Conserved core substructures in the overlay of protein-ligand complexes

The method of conserved core substructure matching (CSM) for the overlay of protein-ligand complexes is described. The method relies upon distance geometry to align structurally similar substructures without regard to sequence similarity onto substructures from a reference protein empirically selected to include key determinants of binding site location and geometry. The error in ligand position is reduced in reoriented ensembles generated with CSM when compared to other overlay methods. Since CSM can only succeed when the selected core substructure is geometrically conserved, misalignments only rarely occur. The method may be applied to reliably overlay large numbers of protein-ligand complexes in a way that optimizes ligand position at a specific binding site or subsite or to align structures from large and diverse protein families where the conserved binding site is localized to only a small portion of either protein. Core substructures may be complex and must be chosen with care. We have created a database of empirically selected core substructures to demonstrate the utility of CSM alignment of ligand binding sites in important drug targets. A Web-based interface can be used to apply CSM to align large collections of protein-ligand complexes for use in drug design using these substructures or to evaluate the use of alternative core substructures that may then be shared with the larger user community. Examples show the benefit of CSM in the practice of structure-based drug design.     

Hydration in drug design. 3. Conserved water molecules at the ligand-binding sites of homologous proteins

Summary Water molecules are known to play an important rôle in mediating protein-ligand interactions. If water molecules are conserved at the ligand-binding sites of homologous proteins, such a finding may suggest the structural importance of water molecules in ligand binding. Structurally conserved water molecules change the conventional definition of binding sites by changing the shape and complementarity of these sites. Such conserved water molecules can be important for site-directed ligand/drug design. Therefore, five different sets of homologous protein/protein-ligand complexes have been examined to identify the conserved water molecules at the ligand-binding sites. Our analysis reveals that there are as many as 16 conserved water molecules at the FAD binding site of glutathione reductase between the crystal structures obtained from human and E. coli. In the remaining four sets of high-resolution crystal structures, 2–4 water molecules have been found to be conserved at the ligand-binding sites. The majority of these conserved water molecules are either bound in deep grooves at the protein-ligand interface or completely buried in cavities between the protein and the ligand. All these water molecules, conserved between the protein/protein-ligand complexes from different species, have identical or similar apolar and polar interactions in a given set. The site residues interacting with the conserved water molecules at the ligand-binding sites have been found to be highly conserved among proteins from different species; they are more conserved compared to the other site residues interacting with the ligand. These water molecules, in general, make multiple polar contacts with protein-site residues.     

C-reactive protein (CRP) aptamer binds to monomeric but not pentameric form of CRP

Native C-reactive protein (CRP) is composed of five identical subunits arranged in a pentameric structure (pCRP). Binding of pCRP to damaged cell membranes produces a second isoform, modified CRP, which has similar antigenicity to isolated monomeric subunits of CRP (mCRP). Emerging evidence indicates that modified CRP plays a role in inflammation and atherosclerosis, however, there are very few techniques that can distinguish the different isoforms of CRP. Here we show that an RNA aptamer binds specifically to mCRP and not to pCRP. Using this aptamer, we describe a simple, fast, and sensitive assay to detect nanomolar concentrations of mCRP using fluorescence anisotropy. In addition, we show that this aptamer can be used to detect mCRP in polyacrylamide gels and bound to a surface using total internal reflection fluorescence microscopy. The biological activity of the mCRP we prepared by heating pCRP with 0.1% sodium dodecyl sulfate was confirmed by observing binding to the complement protein, C1q. This probe provides an important tool for CRP research and has the potential to improve clinical diagnostics that predict risk for cardiovascular disease.     

