Surface‐based protein binding pocket similarity

Protein similarity comparisons may be made on a local or global basis and may consider sequence information or differing levels of structural information. We present a local three‐dimensional method that compares protein binding site surfaces in full atomic detail. The approach is based on the morphological similarity method which has been widely applied for global comparison of small molecules. We apply the method to all‐by‐all comparisons two sets of human protein kinases, a very diverse set of ATP‐bound proteins from multiple species, and three heterogeneous benchmark protein binding site data sets. Cases of disagreement between sequence‐based similarity and binding site similarity yield informative examples. Where sequence similarity is very low, high pocket similarity can reliably identify important binding motifs. Where sequence similarity is very high, significant differences in pocket similarity are related to ligand binding specificity and similarity. Local protein binding pocket similarity provides qualitatively complementary information to other approaches, and it can yield quantitative information in support of functional annotation. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Protein function annotation by local binding site surface similarity

Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex‐PSIM, a previously reported surface‐based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against ～60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that predated query protein biochemical annotation for five out of the eight query proteins. A panel of 12 currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the podorly characterized proteins. Proteins 2014; 82:679–694. © 2013 Wiley Periodicals, Inc.     

Binding similarity network of ligand

The protein and ligand interaction takes an important part in protein function. Both ligand and its binding site are essential components for understanding how the protein-ligand complex functions. Until now, there have been many studies about protein function and evolution, but they usually lacked ligand information. Accordingly, in this study, we tried to answer the following questions: how much ligand and binding site are associated with protein function, and how ligands themselves are related to each other in terms of binding site. To answer the questions, we presented binding similarity network of ligand. Through the network analysis, we attempted to reveal systematic relationship between the ligand and binding site. The results showed that ligand binding site and function were closely related (conservation ratio, 81%). We also showed conservative tendency of function in line with ligand structure similarity with some exceptional cases. In addition, the binding similarity network of ligand revealed scale-free property to some degree like other biological networks. Since most nodes formed highly connected cluster, a clustering coefficient was very high compared with random. All the highly connected ligands (hubs) were involved in various functions forming large cluster and tended to act as a bridge between modular clusters in the network.     

Sua5p a single‐stranded telomeric DNA‐binding protein facilitates telomere replication

In budding yeast Saccharomyces cerevisiae, telomere length maintenance involves a complicated network as more than 280 telomere maintenance genes have been identified in the nonessential gene deletion mutant set. As a supplement, we identified additional 29 telomere maintenance genes, which were previously taken as essential genes. In this study, we report a novel function of Sua5p in telomere replication. Epistasis analysis and telomere sequencing show that sua5Δ cells display progressively shortened telomeres at early passages, and Sua5 functions downstream telomerase recruitment. Further, biochemical, structural and genetic studies show that Sua5p specifically binds single‐stranded telomeric (ssTG) DNA in vitro through a distinct DNA‐binding region on its surface, and the DNA‐binding ability is essential for its telomere function. Thus, Sua5p represents a novel ssTG DNA‐binding protein and positively regulates the telomere length in vivo.     

Predicting binding modes of reversible peptide‐based inhibitors of falcipain‐2 consistent with structure–activity relationships

Falcipain‐2 (FP‐2) is a major hemoglobinase of Plasmodium falciparum, considered an important drug target for the development of antimalarials. A previous study reported a novel series of 20 reversible peptide‐based inhibitors of FP‐2. However, the lack of tridimensional structures of the complexes hinders further optimization strategies to enhance the inhibitory activity of the compounds. Here we report the prediction of the binding modes of the aforementioned inhibitors to FP‐2. A computational approach combining previous knowledge on the determinants of binding to the enzyme, docking, and postdocking refinement steps, is employed. The latter steps comprise molecular dynamics simulations and free energy calculations. Remarkably, this approach leads to the identification of near‐native ligand conformations when applied to a validation set of protein‐ligand structures. Overall, we proposed substrate‐like binding modes of the studied compounds fulfilling the structural requirements for FP‐2 binding and yielding free energy values that correlated well with the experimental data. Proteins 2017; 85:1666–1683. © 2017 Wiley Periodicals, Inc.     

