The PAS fold. A redefinition of the PAS domain based upon structural prediction.

In the postgenomic era it is essential that protein sequences are annotated correctly in order to help in the assignment of their putative functions. Over 1300 proteins in current protein sequence databases are predicted to contain a PAS domain based upon amino acid sequence alignments. One of the problems with the current annotation of the PAS domain is that this domain exhibits limited similarity at the amino acid sequence level. It is therefore essential, when using proteins with low-sequence similarities, to apply profile hidden Markov model searches for the PAS domain-containing proteins, as for the PFAM database. From recent 3D X-ray and NMR structures, however, PAS domains appear to have a conserved 3D fold as shown here by structural alignment of the six representative 3D-structures from the PDB database. Large-scale modelling of the PAS sequences from the PFAM database against the 3D-structures of these six structural prototypes was performed. All 3D models generated (> 5700) were evaluated using prosaii. We conclude from our large-scale modelling studies that the PAS and PAC motifs (which are separately defined in the PFAM database) are directly linked and that these two motifs form the PAS fold. The existing subdivision in PAS and PAC motifs, as used by the PFAM and SMART databases, appears to be caused by major differences in sequences in the region connecting these two motifs. This region, as has been shown by Gardner and coworkers for human PAS kinase (Amezcua, C.A., Harper, S.M., Rutter, J. & Gardner, K.H. (2002) Structure 10, 1349-1361, [1]), is very flexible and adopts different conformations depending on the bound ligand. Some PAS sequences present in the PFAM database did not produce a good structural model, even after realignment using a structure-based alignment method, suggesting that these representatives are unlikely to have a fold resembling any of the structural prototypes of the PAS domain superfamily.     

A structure-similarity-based software for the cardiovascular toxicity prediction of traditional chinese medicine

Although natural medicines are generally considered to be safer than synthetic medicines, some reports on toxicity of many herb drugs, for example Traditional Chinese Medicine (TCM), attract more and more common public attention recently. The reasons for these cardiovascular toxic effects are not always satisfactory. Not all these clinical symptoms can be observed regularly. Cardiovascular toxicity concern is one of the hindrances for TCM to enter international markets. Therefore, we create web-based software for people to evaluate TCM cardiovascular toxicity. The software, TCM Cardiovascular Toxicity Prediction (TCMCardioTox), is based on structure similarity search algorithm (http://rcdd.sysu.edu.cn:8080/home.aspx). When a user enter a TCM name, the system interprets the name as a number of active component structures, and computes the structure similarities of the components against a number of known toxiphores. If one of the similarity is greater than a given similarity threshold, the TCM will be reported as an alert TCM for medical uses.     

Information about the protein secondary structure improves quality of an alignment of protein sequences

All popular algorithms of pair-wise alignment of protein primary structures (e.g. Smith-Waterman (SW), FASTA, BLAST, et al.) utilize only amino acid sequences. The SW-algorithm is the most accurate among them, i.e. it produces alignments that are most similar to the alignments obtained by superposition of protein 3D-structures. But even the SW-algorithm is unable to restore the 3D-based alignment if similarity of amino acid sequences (%id) is below 30%. We have proposed a novel alignment method that explicitly takes into account the secondary structure of the compared proteins. We have shown that it creates significantly more accurate alignments compared to SW-algorithm. In particular, for sequences with %id < 30% the average accuracy of the new method is 58% compared to 35% for SW-algorithm (the accuracy of an algorithmic sequence alignment is the part of restored position of a "golden standard" alignment obtained by superposition of corresponding 3D-structures). The accuracy of the proposed method is approximately identical both for experimental, and for theoretically predicted secondary structures. Thus the method can be applied for alignment of protein sequences even if protein 3D-structure is unknown. The program is available at ftp://194.149.64.196/STRUSWER/.     

