Stereochemical Quality Research Articles

A molecular dynamics and knowledge-based computational strategy to predict native-like structures of polypeptides

One of the main research problems in structural bioinformatics is the prediction of three-dimensional structures (3-D) of polypeptides or proteins. The current rate at which amino acid sequences are identified increases much faster than the 3-D protein structure determination by experimental methods, such as X-ray diffraction and NMR techniques. The determination of protein structures is both experimentally expensive and time consuming. Predicting the correct 3-D structure of a protein molecule is an intricate and arduous task. The protein structure prediction (PSP) problem is, in computational complexity theory, an NP-complete problem. In order to reduce computing time, current efforts have targeted hybridizations between ab initio and knowledge-based methods aiming at efficient prediction of the correct structure of polypeptides. In this article we present a hybrid method for the 3-D protein structure prediction problem. An artificial neural network knowledge-based method that predicts approximated 3-D protein structures is combined with an ab initio strategy. Molecular dynamics (MD) simulation is used to the refinement of the approximated 3-D protein structures. In the refinement step, global interactions between each pair of atoms in the molecule (including non-bond interactions) are evaluated. The developed MD protocol enables us to correct polypeptide torsion angles deviation from the predicted structures and improve their stereo-chemical quality. The obtained results shows that the time to predict native-like 3-D structures is considerably reduced. We test our computational strategy with four mini proteins whose sizes vary from 19 to 34 amino acid residues. The structures obtained at the end of 32.0nanoseconds (ns) of MD simulation were comparable topologically to their correspondent experimental structures.

Adenine Binding Mode Is a Key Factor in Triggering the Early Release of NADH in Coenzyme A-dependent Methylmalonate Semialdehyde Dehydrogenase

Structural dynamics associated with cofactor binding have been shown to play key roles in the catalytic mechanism of hydrolytic NAD(P)-dependent aldehyde dehydrogenases (ALDH). By contrast, no information is available for their CoA-dependent counterparts. We present here the first crystal structure of a CoA-dependent ALDH. The structure of the methylmalonate semialdehyde dehydrogenase (MSDH) from Bacillus subtilis in binary complex with NAD(+) shows that, in contrast to what is observed for hydrolytic ALDHs, the nicotinamide ring is well defined in the electron density due to direct and H(2)O-mediated hydrogen bonds with the carboxamide. The structure also reveals that a conformational isomerization of the NMNH is possible in MSDH, as shown for hydrolytic ALDHs. Finally, the adenine ring is substantially more solvent-exposed, a result that could be explained by the presence of a Val residue at position 229 in helix α(F) that reduces the depth of the binding pocket and the absence of Gly-225 at the N-terminal end of helix α(F). Substitution of glycine for Val-229 and/or insertion of a glycine residue at position 225 resulted in a significant decrease of the rate constant associated with the dissociation of NADH from the NADH/thioacylenzyme complex, thus demonstrating that the weaker stabilization of the adenine ring is a key factor in triggering the early NADH release in the MSDH-catalyzed reaction. This study provides for the first time structural insights into the mechanism whereby the cofactor binding mode is responsible at least in part for the different kinetic behaviors of the hydrolytic and CoA-dependent ALDHs.

Nicotine and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Binding and Access Channel in Human Cytochrome P450 2A6 and 2A13 Enzymes