Fast and automated functional classification with MED‐SuMo: An application on purine‐binding proteins

Ligand–protein interactions are essential for biological processes, and precise characterization of protein binding sites is crucial to understand protein functions. MED‐SuMo is a powerful technology to localize similar local regions on protein surfaces. Its heuristic is based on a 3D representation of macromolecules using specific surface chemical features associating chemical characteristics with geometrical properties. MED‐SMA is an automated and fast method to classify binding sites. It is based on MED‐SuMo technology, which builds a similarity graph, and it uses the Markov Clustering algorithm. Purine binding sites are well studied as drug targets. Here, purine binding sites of the Protein DataBank (PDB) are classified. Proteins potentially inhibited or activated through the same mechanism are gathered. Results are analyzed according to PROSITE annotations and to carefully refined functional annotations extracted from the PDB. As expected, binding sites associated with related mechanisms are gathered, for example, the Small GTPases. Nevertheless, protein kinases from different Kinome families are also found together, for example, Aurora‐A and CDK2 proteins which are inhibited by the same drugs. Representative examples of different clusters are presented. The effectiveness of the MED‐SMA approach is demonstrated as it gathers binding sites of proteins with similar structure‐activity relationships. Moreover, an efficient new protocol associates structures absent of cocrystallized ligands to the purine clusters enabling those structures to be associated with a specific binding mechanism. Applications of this classification by binding mode similarity include target‐based drug design and prediction of cross‐reactivity and therefore potential toxic side effects.     

A novel signal transduction protein: Combination of solute binding and tandem PAS‐like sensor domains in one polypeptide chain

We report the structural and biochemical characterization of a novel periplasmic ligand‐binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from Lake Retba, in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N‐terminal and a tandem PAS‐like sensor domain at the C‐terminal region. SBP domains are found ubiquitously, and their best known function is in solute transport across membranes. PAS‐like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes but have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS‐like domain of the tandem PAS‐like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS‐like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non‐redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.     

OptGraft: A computational procedure for transferring a binding site onto an existing protein scaffold

One of the many challenging tasks of protein design is the introduction of a completely new function into an existing protein scaffold. In this study, we introduce a new computational procedure OptGraft for placing a novel binding pocket onto a protein structure so as its geometry is minimally perturbed. This is accomplished by introducing a two‐level procedure where we first identify where are the most appropriate locations to graft the new binding pocket into the protein fold by minimizing the departure from a set of geometric restraints using mixed‐integer linear optimization. On identifying the suitable locations that can accommodate the new binding pocket, CHARMM energy calculations are employed to identify what mutations in the neighboring residues, if any, are needed to ensure that the minimum energy conformation of the binding pocket conserves the desired geometry. This computational framework is benchmarked against the results available in the literature for engineering a copper binding site into thioredoxin protein. Subsequently, OptGraft is used to guide the transfer of a calcium‐binding pocket from thermitase protein (PDB: 1thm) into the first domain of CD2 protein (PDB:1hng). Experimental characterization of three de novo redesigned proteins with grafted calcium‐binding centers demonstrated that they all exhibit high affinities for terbium (K_d ～ 22, 38, and 55 μM) and can selectively bind calcium over magnesium.     

Uncovering the dynamic evolution of nucleotide‐binding site‐leucine‐rich repeat (NBS‐LRR) genes in Brassicaceae

Plant genomes harbor dozens to hundreds of nucleotide-binding site-leucine-rich repeat(NBS-LRR) genes;however,the long-term evolutionary history of these resistance genes has not been fully understood. This study focuses on five Brassicaceae genomes and the Carica papaya genome to explore changes in NBS-LRR genes that have taken place in this Rosid II lineage during the past 72 million years. Various numbers of NBS-LRR genes were identified from Arabidopsis lyrata(198),A. thaliana(165),Brassica rapa(204),Capsella rubella(127),Thellungiella salsuginea(88),and C. papaya(51). In each genome,the identified NBS-LRR genes were found to be unevenly distributed among chromosomes and most of them were clustered together.Phylogenetic analysis revealed that,before and after Brassicaceae speciation events,both toll/interleukin-1receptor-NBS-LRR(TNL) genes and non-toll/interleukin-1receptor-NBS-LRR(n TNL) genes exhibited a pattern of first expansion and then contraction,suggesting that both subclasses of NBS-LRR genes were responding to pathogen pressures synchronically. Further,by examining the gain/loss of TNL and n TNL genes at different evolutionary nodes,this study revealed that both events often occurred more drastically in TNL genes. Finally,the phylogeny of n TNL genes suggested that this NBS-LRR subclass is composed o two separate ancient gene types: RPW8-NBS-LRR and Coiled-coil-NBS-LRR.     