Structural insights into conserved l-arabinose metabolic enzymes reveal the substrate binding site of a thermophilic l-arabinose isomerase

Structural genomics demonstrates that despite low levels of structural similarity of proteins comprising a metabolic pathway, their substrate binding regions are likely to be conserved. Herein based on the 3D-structures of the α/β-fold proteins involved in the ara operon, we attempted to predict the substrate binding residues of thermophilic Geobacillus stearothermophilusl-arabinose isomerase (GSAI) with no 3D-structure available. Comparison of the structures of l-arabinose catabolic enzymes revealed a conserved feature to form the substrate-binding modules, which can be extended to predict the substrate binding site of GSAI (i.e., D195, E261 and E333). Moreover, these data implicated that proteins in the l-arabinose metabolic pathway might retain their substrate binding niches as the modular structure through conserved molecular evolution even with totally different structural scaffolds.     

大肠杆菌和志贺氏菌O-抗原糖基转移酶与聚合酶的生物信息学研究

利用生物信息学手段对大肠杆菌和志贺氏菌的 1 1 0个O 抗原糖基转移酶与 39个O 抗原聚合酶的序列进行分析 ,探讨这两种酶的序列和结构特点。统计了其序列一致性 ,密码子使用和 (G C) %含量的特点 ;讨论了O 抗原糖基转移酶和聚合酶对底物的特异性 ;推测了 6组糖基转移酶的功能 ;通过对蛋白拓扑结构的预测 ,发现O 抗原聚合酶中广泛存在一个位于细胞周质中的亲水环 (Loop) ,是可能的功能区域 ;通过对蛋白高级结构的预测 ,发现O 抗原糖基转移酶属于两个不同的蛋白超家族。     

Escherichia coli 5S RNA A and B conformers. Characterisation by enzymatic and chemical methods

The structures of the two stable conformers of Escherichia coli 5 S RNA, the and B form, were compared. Information about the structures were obtained using the methods of limited enzymatic hydrolysis and chemical modification of accessible nucleotides. Base-specific modifications were performed for adenosines and cytidines using diethylpyrocarbonate and dimethylsulfate in combination with a strand-scission reaction at the modified site. Base-specific (RNase T1) as well as conformation-specific (nuclease S1, cobra venom nuclease) enzymes were employed for the limited enzymatic hydrolysis. Clear differences in the accessibility of the two 5 S RNA conformers to the enzymes and the chemical reagents were established and the regions with altered reactivities were localized in the 5 S RNA structure. The results are consistent with the disruption of the secondary structural interactions in helix II and partly in helices III and IV during the transition from the A to the B form. (The numbering of the helices is according to the generally accepted Fox and Woese model.) In addition some regions presumably involved in the tertiary structure are distorted. There is evidence, however, for the new formation of structural regions between two distant sites in the 5 S RNA B form. The results enable us to refine the existing 5 S RNA A-form model and provide insight into the structural dynamics that lead to the formation of the 5 S RNA B form.     

Multiple templates-based homology modeling enhances structure quality of AT1 receptor: validation by molecular dynamics and antagonist docking

We present a comparative account on 3D-structures of human type-1 receptor (AT1) for angiotensin II (AngII), modeled using three different methodologies. AngII activates a wide spectrum of signaling responses via the AT1 receptor that mediates physiological control of blood pressure and diverse pathological actions in cardiovascular, renal, and other cell types. Availability of 3D-model of AT1 receptor would significantly enhance the development of new drugs for cardiovascular diseases. However, templates of AT1 receptor with low sequence similarity increase the complexity in straightforward homology modeling, and hence there is a need to evaluate different modeling methodologies in order to use the models for sensitive applications such as rational drug design. Three models were generated for AT1 receptor by, (1) homology modeling with bovine rhodopsin as template, (2) homology modeling with multiple templates and (3) threading using I-TASSER web server. Molecular dynamics (MD) simulation (15 ns) of models in explicit membrane-water system, Ramachandran plot analysis and molecular docking with antagonists led to the conclusion that multiple template-based homology modeling outweighs other methodologies for AT1 modeling.     