Cytochromes P450 (CYP) from the 2A subfamily are known for their roles in the metabolism of nicotine, the addictive agent in tobacco, and activation of the tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Although both the hepatic CYP2A6 and respiratory CYP2A13 enzymes metabolize these compounds, CYP2A13 does so with much higher catalytic efficiency, but the structural basis for this has been unclear. X-ray structures of nicotine complexes with CYP2A13 (2.5 Å) and CYP2A6 (2.3 Å) yield a structural rationale for the preferential binding of nicotine to CYP2A13. Additional structures of CYP2A13 with NNK reveal either a single NNK molecule in the active site with orientations corresponding to metabolites known to form DNA adducts and initiate lung cancer (2.35 Å) or with two molecules of NNK bound (2.1 Å): one in the active site and one in a more distal staging site. Finally, in contrast to prior CYP2A structures with enclosed active sites, CYP2A13 conformations were solved that adopt both open and intermediate conformations resulting from an ∼2.5 Å movement of the F to G helices. This channel occurs in the same region where the second, distal NNK molecule is bound, suggesting that the channel may be used for ligand entry and/or exit from the active site. Altogether these structures provide multiple new snapshots of CYP2A13 conformations that assist in understanding the binding and activation of an important human carcinogen, as well as critical comparisons in the binding of nicotine, one of the most widely used and highly addictive drugs in human use.

A new crystal form of human histidine triad nucleotide-binding protein 1 (hHINT1) in complex with adenosine 5'-monophosphate at 1.38 Å resolution.

Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch of the histidine triad protein superfamily. HINT1 plays an important role in various biological processes and has been found in many species. Here, the structure of the human HINT1-adenosine 5'-monophosphate (AMP) complex at 1.38 Å resolution obtained from a new monoclinic crystal form is reported. The final structure has R(cryst) = 0.1207 (R(free) = 0.1615) and the model exhibits good stereochemical quality. Detailed analysis of the high-resolution data allowed the details of the protein structure to be updated in comparison to the previously published data.

Homology Modeling and Analysis of Structure Predictions of the Bovine Rhinitis B Virus RNA Dependent RNA Polymerase (RdRp)

Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3Dpol). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3Dpol using a combination of different computational tools. Model structures of BRBV 3Dpol were verified for their stereochemical quality and accuracy. The BRBV 3Dpol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP) and pyrophosphate (PPi) bound FMDV 3Dpol (PDB, 1U09 and 2E9Z). The closest proximity of BRBV and FMDV 3Dpol as compared to human rhinovirus type 16 (HRV-16) and rabbit hemorrhagic disease virus (RHDV) 3Dpols is also substantiated by phylogeny analysis and root-mean square deviation (RMSD) between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3Dpol compared to FMDV 3Dpol, indicate that despite a very high homology to FMDV 3Dpol, BRBV 3Dpol may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the design of structure-function interventions and identification of molecular targets for drug design applicable to Aphthovirus RdRps.

Homology Modeling and Docking Studies of Streptomyces peucetius CYP147F1 as Limonene Hydroxylase

Homology modeling of Streptomyces peucetius CYP147F1 was constructed using three cytochrome P450 structures, CYP107L1, CYPVdh, and CYPeryF, as templates. The lowest energy SPCYP147F1 model was then assessed for stereochemical quality and side-chain environment by Accelrys Discovery Studio 3.1 software. Further activesite optimization of the SPCYP147F1 was performed by molecular dynamics to generate the final SPCYP147F1 model. The substrate limonene was then docked into the model. The model-limonene complex was used to validate the active-site architecture, and functionally important residues within the substrate recognition site were identified by subsequent characterization of the secondary structure. The docking of limonene suggested that SPCYP147F1 would have broad specificity with the ligand based on the two different orientations of limonene within the active site facing to the heme. Limonene with C7 facing the heme with distance of 3.4 Angstrom from the Fe was predominant.

Homology Modeling and Molecular Docking Analysis of Streptomyces peucetius CYP125A4 as C26 Monooxygenase

Among 23 cytochrome P450s, CYP125A4 was proposed as a putative monooxygenase based on the high level of amino acid sequence homology (54% identity and 75% similarity) with the well characterized C27-steroid CYP125A1. Utilizing MTBCYP125A1 as a template, homology modeling of SPCYP125A4 was conducted by Accelrys Discovery Studio 3.1 software. The modeled SPCYP125A4 structure with lowest energy value was subsequently assessed for its stereochemical quality and side-chain environment. The final model was generated by showing its active site through the molecular dynamics. The docking of steroids showed broad specificity of SPCYP125A4 with different orientation of ligand within active site facing the heme. One poses of C27-steroid with C26 facing the heme with distance of 3.734 from the Fe were predominant.