Conserved network properties of helical membrane protein structures and its implication for improving membrane protein homology modeling at the twilight zone

Homology modeling techniques remain an important tool for membrane protein studies and membrane protein-targeted drug development. Due to the paucity of available structure data, an imminent challenge in this field is to develop novel computational methods to help improve the quality of the homology models constructed using template proteins with low sequence identity. In this work, we attempted to address this challenge using the network approach developed in our group. First, a structure pair dataset of 27 high-resolution and low sequence identity (7–36%) comparative TM proteins was compiled by analyzing available X-ray structures of helical membrane proteins. Structure deviation between these pairs was subsequently confirmed by calculating their backbone RMSD and comparing their potential energy per residue. Next, this dataset was further studied using the network approach. Results of these analyses indicated that the network measure applied represents a conserved feature of TM domains of similar folds with various sequence identities. Further comparison of this salient feature between high-resolution template structures and their homology models at the twilight zone suggested a useful method to utilize this property for homology model refinement. These findings should be of help for improving the quality of homology models based on templates with low sequence identity, thus broadening the application of homology modeling techniques in TM protein studies.     

Characterization of a conserved "threonine clasp" in CAP-Gly domains: role of a functionally critical OH/pi interaction in protein recognition

XH/pi hydrogen bonds have been predicted to make important contributions to protein structure and function. NMR evidence is presented for an OH/pi interaction between a highly conserved threonine and phenylalanine pair found specifically in CAP-Gly domains associated with mictrotubule plus ends. The functional contribution of this nonclassical hydrogen bond in target peptide recognition is demonstrated via subtle point mutagenesis. The OH/pi interaction is part of a TxFxxxxW motif that comprises a conserved "threonine clasp" that defines function in CAP-Gly domains.     

Conserved motif VIII of murine DNA methyltransferase Dnmt3a is essential for methylation activity

Background

Dnmt3a is a DNA methyltransferase that establishes de novo DNA methylation in mammals. The structure of the Dnmt3a C-terminal domain is similar to the bacterial M. HhaI enzyme, a well-studied prokaryotic DNA methyltransferase. No X-ray structure is available for the complex of Dnmt3a with DNA and the mechanistic details of DNA recognition and catalysis by mammalian Dnmts are not completely understood.

Results

Mutant variants of the catalytic domain of the murine Dnmt3a carrying substitutions of highly conserved N167, R200, and R202 have been generated by site directed mutagenesis and purified. Their methylation activity, DNA binding affinity, ability to flip the target cytosine out of the DNA double helix and covalent complex formation with DNA have been examined. Substitutions of N167 lead to reduced catalytic activity and reduced base flipping. Catalytic activity, base flipping, and covalent conjugate formation were almost completely abolished for the mutant enzymes with substitutions of R200 or R202.

Conclusions

We conclude that R202 plays a similar role in catalysis in Dnmt3a-CD as R232 in M.SssI and R165 in M.HhaI, which could be positioning of the cytosine for nucleophilic attack by a conserved Cys. R200 of Dnmt3a-CD is important in both catalysis and cytosine flipping. Both conserved R200 and R202 are involved in creating and stabilizing of the transient covalent intermediate of the methylation reaction. N167 might contribute to the positioning of the residues from the motif VI, but does not play a direct role in catalysis.

     

Suramin Targets the Conserved Ligand-Binding Pocket of Human Raf1 Kinase Inhibitory Protein

Suramin was initially used to treat African sleeping sickness and has been clinically tested to treat human cancers and HIV infection in the recent years. However, the therapeutic index is low with numerous clinical side-effects, attributed to its diverse interactions with multiple biological macromolecules. Here, we report a novel binding target of suramin, human Raf1 kinase inhibitory protein (hRKIP), which is an important regulatory protein involved in the Ras/Raf1/MEK/ERK (MAPK) signal pathway. Biolayer interference technology showed that suramin had an intermediate affinity for binding hRKIP with a dissociation constant of 23.8 µM. Both nuclear magnetic resonance technology and molecular docking analysis revealed that suramin bound to the conserved ligand-binding pocket of hRKIP, and that residues K113, W173, and Y181 play crucial roles in hRKIP binding suramin. Furthermore, suramin treatment at 160 µM could profoundly increase the ERK phosphorylation level by around 3 times. Our results indicate that suramin binds to hRKIP and prevents hRKIP from binding with hRaf1, thus promoting the MAPK pathway. This work is beneficial to both mechanistically understanding the side-effects of suramin and efficiently improving the clinical applications of suramin.     

