RNA structural motif recognition based on least-squares distance

RNA structural motifs are recurrent structural elements occurring in RNA molecules. RNA structural motif recognition aims to find RNA substructures that are similar to a query motif, and it is important for RNA structure analysis and RNA function prediction. In view of this, we propose a new method known as RNA Structural Motif Recognition based on Least-Squares distance (LS-RSMR) to effectively recognize RNA structural motifs. A test set consisting of five types of RNA structural motifs occurring in Escherichia coli ribosomal RNA is compiled by us. Experiments are conducted for recognizing these five types of motifs. The experimental results fully reveal the superiority of the proposed LS-RSMR compared with four other state-of-the-art methods.     

"Acceptor-donor-acceptor" motifs recognize the Watson-Crick, Hoogsteen and Sugar "donor-acceptor-donor" edges of adenine and adenosine-containing ligands

Nucleotides are among the most extensively exploited chemical moieties in nature and, as a part of a handful of different protein ligands, nucleotides play key roles in energy transduction, enzymatic catalysis and regulation of protein function. We have previously reported that in many proteins with different folds and functions a distinctive adenine-binding motif is involved in the recognition of the Watson-Crick edge of adenine. Here, we show that many proteins do have clear structural motifs that recognize adenosine (and some other nucleotides and nucleotide analogs) not only through the Watson-Crick edge, but also through the sugar and Hoogsteen edges. Each of the three edges of adenosine has a donor-acceptor-donor (DAD) pattern that is often recognized by proteins via a complementary acceptor-donor-acceptor (ADA) motif, whereby three distinct hydrogen bonds are formed: two conventional N-H...O and N-H...N hydrogen bonds, and one weak C-H...O hydrogen bond. The local conformation of the adenine-binding loop is betabetabeta or betabetaalpha and reflects the mode of nucleotide binding. Additionally, we report 21 proteins from five different folds that simultaneously recognize both the sugar edge and the Watson-Crick edge of adenine. In these proteins a unique beta-loop-beta supersecondary structure grasps an adenine-containing ligand between two identical adenine-binding motifs as part of the betaalphabeta-loop-beta fold.     

Prevalent RNA recognition motif duplication in the human genome

The sequence-specific recognition of RNA by proteins is mediated through various RNA binding domains, with the RNA recognition motif (RRM) being the most frequent and present in >50% of RNA-binding proteins (RBPs). Many RBPs contain multiple RRMs, and it is unclear how each RRM contributes to the binding specificity of the entire protein. We found that RRMs within the same RBP (i.e., sibling RRMs) tend to have significantly higher similarity than expected by chance. Sibling RRM pairs from RBPs shared by multiple species tend to have lower similarity than those found only in a single species, suggesting that multiple RRMs within the same protein might arise from domain duplication followed by divergence through random mutations. This finding is exemplified by a recent RRM domain duplication in DAZ proteins and an ancient duplication in PABP proteins. Additionally, we found that different similarities between sibling RRMs are associated with distinct functions of an RBP and that the RBPs tend to contain repetitive sequences with low complexity. Taken together, this study suggests that the number of RBPs with multiple RRMs has expanded in mammals and that the multiple sibling RRMs may recognize similar target motifs in a cooperative manner.     

Ligand recognition by peptidoglycan recognition protein-S (PGRP-S): structure of the complex of camel PGRP-S with heptanoic acid at 2.15 Å resolution

Peptidoglycan recognition proteins (PGRPs) are important components of the innate immune system which provide the first line of defense against invading microbes. There are four members in the family of PGRPs in animals of which PGRP-S is a common domain. It is responsible for the binding to microbial cell wall molecules. In order to understand the mode of binding of PGRP-S to the components of the bacterial cell wall, the structure of the complex of camel PGRP-S (CPGRP-S) with heptanoic acid has been determined at 2.15 Å resolution. The structure determination showed the presence of four crystallographically independent protein molecules which are designated as A, B, C, and D. These four protein molecules associate in the form of two homodimers which are represented as A-B and C-D dimers. The association between molecules A and B gives rise to a shallow cleft on the surface at one end of the dimeric interface. One molecule of heptanoic acid is observed at this binding site in the A-B dimer. The association of C and D molecules results in the formation of a long zig-zag tunnel along with the C-D interface. In the cleft at the C-D interface, three molecules of hydrogen peroxide along with other non-water solvent molecules have been observed. The analysis of the several complexes of CPGRP-S with fatty acids and non-fatty acids such as peptidoglycan, lipopolysaccharide, and lipoteichoic acid shows that the fatty acids bind at the A-B site while non-fatty acids interact through C-D interface.     