Cryo-electron microscopy modeling by the molecular dynamics flexible fitting method.

The increasing power and popularity of cryo-electron microscopy (cryo-EM) in structural biology brought about the development of so-called hybrid methods, which permit the interpretation of cryo-EM density maps beyond their nominal resolution in terms of atomic models. The Cryo-EM Modeling Challenge 2010 is the first community effort to bring together developers of hybrid methods as well as cryo-EM experimentalists. Participating in the challenge, the molecular dynamics flexible fitting (MDFF) method was applied to a number of cryo-EM density maps. The results are described here with special emphasis on the use of symmetry-based restraints to improve the quality of atomic models derived from density maps of symmetric complexes; on a comparison of the stereochemical quality of atomic models resulting from different hybrid methods; and on application of MDFF to electron crystallography data.

Structural studies on the CXCR7 decoy receptor

Motivations. Recent studies have revealed that the chemokine CXCR7 plays an important role in cancer development. However, little is known about the effect of CXCR7 on the process of hepatocellular carcinoma (HCC) cell invasion and angiogenesis. In particular, CXCR7 was overexpressed in HCC tissues. The CXCR7-receptor is a transmembrane protein that binds two chemokines, CXCL11 and CXCL12. It has the same basic structure of the other chemokine receptors composed by 7 helices and some Cys residues within the primary sequence to stabilize the tertiary structure through intermolecular disulfide bonds. The signal transduction of chemokines occurs, usually via the G proteins whereas CXCR7 is a decoy and behaves as the receptor Duffy. In particular, it does not bind to G protein, and then does not activate its metabolic pathway. When CXCR7 binds to CXCL12, it can form homodimers or heterodimers from the moment that tends to bind also to CXCR4. In the case of heterodimer (CXCL12/CXCR4/CXCR7), CXCR7 changes the conformation of the CXCR4/G-protein complex and repealing signaling. The activation of CXCR4 by CXCL12 leads to the activation of signaling through PI3K/AKT, IP3, and MAPK pathways, that promote cell survival, proliferation and chemotaxis. In addition, the pathway of the β-arrestin may be activated by GRK that leads to the internalisation of CXCR4. When CXCR7 alloy CXCL12, the mobilization of intracellular Ca2 + classical does not occur, and the activation of β-arrestin may lead to the scavenging.CXCL12. In tumor cells CXCR7 can also activate the signaling via PLC / MAPK that increases the survival of the cells. Therefore, to study the structure-function relationships in CXCR7, we have modeled its three dimensional structure as well as studied the energetic stability of the protein by molecular dynamics simulations. Finally, its complex with CXCL11 and CXCL12 has been simulated by docking methods. Methods. The three-dimensional model of human CXCR7 was performed by a comparative modeling strategy using as template the structure of the human CXCR4, recently published, and that of bovine rhodopsin using MODELLER9v5 program. Models have been evaluated in terms of stereochemical, structural packing and energetic quality. PatchDock web server was used to model the complexes between CXCR7 and the two ligands, CXCL11 and CXCL12, using for CXCR7 our model obtained by comparative modeling, for CXCL11 and CXCL12 their experimental structures. The complexes were analyzed by ''Cocomaps Server'' to identify the amino acids at the interface and to evaluate their solvent accessibility. The presence of putative H-bonds was calculated with Hbplus program. Results. Our studies show that CXCR7 model presents seven trans-membrane helices, a long N-terminal segment and three loops (i.e. loops 1, 2 and 3) in the extracellular region and, moreover, other three loops and a C-terminal loop in the cytoplasmatic region can also be observed. This model had a Prosa Z-score of -2.19 and 86.54% of residues in most favored regions. In particular, the extracellular loop 2 and the cytoplasmatic C-terminal region comprise two short β-strands and a short helix, respectively, in agreement with the experimental structure of CXCR4. A comparison between the secondary structures of CXCR7 and CXCR4 models shows seven well conserved trans-membrane helices and only few changes in helix length. Furthermore, we have modeled two complexes between CXCR7 and two chemokines, CXCL11 and CXCL12. For these complexes, we evaluated the interaction residues, the related contact maps and the number of inter-chain H-bonds. In general, the analysis of the two complexes shows that the interacting regions are located in the N-terminal region as well as in the loops 2 and 3 of CXCR7. We have also analyzed the physico-chemical properties of interaction residues of CXCR7 in the two complexes: i) some positively and negatively charged residues are present in the N-terminal while in the CXCR7/CXCL11 complex we also found also an aromatic residue; ii) the loop 2 presents a negatively charged residue (Glu) and an aromatic residue (Tyr); iii) the loop 3 shows a single aromatic residue (Phe). These observations suggest that the predominant interactions found in the models between CXCR7 and the two chemokines are on hydrophobic and electrostatic basis. In particular, CXCR7 presents the highest affinity for CXCL12 in terms of binding energy, and of number of H-bonds, of charged and hydrophobic interaction residues, and of salt bridges. Moreover, the presence of stacking interactions is also in good agreement with experimental studies which indicated that the affinity of CXCL12 for CXCR7 is approximately ten times higher than the affinity of CXCL12 for CXCR4.

