Structures of in vitro evolved binding sites on neocarzinostatin scaffold reveal unanticipated evolutionary pathways

We have recently applied in vitro evolution methods to create in Neocarzinostatin a new binding site for a target molecule unrelated to its natural ligand. The main objective of this work was to solve the structure of some of the selected binders in complex with the target molecule: testosterone. Three proteins (1a.15, 3.24 and 4.1) were chosen as representative members of sequence families that came out of the selection process within different randomization schemes. In order to evaluate ligand-induced conformational adaptation, we also determined the structure of one of the proteins (3.24) in the free and complexed forms. Surprisingly, all these mutants bind not one but two molecules of testosterone in two very different ways. The 3.24 structure revealed that the protein spontaneously evolved in the system to bind two ligand molecules in one single binding crevice. These two binding sites are formed by substituted as well as by non-variable side-chains. The comparison with the free structure shows that only limited structural changes are observed upon ligand binding. The X-ray structures of the complex formed by 1a.15 and 4.1 Neocarzinostatin mutants revealed that the two variants form very similar dimers. These dimers were observed neither for the uncomplexed variants nor for wild-type Neocarzinostatin but were shown here to be induced by ligand binding. Comparison of the three complexed forms clearly suggests that these unanticipated structural responses resulted from the molecular arrangement used for the selection experiments.     

Bending strategies of convergently evolved,articulated coralline algae

The evolution of uncalcified genicula in upright calcified corallines has occurred at least three times independently, resulting in articulated corallines within Corallinoideae, Lithophylloideae, and Metagoniolithoideae. Genicula confer flexibility to otherwise rigid thalli, and the localization of bending at discrete intervals amplifies bending stress in genicular tissue. Genicular morphology must, therefore, be balanced between maintaining flexibility while mitigating or resisting stress. Genicula in the three articulated lineages differ in both cellular construction and development, which may result in different constraints on morphology. By studying the interaction between flexibility and morphological variation in multiple species, we investigate whether representatives of convergently evolving clades follow similar strategies to generate mechanically successful articulated fronds. By using computational models to explore different bending strategies, we show that there are multiple ways to generate flexibility in upright corallines but not all morphological strategies are mechanically equivalent. Corallinoids have many joints, lithophylloids have pliant joints, and metagoniolithoids have longer joints—while these strategies can lead to comparable thallus flexibility, they also lead to different levels of stress amplification in bending. Moreover, genicula at greatest risk of stress amplification are typically the strongest, universally mitigating the trade‐off between flexibility and stress reduction.     

Predictable cholesterol binding sites in GPCRs lack consensus motifs

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Plant pathogens convergently evolved to counteract redundant nodes of an NLR immune receptor network

In plants, nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins can form receptor networks to confer hypersensitive cell death and innate immunity. One class of NLRs, known as NLR required for cell death (NRCs), are central nodes in a complex network that protects against multiple pathogens and comprises up to half of the NLRome of solanaceous plants. Given the prevalence of this NLR network, we hypothesised that pathogens convergently evolved to secrete effectors that target NRC activities. To test this, we screened a library of 165 bacterial, oomycete, nematode, and aphid effectors for their capacity to suppress the cell death response triggered by the NRC-dependent disease resistance proteins Prf and Rpi-blb2. Among 5 of the identified suppressors, 1 cyst nematode protein and 1 oomycete protein suppress the activity of autoimmune mutants of NRC2 and NRC3, but not NRC4, indicating that they specifically counteract a subset of NRC proteins independently of their sensor NLR partners. Whereas the cyst nematode effector SPRYSEC15 binds the nucleotide-binding domain of NRC2 and NRC3, the oomycete effector AVRcap1b suppresses the response of these NRCs via the membrane trafficking-associated protein NbTOL9a (Target of Myb 1-like protein 9a). We conclude that plant pathogens have evolved to counteract central nodes of the NRC immune receptor network through different mechanisms. Coevolution with pathogen effectors may have driven NRC diversification into functionally redundant nodes in a massively expanded NLR network.

Plant pathogens have evolved to counteract their hosts’ immune systems. A screen for pathogen effectors that suppress sensor NLR-mediated cell death in tobacco identifies effectors from a cyst nematode and an oomycete that suppress the NRC branch of the immune network to inhibit the immune-related cell death response.     