Interaction of rabbit C-reactive protein with phospholipid monolayers at air/water interface

C-reactive protein (CRP) is a major acute phase reactant in most mammalian species. The structure of CRP has been previously established by crystallography, and the significance of its interaction with lipid membranes is accepted in the literature. However, the nature of the interaction between CRP and phospholipids is not yet well understood. In this paper we use monolayer technique to study the characteristics of the interaction of rabbit C-reactive protein (rCRP) with the phospholipid membranes. The results show that rCRP is surface active and can spontaneously insert into the lipid monolayers. The critical pressure for rCRP inserting into the phospholipid monolayers is about 34.5 mN/m, which is not sensitive to the types of the lipid headgroups and the presence of calcium ions in the subphase. The findings of this paper may provide a clue to the further understanding of the mechanism of the interactions between rCRP and the biological membranes.     

Application of microchip assay system for the measurement of C-reactive protein in human saliva

In the last decade, saliva has been advocated as a non-invasive alternative to blood as a diagnostic fluid. However, use of saliva has been hindered by the inadequate sensitivity of current methods to detect the lower salivary concentrations of many constituents compared to serum. Furthermore, developments in the areas related to lab-on-a-chip systems for saliva-based point of care diagnostics are complicated by the high viscosity and heterogeneous properties associated with this diagnostic fluid. The biomarker C-reactive protein (CRP) is an acute phase reactant and a well-accepted indicator of inflammation. Numerous clinical studies have established elevated serum CRP as a strong, independent risk factor for the development of cardiovascular disease (CVD). CVD has also been associated with oral infections (i.e. periodontal diseases) and there is evidence that systemic CRP may be a link between the two. Clinical measurements of CRP in serum are currently performed with "high sensitivity" CRP (hsCRP) enzyme-linked immunosorbent assay (ELISA) tests that lack the sensitivity for the detection of this important biomarker in saliva. Because measurement of salivary CRP may represent a novel approach for diagnosing and monitoring chronic inflammatory disease, including CVD and periodontal diseases, the objective of this study was to apply an ultra-sensitive microchip assay system for the measurement of CRP in human saliva. Here, we describe this novel lab-on-a-chip system in its first application for the measurement of CRP in saliva and demonstrate its advantages over the traditional ELISA method. The increased sensitivity of the microchip system (10 pg ml(-1) of CRP with 1000-fold dilution of saliva sample) is attributed to its inherent increased signal to noise ratio, resulting from the higher bead surface area available for antigen/antibody interactions and the high stringency washes associated with this approach. Finally, the microchip assay system was utilized in this study to provide direct experimental evidence that chronic periodontal disease may be associated with higher levels of salivary CRP.     

Inositolphosphoglycan Mediators: An Effective Synthesis of the Conserved Linear GPI Anchor Structure*

An effective new preparative synthesis of the conserved linear pseudopentasaccharide structure of the GPI anchors and of the full GPI structure has been carried out that has permitted obtaining both molecules in sufficient quantities as to perform further structural and biologic studies. The synthesis involves a 3+2 block synthesis strategy in which a conveniently protected Man α(1→4) GlcN₃ α(1→6) myo‐Ins building block, previously used in the synthesis of inositolphosphoglycan (IPG) mediators, is glycosylated with a protected Man α(1→2) Man trichloroacetimidate.     