Drug Promiscuity in PDB: Protein Binding Site Similarity Is Key

Drug repositioning applies established drugs to new disease indications with increasing success. A pre-requisite for drug repurposing is drug promiscuity (polypharmacology) – a drug’s ability to bind to several targets. There is a long standing debate on the reasons for drug promiscuity. Based on large compound screens, hydrophobicity and molecular weight have been suggested as key reasons. However, the results are sometimes contradictory and leave space for further analysis. Protein structures offer a structural dimension to explain promiscuity: Can a drug bind multiple targets because the drug is flexible or because the targets are structurally similar or even share similar binding sites? We present a systematic study of drug promiscuity based on structural data of PDB target proteins with a set of 164 promiscuous drugs. We show that there is no correlation between the degree of promiscuity and ligand properties such as hydrophobicity or molecular weight but a weak correlation to conformational flexibility. However, we do find a correlation between promiscuity and structural similarity as well as binding site similarity of protein targets. In particular, 71% of the drugs have at least two targets with similar binding sites. In order to overcome issues in detection of remotely similar binding sites, we employed a score for binding site similarity: LigandRMSD measures the similarity of the aligned ligands and uncovers remote local similarities in proteins. It can be applied to arbitrary structural binding site alignments. Three representative examples, namely the anti-cancer drug methotrexate, the natural product quercetin and the anti-diabetic drug acarbose are discussed in detail. Our findings suggest that global structural and binding site similarity play a more important role to explain the observed drug promiscuity in the PDB than physicochemical drug properties like hydrophobicity or molecular weight. Additionally, we find ligand flexibility to have a minor influence.     

VADAR: a web server for quantitative evaluation of protein structure quality

VADAR (Volume Area Dihedral Angle Reporter) is a comprehensive web server for quantitative protein structure evaluation. It accepts Protein Data Bank (PDB) formatted files or PDB accession numbers as input and calculates, identifies, graphs, reports and/or evaluates a large number (>30) of key structural parameters both for individual residues and for the entire protein. These include excluded volume, accessible surface area, backbone and side chain dihedral angles, secondary structure, hydrogen bonding partners, hydrogen bond energies, steric quality, solvation free energy as well as local and overall fold quality. These derived parameters can be used to rapidly identify both general and residue-specific problems within newly determined protein structures. The VADAR web server is freely accessible at http://redpoll.pharmacy.ualberta.ca/vadar.     

Comparison of proteins based on segments structural similarity

We present here a simple method for fast and accurate comparison of proteins using their structures. The algorithm is based on structural alignment of segments of Calpha chains (with size of 99 or 199 residues). The method is optimized in terms of speed and accuracy. We test it on 97 representative proteins with the similarity measure based on the SCOP classification. We compare our algorithm with the LGscore2 automatic method. Our method has the same accuracy as the LGscore2 algorithm with much faster processing of the whole test set, which is promising. A second test is done using the ToolShop structure prediction evaluation program and shows that our tool is on average slightly less sensitive than the DALI server. Both algorithms give a similar number of correct models, however, the final alignment quality is better in the case of DALI. Our method was implemented under the name 3D-Hit as a web server at http://3dhit.bioinfo.pl/ free for academic use, with a weekly updated database containing a set of 5000 structures from the Protein Data Bank with non-homologous sequences.     

Side-chain conformational entropy in protein folding.

An important, but often neglected, contribution to the thermodynamics of protein folding is the loss of entropy that results from restricting the number of accessible side-chain conformers in the native structure. Conformational entropy changes can be found by comparing the number of accessible rotamers in the unfolded and folded states, or by estimating fusion entropies. Comparison of several sets of results using different techniques shows that the mean conformational free energy change (T delta S) is 1 kcal.mol-1 per side chain or 0.5 kcal.mol-1 per bond. Changes in vibrational entropy appear to be negligible compared to the entropy change resulting from the loss of accessible rotamers. Side-chain entropies can help rationalize alpha-helix propensities, predict protein/inhibitor complex structures, and account for the distribution of side chains on the protein surface or interior.     