Multiple molecular recognition properties of the lipocalin protein family

The lipocalins, a diverse family of small extracellular ligand proteins, display a remarkable range of different molecular properties. While their binding of small hydrophobic molecules, and to a lesser extent their binding to cell surface receptors, is well known, it is shown here that formation of macromolecular complexes is also a common feature of this family. Analysis of known crystallographic structures reveals that the lipocalins process a conserved common structure: an antiparallel β-barrel with a repeated +1 topology. Comparisons show that within this overall similarity the structure of individual proteins is specifically adapted to bind their particular ligands, forming a binding site from an internal cavity (within the barrel) and/or an external loop scaffold, which gives rise to different binding modes that reflects the need to accommodate ligands of different shape, size, and chemical structure. The architecture of the lipocalin fold suggests that the both the ends and sides of this barrel are topologically distinct, differences also apparent in analyses of structural and sequence variation within the family. These different can be linked to experimental evidence suggesting a possible functional dichotomy between the two ends of the lipocalin fold. The structurally invariant end of the molecule may be implicated in general binding small ligands and forming macromolecular complexes via an exposed binding surface.     

A simple motif for protein recognition in DNA secondary structures

DNA in a single-stranded form (ssDNA) exists transiently within the cell and comprises the telomeres of linear chromosomes and the genomes of some DNA viruses. As with RNA, in the single-stranded state, some DNA sequences are able to fold into complex secondary and tertiary structures that may be recognized by proteins and participate in gene regulation. To better understand how such DNA elements might fold and interact with proteins, and to compare recognition features to those of a structured RNA, we used in vitro selection to identify ssDNAs that bind an RNA-binding peptide from the HIV Rev protein with high affinity and specificity. The large majority of selected binders contain a non-Watson-Crick G.T base-pair and an adjacent C:G base-pair and both are essential for binding. This GT motif can be presented in different DNA contexts, including a nearly perfect duplex and a branched three-helix structure, and appears to be recognized in large part by arginine residues separated by one turn of an alpha-helix. Interestingly, a very similar GT motif is necessary also for protein binding and function of a well-characterized model ssDNA regulatory element from the proenkephalin promoter.     

Deciphering common recognition principles of nucleoside mono/di and tri‐phosphates binding in diverse proteins via structural matching of their binding sites

Nucleoside triphosphate (NTP) ligands are of high biological importance and are essential for all life forms. A pre‐requisite for them to participate in diverse biochemical processes is their recognition by diverse proteins. It is thus of great interest to understand the basis for such recognition in different proteins. Towards this, we have used a structural bioinformatics approach and analyze structures of 4677 NTP complexes available in Protein Data Bank (PDB). Binding sites were extracted and compared exhaustively using PocketMatch, a sensitive in‐house site comparison algorithm, which resulted in grouping the entire dataset into 27 site‐types. Each of these site‐types represent a structural motif comprised of two or more residue conservations, derived using another in‐house tool for superposing binding sites, PocketAlign. The 27 site‐types could be grouped further into 9 super‐types by considering partial similarities in the sites, which indicated that the individual site‐types comprise different combinations of one or more site features. A scan across PDB using the 27 structural motifs determined the motifs to be specific to NTP binding sites, and a computational alanine mutagenesis indicated that residues identified to be highly conserved in the motifs are also most contributing to binding. Alternate orientations of the ligand in several site‐types were observed and rationalized, indicating the possibility of some residues serving as anchors for NTP recognition. The presence of multiple site‐types and the grouping of multiple folds into each site‐type is strongly suggestive of convergent evolution. Knowledge of determinants obtained from this study will be useful for detecting function in unknown proteins. Proteins 2017; 85:1699–1712. © 2017 Wiley Periodicals, Inc.     

PIER: protein interface recognition for structural proteomics

Recent advances in structural proteomics call for development of fast and reliable automatic methods for prediction of functional surfaces of proteins with known three-dimensional structure, including binding sites for known and unknown protein partners as well as oligomerization interfaces. Despite significant progress the problem is still far from being solved. Most existing methods rely, at least partially, on evolutionary information from multiple sequence alignments projected on protein surface. The common drawback of such methods is their limited applicability to the proteins with a sparse set of sequential homologs, as well as inability to detect interfaces in evolutionary variable regions. In this study, the authors developed an improved method for predicting interfaces from a single protein structure, which is based on local statistical properties of the protein surface derived at the level of atomic groups. The proposed Protein IntErface Recognition (PIER) method achieved the overall precision of 60% at the recall threshold of 50% at the residue level on a diverse benchmark of 490 homodimeric, 62 heterodimeric, and 196 transient interfaces (compared with 25% precision at 50% recall expected from random residue function assignment). For 70% of proteins in the benchmark, the binding patch residues were successfully detected with precision exceeding 50% at 50% recall. The calculation only took seconds for an average 300-residue protein. The authors demonstrated that adding the evolutionary conservation signal only marginally influenced the overall prediction performance on the benchmark; moreover, for certain classes of proteins, using this signal actually resulted in a deteriorated prediction. Thorough benchmarking using other datasets from literature showed that PIER yielded improved performance as compared with several alignment-free or alignment-dependent predictions. The accuracy, efficiency, and dependence on structure alone make PIER a suitable tool for automated high-throughput annotation of protein structures emerging from structural proteomics projects.     