大豆Em(LEA1)蛋白保守结构域的结构和聚集特性

采用圆二色谱(CD)和核磁共振波谱(NMR)方法研究了大豆Em(LEA1)蛋白保守基序Em-C和Em-2M多肽在不同环境中的结构及聚集行为.研究表明,在水和DMPG溶液中,两种多肽主要以无规结构形式存在.在50％ TFE溶液中,Em-C多肽折叠结构增加,含疏水残基的部分区域可能形成α-螺旋结构,且分子以二聚体形式存在;而Em-2M则以单体形式存在,且有序结构较少.以上结果表明,环境变化可能导致两种多肽的空间结构和聚集行为改变,这有助于理解Em蛋白在不同环境中的结构特点,及其重要区域在全长蛋白中所起的作用.     

Developing column material for the separation of serum amyloid P and C reactive protein from biological sources

In this study, we have investigated the isolation of serum amyloid P (SAP) and C‐reactive protein (CRP) from rainbow trout. It has recently been found that SAP is deposited in atherosclerotic lesions or neurofibrillary tangles, which are related to aging process and Alzheimer's disease. Given the importance of CRP, the CRP level in blood is becoming recognized as a potential means of monitoring cardiovascular risk. These two proteins, members of the pentraxin family of oligomeric serum proteins, were isolated from rainbow trout using N‐methacryloyl‐phosphoserine (MA‐pSer) immobilized poly (2‐hydroxy ethylmethacrylate) (PHEMA) cryogels as a column material in a fast protein liquid chromatography system. The separation process was verified in two steps. First, SAP and CRP proteins were isolated together from serum sample of rainbow trout using MA‐pSer/PHEMA cryogel columns. Second, SAP protein was separated chromatographically from CRP protein using the Ca²⁺ ion immobilized PHEMA cryogel column. According to the data, a new and effective technique has been developed for the isolation of SAP and CRP proteins from a biological source, rainbow trout. Finally, purified SAP and CRP were loaded using sodium dodecyl sulfate–polyacrylamide gel and western blot analysis to investigate the purity of chromatographically isolated SAP and CRP compared with commertial SAP and CRP. Copyright © 2014 John Wiley & Sons, Ltd.     

Why similar protein sequences encode similar three-dimensional structures?

Evolutionarily related proteins have similar sequences. Such similarity is called homology and can be described using substitution matrices such as Blosum 60. Naturally occurring homologous proteins usually have similar stable tertiary structures and this fact is used in so-called homology modeling. In contrast, the artificial protein designed by the Regan group has 50% identical sequence to the B1 domain of Streptococcal IgG-binding protein and a structure similar to the protein Rop. In this study, we asked the question whether artificial similar protein sequences (pseudohomologs) tend to encode similar protein structures, such as proteins existing in nature. To answer this question, we designed sets of protein sequences (pseudohomologs) homologous to sequences having known three-dimensional structures (template structures), same number of identities, same composition and equal level of homology, according to Blosum 60 substitution matrix as the known natural homolog. We compared the structural features of homologs and pseudohomologs by fitting them to the template structure. The quality of such structures was evaluated by threading potentials. The packing quality was measured using three-dimensional homology models. The packing quality of the models was worse for the “pseudohomologs” than for real homologs. The native homologs have better threading potentials (indicating better sequence-structure fit) in the native structure than the designed sequences. Therefore, we have shown that threading potentials and proper packing are evolutionarily more strongly conserved than sequence homology measured using the Blosum 60 matrix. Our results indicate that three-dimensional protein structure is evolutionarily more conserved than expected due to sequence conservation.     