Structural prediction and analysis of VIH-related peptides from selected crustacean species

The tentative elucidation of the 3D-structure of vitellogenesis inhibiting hormone (VIH) peptides is conversely underprivileged by difficulties in gaining enough peptide or protein, diffracting crystals, and numerous extra technical aspects. As a result, no structural information is available for VIH peptide sequences registered in the Genbank. In this situation, it is not surprising that predictive methods have achieved great interest. Here, in this study the molt-inhibiting hormone (MIH) of the kuruma prawn (Marsupenaeus japonicus) is used, to predict the structure of four VIHrelated peptides in the crustacean species. The high similarity of the 3D-structures and the calculated physiochemical characteristics of these peptides suggest a common fold for the entire family.     

Distal conformation of thyroid hormones. The crystal structure of 3,5,3'-triiodothyroacetic acid H-diethanolamine (1:1) complex

In the crystal structure of a complex of the hypocholesteremic thyromimetic agent, 3,5,3′-triiodothyroacetic acid and N-diethanolamine (1:1), the 3′-iodine is observed for the first time in the distal position, away from the α-ring of the diphenyl ether portion of a thyroactive analogue of thyroxine. This result was anticipated from stereochemical and biological activity studies. However, previous observations of structures in which the 3′-iodine was proximal had cast some doubt on the stability of the 3′-distal conformation. The present evidence now suggests that the relative energies of the two conformers are similar and that both conformers are readily accessible in solution.     

Quantification of free ligand conformational preferences by NMR and their relationship to the bioactive conformation

Accurate unbound solution 3D-structures of ligands provide unique opportunities for medicinal chemistry and, in particular, a context to understand binding thermodynamics and kinetics. Previous methods of deriving these 3D-structures have had neither the accuracy nor resolution needed for drug design and have not yet realized their potential. Here, we describe and apply a NMR methodology to the aminoglycoside streptomycin that can accurately quantify accessible 3D-space and rank the occupancy of observed conformers to a resolution that enables medicinal chemistry understanding and design. Importantly, it is based upon conventional small molecule NMR techniques and can be performed in physiologically-relevant solvents. The methodology uses multiple datasets, an order of magnitude more experimental data than previous NMR approaches and a dynamic model during refinement, is independent of computational chemistry and avoids the problem of virtual conformations. The refined set of solution 3D-shapes for streptomycin can be grouped into two major families, of which the most populated is almost identical to the 30S ribosomal subunit bioactive shape. We therefore propose that accurate unbound ligand solution conformations may, in some cases, provide a subsidiary route to bioactive shape without crystallography. This experimental technique opens up new opportunities for drug design and more so when complemented with protein co-crystal structures, SAR data and pharmacophore modeling.     

Conformational comparison of mu-selective endomorphin-2 with its C-terminal free acid in DMSO solution, by 1H NMR spectroscopy and molecular modeling calculation.

In order to make clear the structural role of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH2), an endogenous mu-receptor ligand, in the biological function, the solution conformations of endomorphin-2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH), studied using two-dimensional 1H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr-Pro omega bond in a population ratio of approximately/= 1:2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton-proton distance constraints derived from the ROE cross-peaks. These energy-minimized conformers, which were all in the phi torsion angles estimated from J(NHCalphaH) coupling constants within +/- 30 degrees, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro2-Phe3 moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the 'bow'-shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C- and N-terminal groups. These results indicate clearly that the substitution of carboxyl group for C-terminal amide group makes the peptide flexible. The conformational requirement for mu-receptor activation has been discussed based on the active form proposed for endomorphin-1 and by comparing conformational features of EM2 and EM2OH.     

The acylhalocapnines of halophilic bacteria: structural details of unusual sulfonate sphingoids

Sulfonate sphingoids or sulfonolipids are bioactive unusual compounds found in members of the Bacteroidetes family. The present report describes the structures of sulfonolipids of halophilic bacteria, sharing structural similarity with compounds of fungal origin inhibiting the serine palmitoyl transferase and with capnines, known as antagonists of von Willebrandt factor. Two sulfonolipids (SL1 and SL2) were isolated from the lipid extract of the halophile Salisaeta longa and analyzed by ESI-MS/MS. SL1 and SL2 structures have in common the long chain aminosulfonate 2-carboxy-2-amino-3,4-hydroxy-17 methyloctadec-5-ene-1-sulfonic for which the common name of halocapnine is suggested. The hydroxyl group on carbon 3 of aminosulfonate moiety is acylated: iso C15 and iso hydroxy C15 chains are present in SL1 and SL2, respectively. The levels of the two different sulfonolipids in the bacterium were found to be modulated by the proportion of sodium and magnesium ions in the environment.     