Novel structural tree of β-proteins containing abcd units

A database of 528 β-proteins and β-domains containing abcd units, including 244 nonhomologous ones, was compiled from the Protein Data Bank (total 1511 PDB entries). A novel structural tree of this class of proteins was constructed to include 153 possible polypeptide chain folds. A structural classification of β-proteins containing abcd units was proposed on the basis of the tree. The database and the structural tree are available at http://strees.protres.ru/.     

Crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 reveals a new protein family with an RNA recognition motif-like domain

We have determined the crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 at 1.9 A resolution. This protein is a member of the Escherichia coli ygcH sequence family, which contains approximately 15 sequence homologs of bacterial origin. These homologs have a high isoelectric point. The crystal structure reveals that TTHB192 consists of two independently folded domains, and that each domain exhibits a ferredoxin-like fold with a four-stranded antiparallel beta-sheet packed on one side by alpha-helices. These two tandem domains face each other to generate a beta-sheet platform. TTHB192 displays overall structural similarity to Sex-lethal protein and poly(A)-binding protein fragments. These proteins have RNA binding activity which is supported by a beta-sheet platform formed by two tandem repeats of an RNA recognition motif domain with signature sequence motifs on the beta-sheet surface. Although TTHB192 does not have the same signature sequence motif as the RNA recognition motif domain, the presence of an evolutionarily conserved basic patch on the beta-sheet platform could be functionally relevant for nucleic acid-binding. This report shows that TTHB192 and its sequence homologs adopt an RNA recognition motif-like domain and provides the first testable functional hypothesis for this protein family.     

The fragment transformation method to detect the protein structural motifs

To identify functional structural motifs from protein structures of unknown function becomes increasingly important in recent years due to the progress of the structural genomics initiatives. Although certain structural patterns such as the Asp-His-Ser catalytic triad are easy to detect because of their conserved residues and stringently constrained geometry, it is usually more challenging to detect a general structural motifs like, for example, the betabetaalpha-metal binding motif, which has a much more variable conformation and sequence. At present, the identification of these motifs usually relies on manual procedures based on different structure and sequence analysis tools. In this study, we develop a structural alignment algorithm combining both structural and sequence information to identify the local structure motifs. We applied our method to the following examples: the betabetaalpha-metal binding motif and the treble clef motif. The betabetaalpha-metal binding motif plays an important role in nonspecific DNA interactions and cleavage in host defense and apoptosis. The treble clef motif is a zinc-binding motif adaptable to diverse functions such as the binding of nucleic acid and hydrolysis of phosphodiester bonds. Our results are encouraging, indicating that we can effectively identify these structural motifs in an automatic fashion. Our method may provide a useful means for automatic functional annotation through detecting structural motifs associated with particular functions.     

Arg149 is involved in switching the low affinity, open state of the binding protein AfProX into its high affinity, closed state

The substrate binding protein AfProX from the Archaeoglobus fulgidus ProU ATP binding cassette transporter is highly selective for the compatible solutes glycine betaine (GB) and proline betaine, which confer thermoprotection to this hyperthermophilic archaeon. A detailed mutational analysis of the substrate binding site revealed the contribution of individual amino acids for ligand binding. Replacement of Arg149 by an Ala residue displayed the largest impact on substrate binding. The structure of a mutant AfProX protein (substitution of Tyr111 with Ala) in complex with GB was solved in the open liganded conformation to gain further insight into ligand binding. In this crystal structure, GB is bound differently compared to the GB closed liganded structure of the wild-type AfProX protein. We found that a network of amino acid side chains communicates the presence of GB toward Arg149, which increases ligand affinity and induces domain closure of AfProX. These results were corroborated by molecular dynamics studies and support the view that Arg149 finalizes the high-affinity state of the AfProX substrate binding protein.     