HomologyPlot: Searching for homology to a family of proteins using a database of unique conserved patterns

Summary A new database of conserved amino acid residues is derived from the multiple sequence alignment of over 84 families of protein sequences that have been reported in the literature. This database contains sequences of conserved hydrophobic core patterns which are probably important for structure and function, since they are conserved for most sequences in that family. This database differs from other single-motif or signature databases reported previously, since it contains multiple patterns for each family. The new database is used to align a new sequence with the conserved regions of a family. This is analogous to reports in the literature where multiple sequence alignments are used to improve a sequence alignment. A program called Homology-Plot (suitable for IBM or compatible computers) uses this database to find homology of a new sequence to a family of protein sequences. There are several advantages to using multiple patterns. First, the program correctly identifies a new sequence as a member of a known family. Second, the search of the entire database is rapid and requires less than one minute. This is similar to performing a multiple sequence alignment of a new sequence to all of the known protein family sequences. Third, the alignment of a new sequence to family members is reliable and can reproduce the alignment of conserved regions already described in the literature. The speed and efficiency of this method is enhanced, since there is no need to score for insertions or deletions as is done in the more commonly used sequence alignment methods. In this method only the patterns are aligned. HomologyPlot also provides general information on each family, as well as a listing of patterns in a family.     

Computing motif correlations in proteins

Protein motifs, which are specific regions and conserved regions, are found by comparing multiple protein sequences. These conserved regions in general play an important role in protein functions and protein folds, for example, for their binding properties or enzymatic activities. The aim here is to find the existence correlations of protein motifs. The knowledge of protein motif/domain sharing should be important in shedding new light on the biologic functions of proteins and offering a basis in analyzing the evolution in the human genome or other genomes. The protein sequences used here are obtained from the PIR-NREF database and the protein motifs are retrieved from the PROSITE database. We apply data mining approach to discover the occurrence correlations of motif in protein sequences. The correlation of motifs mined can be used in evolution analyses and protein structure prediction. We discuss the latter, i.e., protein structure prediction in this study. The correlations mined are stored and maintained in a database system. The database is now available at http://bioinfo.csie.ncu.edu.tw/ProMotif/.     

Protein-protein binding-sites prediction by protein surface structure conservation

A new algorithm to predict protein-protein binding sites using conservation of both protein surface structure and physical-chemical properties in structurally similar proteins is developed. Binding-site residues in proteins are known to be more conserved than the rest of the surface, and finding local surface similarities by comparing a protein to its structural neighbors can potentially reveal the location of binding sites on this protein. This approach, which has previously been used to predict binding sites for small ligands, is now extended to predict protein-protein binding sites. Examples of binding-site predictions for a set of proteins, which have previously been studied for sequence conservation in protein-protein interfaces, are given. The predicted binding sites and the actual binding sites are in good agreement. Our algorithm for finding conserved surface structures in a set of similar proteins is a useful tool for the prediction of protein-protein binding sites.     

Common Fibril Structures Imply Systemically Conserved Protein Misfolding Pathways In Vivo

Systemic amyloidosis is caused by the misfolding of a circulating amyloid precursor protein and the deposition of amyloid fibrils in multiple organs. Chemical and biophysical analysis of amyloid fibrils from human AL and murine AA amyloidosis reveal the same fibril morphologies in different tissues or organs of one patient or diseased animal. The observed structural similarities concerned the fibril morphology, the fibril protein primary and secondary structures, the presence of post-translational modifications and, in case of the AL fibrils, the partially folded characteristics of the polypeptide chain within the fibril. Our data imply for both analyzed forms of amyloidosis that the pathways of protein misfolding are systemically conserved; that is, they follow the same rules irrespective of where inside one body fibrils are formed or accumulated.     

Structural,functional, and evolutionary analysis of late embryogenesis abundant proteins (LEA) in Triticum aestivum: A detailed molecular level biochemistry using in silico approach