RNANetMotif: Identifying sequence-structure RNA network motifs in RNA-protein binding sites

RNA molecules can adopt stable secondary and tertiary structures, which are essential in mediating physical interactions with other partners such as RNA binding proteins (RBPs) and in carrying out their cellular functions. In vivo and in vitro experiments such as RNAcompete and eCLIP have revealed in vitro binding preferences of RBPs to RNA oligomers and in vivo binding sites in cells. Analysis of these binding data showed that the structure properties of the RNAs in these binding sites are important determinants of the binding events; however, it has been a challenge to incorporate the structure information into an interpretable model. Here we describe a new approach, RNANetMotif, which takes predicted secondary structure of thousands of RNA sequences bound by an RBP as input and uses a graph theory approach to recognize enriched subgraphs. These enriched subgraphs are in essence shared sequence-structure elements that are important in RBP-RNA binding. To validate our approach, we performed RNA structure modeling via coarse-grained molecular dynamics folding simulations for selected 4 RBPs, and RNA-protein docking for LIN28B. The simulation results, e.g., solvent accessibility and energetics, further support the biological relevance of the discovered network subgraphs.     

CAP binding sites reveal pyrimidine-purine pattern characteristic of DNA bending

To investigate the intrinsic bending of DNA at sites where proteins bind, we analyzed catabolite gene activator protein (CAP) binding sites and various operators from the viewpoint of DNA bending flexibility. Theoretical conformational analysis. DNase I digestion and x-ray crystallography data indicate that bending of B-DNA is highly anisotropic and sequence-dependent. Certain dimers prefer to bend into the major groove ("major-philic") and others prefer to bend into the minor groove ("minor-philic" dimers). From these data we considered TA, CG, CA:TG and GG:CC as major-philic dimers and AT,AA:TT and GT:AC as minor-philic ones. Analysis of 31 CAP binding sites has identified strong major-philic tendencies 5-7 base pairs (bp) away from the center. In addition, we found minor-philic poly-A tracts extending 4-5 bp away from the proposed major-philic bends. Finally, to analyze the central regions we followed the lead of Shumilov and classified the DNA sites by their spacer lengths [V.Y. Shumilov, Mol. Biol. (Mosk) 21, 168-187 (1987)]. In this way, we identified two subsets of CAP binding sites: one with 6 bp between the TGTGA:TCACA consensus boxes (N6-set) and one with 8 central bp (N8-set). We discovered that the dimer at the center of an N6-set site was usually major-philic, whereas at the center of an N8-set site more often minor-philic. Analysis of phages 434, P22 lambda and trp operators revealed similar results. In conclusion, our data show that CAP binding sites have major-philic and minor-philic dimers at specific positions; the location of these dimers may facilitate wrapping of DNA around CAP. A similar pattern is seen in nucleosomes.     

Composite structural motifs of binding sites for delineating biological functions of proteins

Most biological processes are described as a series of interactions between proteins and other molecules, and interactions are in turn described in terms of atomic structures. To annotate protein functions as sets of interaction states at atomic resolution, and thereby to better understand the relation between protein interactions and biological functions, we conducted exhaustive all-against-all atomic structure comparisons of all known binding sites for ligands including small molecules, proteins and nucleic acids, and identified recurring elementary motifs. By integrating the elementary motifs associated with each subunit, we defined composite motifs that represent context-dependent combinations of elementary motifs. It is demonstrated that function similarity can be better inferred from composite motif similarity compared to the similarity of protein sequences or of individual binding sites. By integrating the composite motifs associated with each protein function, we define meta-composite motifs each of which is regarded as a time-independent diagrammatic representation of a biological process. It is shown that meta-composite motifs provide richer annotations of biological processes than sequence clusters. The present results serve as a basis for bridging atomic structures to higher-order biological phenomena by classification and integration of binding site structures.     

Automatic generation of 3D motifs for classification of protein binding sites

Background  

Since many of the new protein structures delivered by high-throughput processes do not have any known function, there is a need for structure-based prediction of protein function. Protein 3D structures can be clustered according to their fold or secondary structures to produce classes of some functional significance. A recent alternative has been to detect specific 3D motifs which are often associated to active sites. Unfortunately, there are very few known 3D motifs, which are usually the result of a manual process, compared to the number of sequential motifs already known. In this paper, we report a method to automatically generate 3D motifs of protein structure binding sites based on consensus atom positions and evaluate it on a set of adenine based ligands.     