The RNA recognition motif domains of RBM5 are required for RNA binding and cancer cell proliferation inhibition

RBM5 is a known putative tumor suppressor gene that has been shown to function in cell growth inhibition by modulating apoptosis. RBM5 also plays a critical role in alternative splicing as an RNA binding protein. However, it is still unclear which domains of RBM5 are required for RNA binding and related functional activities. We hypothesized the two putative RNA recognition motif (RRM) domains of RBM5 spanning from amino acids 98–178 and 231–315 are essential for RBM5-mediated cell growth inhibition, apoptosis regulation, and RNA binding. To investigate this hypothesis, we evaluated the activities of the wide-type and mutant RBM5 gene transfer in low-RBM5 expressing A549 cells. We found that, unlike wild-type RBM5 (RBM5-wt), a RBM5 mutant lacking the two RRM domains (RBM5-ΔRRM), is unable to bind RNA, has compromised caspase-2 alternative splicing activity, lacks cell proliferation inhibition and apoptosis induction function in A549 cells. These data provide direct evidence that the two RRM domains of RBM5 are required for RNA binding and the RNA binding activity of RBM5 contributes to its function on apoptosis induction and cell growth inhibition.     

Classification and annotation of the relationship between protein structural change and ligand binding

The causal relationship between protein structural change and ligand binding was classified and annotated for 839 nonredundant pairs of crystal structures in the Protein Data Bank—one with and the other without a bound low-molecular-weight ligand molecule. Protein structural changes were first classified into either domain or local motions depending on the size of the moving protein segments. Whether the protein motion was coupled with ligand binding was then evaluated based on the location of the ligand binding site and by application of the linear response theory of protein structural change. Protein motions coupled with ligand binding were further classified into either closure or opening motions. This classification revealed the following: (i) domain motions coupled with ligand binding are dominated by closure motions, which can be described by the linear response theory; (ii) local motions frequently accompany order-disorder or α-helix-coil conformational transitions; and (iii) transferase activity (Enzyme Commission   number 2) is the predominant function among coupled domain closure motions. This could be explained by the closure motion acting to insulate the reaction site of these enzymes from environmental water.     

A structural pattern-based method for protein fold recognition

A method (SPREK) was developed to evaluate the register of a sequence on a structure based on the matching of structural patterns against a library derived from the protein structure databank. The scores obtained were normalized against random background distributions derived from sequence shuffling and permutation methods. 'Random' structures were also used to evaluate the effectiveness of the method. These were generated by a simple random-walk and a more sophisticated structure prediction method that produced protein-like folds. For comparison with other methods, the performance of the method was assessed using collections of models including decoys and models from the CASP-5 exercise. The performance of SPREK on the decoy models was equivalent to (and sometimes better than) those obtained with more complex approaches. An exception was the two smallest proteins, for which SPREK did not perform well due to a lack of patterns. Using the best parameter combination from trials on decoy models, the CASP models of intermediate difficulty were evaluated by SPREK and the quality of the top scoring model was evaluated by its CASP ranking. Of the 14 targets in this class, half lie in the top 10% (out of around 140 models for each target). The two worst rankings resulted from the selection by our method of a well-packed model that was based on the wrong fold. Of the other poor rankings, one was the smallest protein and the others were the four largest (all over 250 residues).     

Towards a structural classification of phosphate binding sites in protein-nucleotide complexes: an automated all-against-all structural comparison using geometric matching

A method is described for the rapid comparison of protein binding sites using geometric matching to detect similar three-dimensional structure. The geometric matching detects common atomic features through identification of the maximum common sub-graph or clique. These features are not necessarily evident from sequence or from global structural similarity giving additional insight into molecular recognition not evident from current sequence or structural classification schemes. Here we use the method to produce an all-against-all comparison of phosphate binding sites in a number of different nucleotide phosphate-binding proteins. The similarity search is combined with clustering of similar sites to allow a preliminary structural classification. Clustering by site similarity produces a classification of binding sites for the 476 representative local environments producing ten main clusters representing half of the representative environments. The similarities make sense in terms of both structural and functional classification schemes. The ten main clusters represent a very limited number of unique structural binding motifs for phosphate. These are the structural P-loop, di-nucleotide binding motif [FAD/NAD(P)-binding and Rossman-like fold] and FAD-binding motif. Similar classification schemes for nucleotide binding proteins have also been arrived at independently by others using different methods.     

Location and nature of the residues important for ligand recognition in G-protein coupled receptors

The overall structure of the biogenic amine subclass of the G-protein-coupled receptors, and of their ligand binding sites, is discussed with the aim of highlighting the major structural features of these receptors that are responsible for ligand recognition. A comparison is made between biogenic amine receptors, peptide receptors of the rhodopsin class, and the secretin receptors which all have peptide ligands. The question of where the peptide ligands bind, whether at extracellular sites or within the transmembrane helix bundle, is discussed. The suitability of the rhodopsin crystal structure as a template for construction of homology models is discussed and it is concluded that there are many reasons why a caution should be issued against using it uncritically.