LEA (Late Embryogenesis Abundant) proteins are abundant in plants and play a crucial role in abiotic stress tolerance. In our work, we primarily focused on the variations in physiochemical properties, conserved domains, secondary structure, gene ontology and evolutionary relationships among 40 LEA proteins of Triticum aestivum (common wheat). Wheat LEA protein belongs to first 6 classes out of the 13 classes present in LEApdB, the comprehensive database for LEA proteins. Proteins belonging to each LEApdB class have structures and functions distinguished from other classes. The study found three different conserved LEA domains in Triticum aestivum. One important domain was dehydrin, present in wheat proteins of classes 1, 2 and 4, though varied in sequence level, have similar biological processes. The study also found sequence level and phylogenetic similarity between dehydrin domains of class 1 and 4, but distinct from that of LEApdB class 2. This study also demonstrated functional diversity in two class 6 proteins occurred due to many destabilizing mutations in the LEA4 domain that caused alteration of ligand binding and conformational shift from 3₁₀-helix → turn within the domain. The LEA4 domains of these proteins also showed functional similarity and evolutionary relatedness with three other proteins of genus Aegilops, denoting that these proteins in Triticum aestivum were derived from its ancestor Aegilops. The study also assigned LEApdB class 4 to an unclassified LEA protein ‘WZY2-1’ based on amino acid composition, conserved domain, motif architecture and phylogenetic relatedness with class 4 proteins. Our study has revealed a detailed analysis of LEA proteins in Triticum aestivum and can serve as a pillar for further investigations and comparative analysis of wheat LEA proteins with other cereal or plant types.     

The role of the putative catalytic base in the phosphoryl transfer reaction in a protein kinase: first-principles calculations

Protein kinases are important enzymes controlling the majority of cellular signaling events via a transfer of the gamma-phosphate of ATP to a target protein. Even after many years of study, the mechanism of this reaction is still poorly understood. Among many factors that may be responsible for the 1011-fold rate enhancement due to this enzyme, the role of the conserved aspartate (Asp166) has been given special consideration. While the essential presence of Asp166 has been established by mutational studies, its function is still debated. The general base catalyst role assigned to Asp166 on the basis of its position in the active site has been brought into question by the pH dependence of the reaction rate, isotope measurements, and pre-steady-state kinetics. Recent semiempirical calculations have added to the controversy surrounding the role of Asp166 in the catalytic mechanism. No major role for Asp166 has been found in these calculations, which have predicted the reaction process consisting of an early transfer of a substrate proton onto the phosphate group. These conclusions were inconsistent with experimental observations. To address these differences between experimental results and theory with a more reliable computational approach and to provide a theoretical platform for understanding catalysis in this important enzyme family, we have carried out first-principles structural and dynamical calculations of the reaction process in cAPK kinase. To preserve the essential features of the reaction, representations of all of the key conserved residues (82 atoms) were included in the calculation. The structural calculations were performed using the local basis density functional (DFT) approach with both hybrid B3LYP and PBE96 generalized gradient approximations. This kind of calculation has been shown to yield highly accurate structural information for a large number of systems. The optimized reactant state structure is in good agreement with X-ray data. In contrast to semiempirical methods, the lowest energy product state places the substrate proton on Asp166. First-principles molecular dynamics simulations provide additional support for the stability of this product state. The latter also demonstrate that the proton transfer to Asp166 occurs at a point in the reaction where bond cleavage at the PO bridging position is already advanced. This mechanism is further supported by the calculated structure of the transition state in which the substrate hydroxyl group is largely intact. A metaphoshate-like structure is present in the transition state, which is consistent with the X-ray structures of transition state mimics. On the basis of the calculated structure of the transition state, it is estimated to be 85% dissociative. Our analysis also indicates an increase in the hydrogen bond strength between Asp166 and substrate hydroxyl and a small decrease in the bond strength of the latter in the transition state. In summary, our calculations demonstrate the importance of Asp166 in the enzymatic mechanism as a proton acceptor. However, the proton abstraction from the substrate occurs late in the reaction process. Thus, in the catalytic mechanism of cAPK protein kinase, Asp166 plays a role of a "proton trap" that locks the transferred phosphoryl group to the substrate. These results resolve prior inconsistencies between theory and experiment and bring new understanding of the role of Asp166 in the protein kinase catalytic mechanism.