Near identity of 3′ RNA secondary structure in bromoviruses and cucumber mosaic virus

The 3′ terminal sequences of RNAs 1, 2, 3 and 4 from each of the three bromoviruses (brome mosaic, cowpea chlorotic mottle and broad bean mottle viruses) and also from cucumber mosaic virus display interviral sequence similarity in addition to strong intraviral homology. Interviral similarity is much more evident when RNA secondary, rather than primary, structures are compared. The last 190 bases of the various RNAs can fold into strikingly similar, extensively base-paired secondary structures whose common features are supported by RNA structure mapping. The extreme 3′ end of each viral RNA can base-pair in two distinct configurations. Bromovirus RNA 3s each contain an unusually accessible internal oligo(A) sequence which, in brome mosaic virus at least, is located in the intercistronic noncoding region. Functional implications of these structural features are discussed.     

Conformational interconversion in compstatin probed with molecular dynamics simulations

Compstatin is a 13-residue cyclic peptide that has the potential to become a therapeutic agent against unregulated complement activation. In our effort to understand the structural and dynamic characteristics of compstatin that form the basis for rational and combinatorial optimization of structure and activity, we performed 1-ns molecular dynamics (MD) simulations. We used as input in the MD simulations the ensemble of 21 lowest energy NMR structures, the average minimized structure, and a global optimization structure. At the end of the MD simulations we identified five conformations, with populations ranging between 9% and 44%. These conformations are as follows: 1) coil with alphaR-alphaR beta-turn, as was the conformation of the initial ensemble of NMR structures; 2) beta-hairpin with epsilon-alphaR beta-turn; 3) beta-hairpin with alphaR-alphaR beta-turn; 4) beta-hairpin with alphaR-beta beta-turn; and 5) alpha-helical. Conformational switch was possible with small amplitude backbone motions of the order of 0.1-0.4 A and free energy barrier crossing of 2-11 kcal/mol. All of the 21 MD structures corresponding to the NMR ensemble possessed a beta-turn, with 14 structures retaining the alphaR-alphaR beta-turn type, but the average minimized structure and the global optimization structures were converted to alpha-helical conformations. Overall, the MD simulations have aided to gain insight into the conformational space sampled by compstatin and have provided a measure of conformational interconversion. The calculated conformers will be useful as structural and possibly dynamic templates for optimization in the design of compstatin using structure-activity relations (SAR) or dynamics-activity relations (DAR).     

Application of biasing‐potential replica‐exchange simulations for loop modeling and refinement of proteins in explicit solvent

Comparative or homology modeling of a target protein based on sequence similarity to a protein with known structure is widely used to provide structural models of proteins. Depending on the target‐template similarity these model structures may contain regions of limited structural accuracy. In principle, molecular dynamics (MD) simulations can be used to refine protein model structures and also to model loop regions that connect structurally conserved regions but it is limited by the currently accessible simulation time scales. A recently developed biasing potential replica exchange (BP‐REMD) method was used to refine loops and complete decoy protein structures at atomic resolution including explicit solvent. In standard REMD simulations several replicas of a system are run in parallel at different temperatures allowing exchanges at preset time intervals. In a BP‐REMD simulation replicas are controlled by various levels of a biasing potential to reduce the energy barriers associated with peptide backbone dihedral transitions. The method requires much fewer replicas for efficient sampling compared with T‐REMD. Application of the approach to several protein loops indicated improved conformational sampling of backbone dihedral angle of loop residues compared to conventional MD simulations. BP‐REMD refinement simulations on several test cases starting from decoy structures deviating significantly from the native structure resulted in final structures in much closer agreement with experiment compared to conventional MD simulations. Proteins 2010. © 2010 Wiley‐Liss, Inc.