Cloning, In Silico Characterization and Prediction of Three Dimensional Structure of SbDof1, SbDof19, SbDof23 and SbDof24 Proteins from Sorghum [Sorghum bicolor (L.) Moench

In the present study, four full-length Dof (DNA-binding with one finger) genes from Sorghum bicolor namely SbDof1, SbDof19, SbDof23, and SbDof24 were PCR amplified, gel eluted, cloned, and sequenced (accession number HQ540084, HQ540085, HQ540086, and HQ540087, respectively). These sequences were further characterized in silico by subjecting them to homology search, multiple sequence alignment, phylogenetic tree construction, and protein functional analysis, revealing their identity to Dof like proteins. Phylogenetic analysis of cloned SbDof genes along with other reported Dof proteins revealed existence of two major groups A and B, while group A was further bifurcated into two sub-groups (viz., I and II). Motif scan analysis of SbDof proteins revealed the presence of glycine- and alanine-rich profiles in SbDof1, while proline-rich profile was observed in SbDof23. Asparagines, methionine, and serine-rich profiles were common in case of both SbDof19 and SbDof24 proteins. The three dimensional structures of SbDof proteins were predicted by I-TASSER server based on multiple threading method. The modeled structures were refined by energy minimization and their stereo chemical qualities were validated by PROCHECK and QMEAN server indicating the acceptability of the predicted models. The final models were submitted to PMDB database with assigned PMDB IDs, i.e., PM0077395, PM0077396, PM0077397, PM0077398, and PM0076448 for SbDof1, SbDof19, SbDof23, SbDof24, and Dof domain, respectively. Based on gene ontology (GO) terms in I-TASSER server putative functions of modeled SbDof proteins were also predicted.

