDI between the reduced and oxidized forms, and the b' domain provides a primary binding site for substrate proteins. Although the structures and functions of PDI have studied, it is still argued whether the overall conformation of PDI depends on the redox state of the active site cysteine pair. Here, we report redox-dependent conformational and solvation changes of PDI from a thermophilic fungus elucidated by small-angle X-ray scattering (SAXS) analysis. The redox state and secondary structures of PDI were also characterized by nuclear magnetic resonance and circular dichroic spectroscopy, respectively. The oxidized form of PDI showed SAXS differences from the reduced form, and the low-resolution molecular models restored from the SAXS profiles differed between the two forms regarding the positions of the a'-c region relative to the a-b-b' region. The normal mode analysis of the crystal structure of yeast PDI revealed that the inherent motions of the a-b-b' and a'-c regions expose the substrate binding surface of the b' domain. The apparent molecular weight of the oxidized form estimated from SAXS was 1.1 times larger than that of the reduced form, whereas the radius of gyration (ca. 33 A) was nearly independent of the redox state. These results suggest that the conformation of PDI is controlled by the redox state of the active site cysteine residues in the a and a' domains and that the conformational alternation accompanies solvation changes in the active site cleft formed by the a, b, b', and a' domains. On the basis of the results presented here, we propose a mechanism explaining the observed redox-dependent conformational and solvation changes of PDI.

SummaryE2 ubiquitin-conjugating enzymes catalyze the attachment of ubiquitin to lysine residues of target proteins. The UbcH5b E2 enzyme has been shown to play a key role in the initiation of the ubiquitination of substrate proteins upon action of several E3 ligases. Here we have determined the 2.2 Å crystal structure of an intermediate of UbcH5b∼ubiquitin (Ub) conjugate, which is assembled into an infinite spiral through the backside interaction. This active complex may provide multiple E2 active sites, enabling efficient ubiquitination of substrates. Indeed, biochemical assays support a model in which the self-assembled UbcH5b∼Ub can serve as a bridge for the gap between the lysine residue of the substrate and the catalytic cysteine of E2.

Although the multi-dimensional HPLC maps of neutral, sialyl, and sulfated N-glycans have been reported and widely used for glycosylation profiling, those of glucuronyl oligosaccharides have not yet been available. In the present study, by in vitro enzymatic reactions, we prepared 55 different glucuronyl PA-oligosaccharides that include 6 kinds of HNK-1-containing N-glycans, and established their HPLC map. Furthermore, we applied this map to the characterization of branch specificity in glucuronylation reaction catalyzed by human GlcAT-S, revealing that this enzyme transfers the glucuronyl residues preferentially onto the Gal 1�4GlcNAc 1�4Man 1�3 and Gal 1�4GlcNAc 1�2Man 1�3 branches of a galactose-terminated tri-antennary oligosaccharide. The HPLC map developed in the present study will be a useful glycomics tool for identification and profiling of glucuronyl N-glycans expressed in the neural and other biological systems.

Eukaryotic 20S proteasomes are composed of two alpha-rings and two beta-rings, which form an alphabetabetaalpha stacked structure. Here we describe a proteasome-specific chaperone complex, designated Dmp1-Dmp2, in budding yeast. Dmp1-Dmp2 directly bound to the alpha5 subunit to facilitate alpha-ring formation. In Deltadmp1 cells, alpha-rings lacking alpha4 and decreased formation of 20S proteasomes were observed. Dmp1-Dmp2 interacted with proteasome precursors early during proteasome assembly and dissociated from the precursors before the formation of half-proteasomes. Notably, the crystallographic structures of Dmp1 and Dmp2 closely resemble that of PAC3-a mammalian proteasome-assembling chaperone; nonetheless, neither Dmp1 nor Dmp2 showed obvious sequence similarity to PAC3. The structure of the Dmp1-Dmp2-alpha5 complex reveals how this chaperone functions in proteasome assembly and why it dissociates from proteasome precursors before the beta-rings are assembled.

Although biological importance of intrinsically disordered proteins is becoming recognized, NMR analyses of this class of proteins remain as tasks with more challenge because of poor chemical shift dispersion. It is expected that ultra-high field NMR spectroscopy offers improved resolution to cope with this difficulty. Here, we report an ultra-high field NMR study of alpha-synuclein, an intrinsically disordered protein identified as the major component of the Lewy bodies. Based on NMR spectral data collected at a 920 MHz proton frequency, we performed epitope mapping of an anti-alpha-synuclein monoclonal antibody, and furthermore, characterized conformational effects of phosphorylation at Ser129 of alpha-synuclein.

Group II chaperonin (CPN) cooperates with prefoldin (PFD), which forms a jellyfish-shaped heterohexameric complex with a molecular mass of 87 kDa. PFD captures an unfolded protein with the tentacles and transfers it to the cavity of CPN. Although X-ray crystal structures of CPN and PFD have been reported, no structural information has been so far available for the terminal regions of the PFD tentacles nor for the C-terminal segments of CPNs, which were regarded to be functionally significant in the previous studies. Here we report 13C NMR analyses on archaeal PFD, CPN, and their complex, focusing on those structurally uncharacterized regions. The PFD and CPN complexes selectively labeled with 13C at methionyl carbonyl carbons were separately and jointly subjected to NMR measurements. 13C NMR spectral data demonstrated that the N-terminal segment of the α and β subunits of PFD as well as the C-terminal segments of the CPN hexadecamer retain significant degrees of freedom in internal motion even in the complex with a molecular mass of 1.1 MDa. Proteins 2008. © 2007 Wiley-Liss, Inc.