The Caulobacter crescentus chromosome replication origin evolved two classes of weak DnaA binding sites

The Caulobacter crescentus replication initiator DnaA and essential response regulator CtrA compete to control chromosome replication. The C. crescentus replication origin (Cori) contains five strong CtrA binding sites but only two apparent DnaA boxes, termed G-boxes (with a conserved second position G, TGATCCACA). Since clusters of DnaA boxes typify bacterial replication origins, this discrepancy suggested that C. crescentus DnaA recognizes different DNA sequences or compensates with novel DNA-binding proteins. We searched for novel DNA sites by scanning mutagenesis of the most conserved Cori DNA. Autonomous replication assays showed that G-boxes and novel W-boxes (TCCCCA) are essential for replication. Further analyses showed that C. crescentus DnaA binds G-boxes with moderate and W-boxes with very weak affinities significantly below DnaA's capacity for high-affinity Escherichia coli-boxes (TTATCCACA). Cori has five conserved W-boxes. Increasing W-box affinities increases or decreases autonomous replication depending on their strategic positions between the G-boxes. In vitro, CtrA binding displaces DnaA from proximal G-boxes and from distal W-boxes implying CtrA-DnaA competition and DnaA-DnaA cooperation between G-boxes and W-boxes. Similarly, during cell cycle progression, CtrA proteolysis coincides with DnaA binding to Cori. We also observe highly conserved W-boxes in other replication origins lacking E. coli-boxes. Therefore, strategically weak DnaA binding can be a general means of replication control.     

Fluorescent inhibitors reveal solvent-dependent micropolarity in the lipid binding sites of lipases

Triacylglycerol analogue p-nitrophenyl phosphonates specifically react with the active-site serine of lipolytic enzymes to give covalent lipase-inhibitor complexes, mimicking the first transition state which is involved in lipase-mediated ester hydrolysis. Here we report on a new type of phosphonate inhibitors containing a polarity-sensitive fluorophore to monitor micropolarity around the active site of the enzyme in different solvents. The respective compounds are hexyl and methyl dimethylamino-naphthalenecarbonylethylmercaptoethoxy-phosphonates. The hexyl phosphonate derivative was reacted with lipases from Rhizopus oryzae (ROL), Chromobacterium viscosum (CVL), and Pseudomonas cepacia (PCL). The resulting lipid-protein complexes were characterized in solution with respect to water penetration into the lipid binding site and the associated conformational changes of the proteins as a consequence of solvent polarity changes. We found that the accessibility of the lipid-binding site in all lipases studied was lowest in water. It was much higher when the protein was dissolved in aqueous ethanol. These biophysical effects may contribute to the previously observed dramatic changes of enzyme functions such as activity and stereoselectivity depending on the respective solvents.     

SiteMotif: A graph-based algorithm for deriving structural motifs in Protein Ligand binding sites

Studying similarities in protein molecules has become a fundamental activity in much of biology and biomedical research, for which methods such as multiple sequence alignments are widely used. Most methods available for such comparisons cater to studying proteins which have clearly recognizable evolutionary relationships but not to proteins that recognize the same or similar ligands but do not share similarities in their sequence or structural folds. In many cases, proteins in the latter class share structural similarities only in their binding sites. While several algorithms are available for comparing binding sites, there are none for deriving structural motifs of the binding sites, independent of the whole proteins. We report the development of SiteMotif, a new algorithm that compares binding sites from multiple proteins and derives sequence-order independent structural site motifs. We have tested the algorithm at multiple levels of complexity and demonstrate its performance in different scenarios. We have benchmarked against 3 current methods available for binding site comparison and demonstrate superior performance of our algorithm. We show that SiteMotif identifies new structural motifs of spatially conserved residues in proteins, even when there is no sequence or fold-level similarity. We expect SiteMotif to be useful for deriving key mechanistic insights into the mode of ligand interaction, predict the ligand type that a protein can bind and improve the sensitivity of functional annotation.     