Analyzing a potential drug target N-myristoyltransferase of Plasmodium falciparum through in silico approaches

Background:Despite concerted global efforts to combat malaria, malaria elimination is still a remote dream. Fast evolution rate of malarial parasite along with its ability to respond quickly to any drug resulting in partial or complete resistance has been a cause of concern among researcher communities.Materials and Methods:Molecular modeling approach was adopted to gain insight about the structure and various analyses were performed. Modeller 9v3, Protparam, Protscale, MEME, NAMD and other tools were employed for this study. PROCHECK and other tools were used for stereo-chemical quality evaluation.Results and Conclusion:It was observed during the course of study that this protein contains 32.2% of aliphatic amino acids among which Leucine (9.5%) is predominant. Theoretical pI of 8.39 identified the protein as basic in nature and most of the amino acids present in N-Myristoyltransferase are hydrophobic (46.1%). Secondary structure analysis shows predominance of alpha helices and random coils. Motif analyses revealed that this target protein contains 2 signature motifs, i.e., EVNFLCVHK and KFGEGDG. Apart from motif search, three-dimensional model was generated and validated and the stereo-chemical quality check confirmed that 97.7% amino acid residues fall in the core region of Ramachandran plot. Molecular dynamics simulation resulted in maximum 1.3 Å Root Mean Square Deviation (RMSD) between the initial structure and the trajectories obtained later on. The template and the target molecule has shown 1.5 Å RMSD for the C alpha trace. A docking study was also conducted with various ligand molecules among which specific benzofuran compounds turned out to be effective. This derived information will help in designing new inhibitor molecules for this target protein as well in better understanding the parasite protein.

MOLECULAR MODELLING OF RNASES FROM ALMOND INVOLVED IN SELF-INCOMPATIBILITY

MOLECULAR MODELLING OF RNASES FROM ALMOND INVOLVED IN SELF-INCOMPATIBILITY

Homology modeling and docking study of recent SHV type β-lactamses with traditional and novel inhibitors: an in silico approach to combat problem of multiple drug resistance in various infections

Extended-spectrum β-lactamases (ESBLs) are enzymes produced by bacteria, which impart resistance against advanced generation-cephalosporins. Sulfhydryl reagent variable (SHV) enzymes are among the most prevalent ESBLs. The molecular interactions involved in the binding of recent SHV-variants like SHV-48, SHV-61, SHV-89, SHV-95 or SHV-105 to β-lactamase inhibitors have not been reported yet. The crystal structures of all SHV enzymes taken in this study are not available; homology modeling was used to generate three dimensional structure of these SHV enzymes, the models generated were subjected for model validation to confirm the stereo chemical quality of these models. Some of the most effective novel and traditional β-lactamase inhibitors were selected to target the active site amino acid residues of these modeled SHV enzymes for predicting comparative efficacies of these inhibitors against the said enzymes on the basis of interaction energies of docking. All SHV-variants were found to be interacted with clavulanic acid through A237, T235, K73, and S70 as the common residues in this study. Out of 20 docking interactions studied, only residues A237 and T235 were found crucial for the correct positioning of inhibitors within the binding site of SHV enzymes in 13 and 10 instances, respectively. On the basis of interaction energy and Ki (inhibitor constant) calculations, Ln-1255 appeared as the most efficient inhibitor against all the studied bacterial enzymes except for SHV-61, which was best inhibited by Penem2, and which was also found to be a promising inhibitor against SHV type betalactamases enzymes. Furthermore, this study provides new insights to understand crucial amino acids residues for ‘SHV–inhibitor’ interactions, which might be useful in designing new versatile SHV-inhibitors. Moreover, empirical treatment can be improved by knowing the efficacies of specific inhibitor against different variants.

Homology modeling, molecular dynamics, and molecular docking studies of Trichomonas vaginalis carbamate kinase

Carbamate kinase catalyzes the reversible reaction of carbamoyl phosphate, ADP to ATP and ammonium phosphate which is then hydrolyzed to ammonia and carbonate. In this study, the three-dimensional model of TvCK has been constructed by molecular modeling studies. The models were further energy minimized and refined. The best model was selected based on the stereochemical quality (PROCHECK, ProSA, and Verify-3D). High throughput virtual screening was carried out against TOS Lab and MayBridge database. The 10 hit compounds were selected for further rigid and flexible docking studies. The compound ID’s 13288 (TOS Lab collections), 6843 and 10949 (MayBridge) show the best docking scores and binds within the predicted active site of TvCK. The in silico ADME and biological activity properties of these compounds are in acceptable range. Thus, these types of compounds could bind to the active site of TvCK. Hence, this study may play a guiding role in in vitro and in vivo studies.