Rapid reprogramming of haemoglobin structure‐function exposes multiple dual‐antimicrobial potencies

The intrinsic cytotoxicity of cell‐free haemoglobin (Hb) has hampered the development of reliable Hb‐based blood substitutes for over seven decades. Notably, recent evidence shows that the Hb deploys this cytotoxic attack against invading microbes, albeit, through an unknown mechanism. Here, we unraveled a rapid molecular reprogramming of the Hb structure‐function triggered by virulent haemolytic pathogens that feed on the haem‐iron. On direct contact with the microbe, the Hb unveils its latent antimicrobial potency, where multiple antimicrobial fragments are released, each harbouring coordinated ‘dual‐action centres’: microbe binding and pseudoperoxidase (POX) cycle activity. The activated Hb fragments anchor onto the microbe while the juxtaposed POX instantly unleashes a localized oxidative shock, killing the pathogen‐in‐proximity. This concurrent action conceivably restricts the diffusion of free radicals. Furthermore, the host astutely protects itself from self‐cytotoxicity by simultaneously releasing endogenous antioxidants. We found that this decryption mechanism of antimicrobial potency is conserved in the ancient invertebrate respiratory protein, indicating its fundamental significance. Our definition of dual‐antimicrobial centres in the Hb provides vital clues for designing a safer Hb‐based oxygen carrier blood substitute.     

The ligand‐binding b′ domain of human protein disulphide‐isomerase mediates homodimerization

Purified preparations of the recombinant b′x domain fragment of human protein‐disulphide isomerase (PDI), which are homogeneous by mass spectrometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis, comprise more than one species when analyzed by ion‐exchange chromatography and nondenaturing polyacrylamide gel electrophoresis. These species were resolved and shown to be monomer and dimer by analytical ultracentrifugation and analytical size‐exclusion chromatography. Spectroscopic properties indicate that the monomeric species corresponds to the “capped” conformation observed in the x‐ray structure of the I272A mutant of b′x (Nguyen, Wallis, Howard, Haapalainen, Salo, Saaranen, Sidhu, Wierenga, Freedman, Ruddock, and Williamson, J Mol Biol 2008;383:1144‐1155) in which the x region binds to a hydrophobic patch on the surface of the b′ domain; conversely, the dimeric species has an “open” or “uncapped” conformation in which the x region does not bind to this surface. The larger bb′x fragment of human PDI shows very similar behavior to b′x and can be resolved into a capped monomeric species and an uncapped dimer. Preparations of recombinant b′ domain of human PDI and of the bb′ domain pair are found exclusively as dimers. Full‐length PDI is known to comprise a mixture of monomeric and dimeric species, whereas the isolated a , b , and a′ domains of PDI are found exclusively as monomers. These results show that the b′ domain of human PDI tends to form homodimers—both in isolation and in other contexts—and that this tendency is moderated by the adjacent x region, which can bind to a surface patch on the b′ domain.     

Database of protein complexes with multivalent binding ability: Bival‐bind

Phenomena of multivalent binding of ligands with receptors are ubiquitous in biology and of growing interest in material sciences. Multivalency can enhance binding affinity dramatically. To understand the mechanism of multivalent binding in more detail model systems of bi‐ and multivalent receptors are needed, but are difficult to find. Furthermore it is useful to know about multivalent receptors, which can serve as targets to design multivalent drugs. The present contribution tries to close this gap. The Bival‐Bind database ( http://agknapp.chemie.fu‐berlin.de/bivalbind ) provides a relatively complete list – 2073 protein complexes with less than 90% sequence identity – out of the protein database, which can serve as bi‐ or multivalent receptors. Steric clashes of molecular spacers – necessary to connect the monomeric ligand units – with the receptor surface can diminish binding affinity dramatically and, thus, abolish the expected enhancement of binding affinity due to the multivalency. The potential multivalent receptors in the Bival‐Bind database are characterized with respect to the receptor surface topography. A height profile between the receptor binding pockets is provided, which is an important information to estimate the influence of unfavorable spacer receptor interaction. Proteins 2014; 82:744–751. © 2013 Wiley Periodicals, Inc.     