Tissue-specific splicing of disordered segments that embed binding motifs rewires protein interaction networks

Alternative inclusion of exons increases the functional diversity of proteins. Among alternatively spliced exons, tissue-specific exons play a critical role in maintaining tissue identity. This raises the question of how tissue-specific protein-coding exons influence protein function. Here we investigate the structural, functional, interaction, and evolutionary properties of constitutive, tissue-specific, and other alternative exons in human. We find that tissue-specific protein segments often contain disordered regions, are enriched in posttranslational modification sites, and frequently embed conserved binding motifs. Furthermore, genes containing tissue-specific exons tend to occupy central positions in interaction networks and display distinct interaction partners in the respective tissues, and are enriched in signaling, development, and disease genes. Based on these findings, we propose that tissue-specific inclusion of disordered segments that contain binding motifs rewires interaction networks and signaling pathways. In this way, tissue-specific splicing may contribute to functional versatility of proteins and increases the diversity of interaction networks across tissues.     

Flexible interaction of Drosophila Smad complexes with bipartite binding sites

A subset of BMP-responsive enhancer elements are characterized by pairing of a GC-rich Smad1 binding site and an SBE-type Smad4 binding site. Such paired, or bipartite, sites are in some cases just 5 bp apart and thus might be contacted by a single Smad1-Smad4 complex. Other potential pairings are separated as much as 60 bp but it is not known whether such longer distances can be spanned by a Smad1-Smad4 complex, indeed binding of native Smad1-Smad4 complexes to any of these bipartite elements has yet to be reported. Here we report that a complex of the homologous Drosophila Smad proteins, Mad and Medea, is capable of concerted binding to GC-rich and SBE sites separated by as much as 20 bp. The wider the separation, the more severely binding affinity was reduced by shortening of the linker region that tethers the DNA binding domain of Medea. In contrast, length of the Mad linker did not affect the allowed distance between paired sites, rather it contributes specifically to Mad contact with the GC-rich site. Finally, we show that Smad1 and Smad4 can participate in binding to bipartite sites.     

SuperStar: improved knowledge-based interaction fields for protein binding sites

SuperStar is an empirical method for identifying interaction sites in proteins, based entirely on experimental information about non-bonded interactions occurring in small-molecule crystal structures, taken from the IsoStar database. We describe recent modifications and additions to SuperStar, validating the results on a test set of 122 X-ray structures of protein-ligand complexes. In this validation, propensity maps are generated for all the binding sites of these proteins, using four different probes: a charged NH(+)(3) nitrogen atom, a carbonyl oxygen atom, a hydroxyl oxygen atom and a methyl carbon atom. Next, the maps are compared with the experimentally observed positions of ligand atoms of these types. A peak-searching algorithm is introduced that highlights potential interaction hot spots. For the three hydrogen-bonding probes - NH(+)(3) nitrogen atom, carbonyl oxygen atom and hydroxyl oxygen atom - the average distance from the ligand atom to the nearest SuperStar peak is 1.0-1.2 A (0.8-1.0 A for solvent-inaccessible ligand atoms). For the methyl carbon atom probe, this distance is about 1.5 A, probably because interactions to methyl groups are much less directional.The most important addition to SuperStar is the enabling of propensity maps around metal centres - Ca(2+), Mg(2+) and Zn(2+) - in protein binding sites. The results are validated on a test set of 24 protein-ligand complexes that have a metal ion in their binding site. Coordination geometries are derived automatically, using only the protein atoms that coordinate to the metal ion. The correct coordination geometry is derived in approximately 75 % of the cases. If the derived geometry is assumed during the SuperStar calculation, the average distance from a ligand atom coordinating to the metal ion to the nearest peak in the propensity map for an oxygen probe is 0.87(7) A. If the correct coordination geometry is imposed, this distance reduces to 0.59(7)A. This indicates that the SuperStar predictions around metal-binding sites are at least as good as those around other protein groups. Using clustering techniques, a non-redundant set of probes is selected from the set of probes available in the IsoStar database. The performance in SuperStar of all these probes is tested on the test set of protein-ligand complexes. With the exception of the "ether oxygen" probe and the "any NH(+)" probe, all new probes perform as well as the four probes introduced first.