High-resolution X-ray structure of the rabbit histidine triad nucleotide-binding protein 1 (rHINT1)–adenosine complex at 1.10 Å resolution

Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch in the histidine-triad protein superfamily. HINT1 plays an important role in various biological processes and has been found in many species. Here, the first complete structure of the rabbit HINT1-adenosine complex is reported at 1.10 Å resolution, which is one of the highest resolutions obtained for a HINT1 structure. The final structure has an R(cryst) of 14.25% (R(free) = 16.77%) and the model exhibits good stereochemical qualities. A detailed analysis of the atomic resolution data allowed an update of the details of the protein structure in comparison to previously published data.

Identification of B Cell Epitopes of Alcohol Dehydrogenase Allergen of Curvularia lunata

Background/ObjectiveEpitope identification assists in developing molecules for clinical applications and is useful in defining molecular features of allergens for understanding structure/function relationship. The present study was aimed to identify the B cell epitopes of alcohol dehydrogenase (ADH) allergen from Curvularia lunata using in-silico methods and immunoassay.MethodB cell epitopes of ADH were predicted by sequence and structure based methods and protein-protein interaction tools while T cell epitopes by inhibitory concentration and binding score methods. The epitopes were superimposed on a three dimensional model of ADH generated by homology modeling and analyzed for antigenic characteristics. Peptides corresponding to predicted epitopes were synthesized and immunoreactivity assessed by ELISA using individual and pooled patients' sera.ResultThe homology model showed GroES like catalytic domain joined to Rossmann superfamily domain by an alpha helix. Stereochemical quality was confirmed by Procheck which showed 90% residues in most favorable region of Ramachandran plot while Errat gave a quality score of 92.733%. Six B cell (P1–P6) and four T cell (P7–P10) epitopes were predicted by a combination of methods. Peptide P2 (epitope P2) showed E(X)2GGP(X)3KKI conserved pattern among allergens of pathogenesis related family. It was predicted as high affinity binder based on electronegativity and low hydrophobicity. The computational methods employed were validated using Bet v 1 and Der p 2 allergens where 67% and 60% of the epitope residues were predicted correctly. Among B cell epitopes, Peptide P2 showed maximum IgE binding with individual and pooled patients' sera (mean OD 0.604±0.059 and 0.506±0.0035, respectively) followed by P1, P4 and P3 epitopes. All T cell epitopes showed lower IgE binding.ConclusionFour B cell epitopes of C. lunata ADH were identified. Peptide P2 can serve as a potential candidate for diagnosis of allergic diseases.

Protein-protein docking on molecular models of Aspergillus niger RNase and human actin: novel target for anticancer therapeutics

The 3D models of human actin protein and A.niger RNase were designed using the templates ACTBIND (PDB ID: 3D3Z) and crystalline profilin-beta-actin (PDB ID: 2BTF), respectively in Modeller9v5. These models are testified using several validation methods including PROCHECK, ERRAT, WHAT-IF, PROSA2003 and VERIFY-3D. The stereo-chemical quality of the models was judged by Ramachandran plot with PROCHECK. The total quality G-factor -0.2, shows a good quality model. The ERRAT score for the human actin and A.niger RNase models are 86.104 and 84.615, respectively, fit well within the range of a high quality model. The ERRAT score for the templates 2BTF and 3D3Z are 91.111 and 97.391, respectively. The WHAT-IF evaluation justifies a reasonable homology model structure as none of the scores for each residue in the homology model is lower than -5.0. The energy-minimized model of human actin with PROSA reveals the Z-score value -10.52 between native conformations of the crystal structures. The VERIFY 3D average score is 0.36. All evidence suggests that the geometric quality of the backbone conformation, the residue interaction, the residue contact and the energy profile of the structures were well within the limits of reliable structures. The interaction energy of docking was calculated using the HEX server. The Etotal, lowest docked energy, and calculated RMSD values were -1.608 kcal mol(-1), -8.369 kcal mol(-1) and 0.617 Å, respectively. The study presented in the current project may be useful to design molecules that may have anticancer activity.