Expression pattern and ligand‐binding properties of odorant‐binding protein 13 from Monochamus alternatus hope

Odorant‐binding proteins (OBPs) are believed to play an important role in olfactory recognition. In this study, expression pattern and fluorescence binding characteristics of MaltOBP13 from the Japanese pine sawyer beetle, Monochamus alternatus Hope, were investigated via qPCR analysis of MaltOBP13 mRNA level and binding assay of MaltOBP13 and ligands. qPCR monitoring indicated MaltOBP13 mainly expressed in newly emerged males, particularly highly expressed in the last abdominal segment of males, and the expression level was significantly higher in 13‐day‐old mated adults than those of other stages. To further understand the function of the MaltOBP13 protein in odorant reception, the binding affinity of recombinant MaltOBP13 to ligands was tested by fluorescence binding assays with N‐phenyl‐1‐naphthylamine as a fluorescent probe. The results of this assay indicated that MaltOBP13 exhibited a high binding affinity for pine volatiles and binding capacity was higher in acidic conditions than in neutral environment, indicating a possible role in finding host plants.     

RBRDetector: Improved prediction of binding residues on RNA‐binding protein structures using complementary feature‐ and template‐based strategies

Computational prediction of RNA‐binding residues is helpful in uncovering the mechanisms underlying protein‐RNA interactions. Traditional algorithms individually applied feature‐ or template‐based prediction strategy to recognize these crucial residues, which could restrict their predictive power. To improve RNA‐binding residue prediction, herein we propose the first integrative algorithm termed RBRDetector (RNA‐Binding Residue Detector) by combining these two strategies. We developed a feature‐based approach that is an ensemble learning predictor comprising multiple structure‐based classifiers, in which well‐defined evolutionary and structural features in conjunction with sequential or structural microenvironment were used as the inputs of support vector machines. Meanwhile, we constructed a template‐based predictor to recognize the putative RNA‐binding regions by structurally aligning the query protein to the RNA‐binding proteins with known structures. The final RBRDetector algorithm is an ingenious fusion of our feature‐ and template‐based approaches based on a piecewise function. By validating our predictors with diverse types of structural data, including bound and unbound structures, native and simulated structures, and protein structures binding to different RNA functional groups, we consistently demonstrated that RBRDetector not only had clear advantages over its component methods, but also significantly outperformed the current state‐of‐the‐art algorithms. Nevertheless, the major limitation of our algorithm is that it performed relatively well on DNA‐binding proteins and thus incorrectly predicted the DNA‐binding regions as RNA‐binding interfaces. Finally, we implemented the RBRDetector algorithm as a user‐friendly web server, which is freely accessible at http://ibi.hzau.edu.cn/rbrdetector . Proteins 2014; 82:2455–2471. © 2014 Wiley Periodicals, Inc.     

Molecular characterization,expression pattern and ligand‐binding properties of the pheromone‐binding protein gene from Cyrtotrachelus buqueti

Pheromone‐binding proteins (PBPs) play important roles in the information exchange between insect sexes, specifically in the process of transporting fat‐soluble odour molecules from the external environment to olfactory receptors through the olfactory sensillum lymph. The PBP functions in this process may explain the sex pheromone identification mechanism used by insects, laying a theoretical foundation for the prevention and control of pests by interfering with olfactory recognition. In the present study, a PBP gene of Cyrtotrachelus buqueti (GenBank accession number: KU845733) is cloned for prokaryotic expression. Using N‐phenyl‐1‐naphthylamine as the fluorescent probe in a competitive binding assay, the ability of CbuqPBP1 to bind 12 sex pheromone analogues and three volatiles of Neosinocalamus affinis shoots is examined. Of the 12 C. buqueti sex pheromone analogues, dibutyl phthalate gives the greatest displacement (inhibitory constant value of 11.1 μm ), whereas the other sex pheromone components show much smaller displacements. Consistent with other PBPs, the three plant volatiles (linalool, benzaldehyde and indole) show only a limited displacement of CbuqPBP1. However, the binding abilities of 1 : 1 ratios of each of the three plant volatiles with dibutyl phthalate show increases of 62.3%, 65.1% and 51.7% over the binding abilities of the three plant volatiles alone. CbuqPBP1 has dual roles in the processes of sensing sex pheromones and plant volatiles.     

Crystal structure of AFV1‐102, a protein from the acidianus filamentous virus 1

Viruses infecting hyperthermophilic archaea have intriguing morphologies and genomic properties. The vast majority of their genes do not have homologs other than in other hyperthermophilic viruses, and the biology of these viruses is poorly understood. As part of a structural genomics project on the proteins of these viruses, we present here the structure of a 102 amino acid protein from acidianus filamentous virus 1 (AFV1‐102). The structure shows that it is made of two identical motifs that have poor sequence similarity. Although no function can be proposed from structural analysis, tight binding of the gateway tag peptide in a groove between the two motifs suggests AFV1‐102 is involved in protein protein interactions.