Comparative modeling of 25-hydroxycholesterol-7α-hydroxylase (CYP7B1): ligand binding and analysis of hereditary spastic paraplegia type 5 CYP7B1 mutations

CYP7B1 mutations have been linked directly with the neurodegenerative disease hereditary spastic paraplegia (HSP), with mutations in the CYP7B1 gene identified as being directly responsible for autosomal recessive HSP type 5A (SPG5). To evaluate the potential impact of CYP7B1 mutations identified in SPG5 on binding and protein function, a comparative model of cytochrome P450 7B1 (CYP7B1) was constructed using human CYP7A1 as a template during model construction. The secondary structure was predicted using the PSIPRED and GOR4 prediction methods, the lowest energy CYP7B1 model was generated using MOE, and then this model was assessed in terms of stereochemical quality and the side chain environment using RAMPAGE, Verify3D and ProSA. Evaluation of the active site residues of the CYP7B1 model and validation of the active site architecture were performed via molecular docking experiments: the docking of the substrates 25-hydroxycholesterol and 27-hydroxycholesterol and the inhibitor 3α-Adiol identified structurally and functionally important residues. Mutational analysis of CYP7B1 amino acid mutations related to hereditary spastic paraplegia type 5 considered phosphorylation, ligand/substrate binding and the structural roles of mutated amino acid residues, with R112, T297 and S363 mutations expected to have a direct impact on ligand binding, while mutations involving R417 would indirectly affect ligand binding as a result of impairment in catalytic function.

Solution Structures of Ca2+-CIB1 and Mg2+-CIB1 and Their Interactions with the Platelet Integrin αIIb Cytoplasmic Domain

The calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous Ca(2+)-binding protein and a specific binding partner for the platelet integrin αIIb cytoplasmic domain, which confers the key role of CIB1 in hemostasis. CIB1 is also known to be involved in apoptosis, embryogenesis, and the DNA damage response. In this study, the solution structures of both Ca(2+)-CIB1 and Mg(2+)-CIB1 were determined using solution-state NMR spectroscopy. The methyl groups of Ile, Leu, and Val were selectively protonated to compensate for the loss of protons due to deuteration. The solution structure of Ca(2+)-CIB1 possesses smaller opened EF-hands in its C-domain compared with available crystal structures. Ca(2+)-CIB1 and Mg(2+)-CIB1 have similar structures, but the N-lobe of Mg(2+)-CIB1 is slightly more opened than that of Ca(2+)-CIB1. Additional NMR experiments, such as chemical shift perturbation and methyl group solvent accessibility as measured by a nitroxide surface probe, were carried out to further characterize the structures of Ca(2+)-CIB1 and Mg(2+)-CIB1 as well as their interactions with the integrin αIIb cytoplasmic domain. NMR measurements of backbone amide proton slow motion (microsecond to millisecond) dynamics confirmed that the C-terminal helix of Ca(2+)-CIB1 is displaced upon αIIb binding. The EF-hand III of both Ca(2+)-CIB1 and Mg(2+)-CIB1 was identified to be directly involved in the interaction of CIB1 with αIIb. Together, these data illustrate that CIB1 behaves quite differently from related EF-hand regulatory calcium-binding proteins, such as calmodulin or neuronal calcium sensor proteins.

Structures of Human Golgi-resident Glutaminyl Cyclase and Its Complexes with Inhibitors Reveal a Large Loop Movement upon Inhibitor Binding

Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05-1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC.