PONDR-FIT: A meta-predictor of intrinsically disordered amino acids

Protein intrinsic disorder is becoming increasingly recognized in proteomics research. While lacking structure, many regions of disorder have been associated with biological function. There are many different experimental methods for characterizing intrinsically disordered proteins and regions; nevertheless, the prediction of intrinsic disorder from amino acid sequence remains a useful strategy especially for many large-scale proteomic investigations. Here we introduced a consensus artificial neural network (ANN) prediction method, which was developed by combining the outputs of several individual disorder predictors. By eight-fold cross-validation, this meta-predictor, called PONDR-FIT, was found to improve the prediction accuracy over a range of 3 to 20% with an average of 11% compared to the single predictors, depending on the datasets being used. Analysis of the errors shows that the worst accuracy still occurs for short disordered regions with less than ten residues, as well as for the residues close to order/disorder boundaries. Increased understanding of the underlying mechanism by which such meta-predictors give improved predictions will likely promote the further development of protein disorder predictors. Access to PONDR-FIT is available at www.disprot.org.     

Quantification of the elevated rate of domain rearrangements in metazoa

Most eukaryotic proteins consist of multiple domains created through gene fusions or internal duplications. The most frequent change of a domain architecture (DA) is insertion or deletion of a domain at the N or C terminus. Still, the mechanisms underlying the evolution of multidomain proteins are not very well studied.Here, we have studied the evolution of multidomain architectures (MDA), guided by evolutionary information in the form of a phylogenetic tree. Our results show that Pfam domain families and MDAs have been created with comparable rates (0.1-1 per million years (My)). The major changes in DA evolution have occurred in the process of multicellularization and within the metazoan lineage. In contrast, creation of domains seems to have been frequent already in the early evolution. Furthermore, most of the architectures have been created from older domains or architectures, whereas novel domains are mainly found in single-domain proteins. However, a particular group of exon-bordering domains may have contributed to the rapid evolution of novel multidomain proteins in metazoan organisms. Finally, MDAs have evolved predominantly through insertions of domains, whereas domain deletions are less common.In conclusion, the rate of creation of multidomain proteins has accelerated in the metazoan lineage, which may partly be explained by the frequent insertion of exon-bordering domains into new architectures. However, our results indicate that other factors have contributed as well.     

Intrinsically disordered Meningioma-1 stabilizes the BAF complex to cause AML

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Synthesis of novel benzbromarone derivatives designed to avoid metabolic activation

We synthesized six novel BBR derivatives that were designed to avoid metabolic activation via ipso-substitution and evaluated for their degree of toxicity and hURAT1 inhibition. It was found that all of the derivatives demonstrate lower cytotoxicity in mouse hepatocytes and lower levels of metabolic activation than BBR, while maintaining their inhibitory activity toward the uric acid transporter. We propose that these derivatives could serve as effective uricosuric agents that have much better safety profiles than BBR.     

CDF it all: Consensus prediction of intrinsically disordered proteins based on various cumulative distribution functions

Many biologically active proteins are intrinsically disordered. A reasonable understanding of the disorder status of these proteins may be beneficial for better understanding of their structures and functions. The disorder contents of disordered proteins vary dramatically, with two extremes being fully ordered and fully disordered proteins. Often, it is necessary to perform a binary classification and classify a whole protein as ordered or disordered. Here, an improved error estimation technique was applied to develop the cumulative distribution function (CDF) algorithms for several established disorder predictors. A consensus binary predictor, based on the artificial neural networks, NN-CDF, was developed by using output of the individual CDFs. The consensus method outperforms the individual predictors by 4-5% in the averaged accuracy.     

RGG/RG Motif Regions in RNA Binding and Phase Separation

RGG/RG motifs are RNA binding segments found in many proteins that can partition into membraneless organelles. They occur in the context of low-complexity disordered regions and often in multiple copies. Although short RGG/RG-containing regions can sometimes form high-affinity interactions with RNA structures, multiple RGG/RG repeats are generally required for high-affinity binding, suggestive of the dynamic, multivalent interactions that are thought to underlie phase separation in formation of cellular membraneless organelles. Arginine can interact with nucleotide bases via hydrogen bonding and π-stacking; thus, nucleotide conformers that provide access to the bases provide enhanced opportunities for RGG interactions. Methylation of RGG/RG regions, which is accomplished by protein arginine methyltransferase enzymes, occurs to different degrees in different cell types and may regulate the behavior of proteins containing these regions.     

Power Law Distribution Defines Structural Disorder as a Structural Element Directly Linked with Function

Although intrinsically disordered proteins are prevalent and functionally important, it has never been asked whether structural disorder should be considered as a separate structural category on its own or merely as a lack of secondary and/or tertiary structure. We address this issue by showing that its length distribution in the human proteome follows a power law, with many short regions but also a significant incidence of very long disordered regions. This behavior is in sharp contrast with that of conventional secondary structural elements and is highly reminiscent of the distribution of tertiary structural units in proteins. We interpret this finding by the direct functional involvement of disorder, which distinguishes it from secondary structural elements and endows it with tertiary structural attributes.     

Mutation mapping of apolipoprotein A-I structure assisted with the putative cholesterol recognition regions

Twenty-nine from 52 missense mutations in apoA-I gene are predicted to be deleterious by both SIFT and PolyPhen-2 algorithms. Among those, eight mutations with a prominent change in structure stability as modeled by the SDM tool for both lipid-free (Mei and Atkinson (2011) PDB ID: 3R2P) and HDL-bound (Wu et al. (2009) PDB ID: 3K2S) apoA-I, are referred as structural. The remaining mutations with a preferential location in a long intrinsically disordered region, predicted by the SPINE-D and DNdisorder tools, may influence the functional sites. Among structural mutations, five amyloidosis-only-related mutations, significant in a lipid-free structure, are located in 1–90 region. Six amyloidosis- and hypoalphalipoproteinemia-associated mutations, differently significant in two chains of lipid-bound apoA-I, are distributed among the N-domain. Six cholesterol recognition putative motifs (5 CRAC/1 CCM) in apoA-I structure are suggested to interact with cholesterol. Among those, the K40-W50 partially conserved CCM sequence with a putative recognition feature, predicted by the MoRF tool, may underlie cholesterol binding to lipid-free apoA-I, the binding triggering the disorder-to-order transition within MoRF. Thus, the impairment of helix formation and accelerated protein aggregation may underlie the amyloidogenic effect of W50R substitution. Also, D102H substitution in conserved CRAC2 V97-K106 sequence may be harmful in reverse cholesterol transport. With PDBe Motifs and Sites algorithm, cholesterol is a ligand for L101, F104 and W108 residues in HDL-bound apoA-I. The influence of specific mutation on apoA-I structure and mutated apolipoprotein switch between different pathologies is suggested to depend on the surrounding phase properties.     

Substrate preference of transglutaminase 2 revealed by logistic regression analysis and intrinsic disorder examination

Tissue transglutaminase (TG2) catalyzes the Ca²⁺-dependent posttranslational modification of proteins via formation of isopeptide bonds between their glutamine and lysine residues. Although substrate specificity of TG2 has been studied repeatedly at the sequence level, no clear consensus sequences have been determined so far. With the use of the extensive structural information on TG2 substrate proteins listed in TRANSDAB Wiki database†, a slight preference of TG2 for glutamine and lysine residues situated in turns could be observed. When the spatial environment of the favored glutamine and lysine residues was analyzed with logistic regression, the presence of specific amino acid patterns was identified. By using the occurrence of the predictor amino acids as selection criteria, several polypeptides were predicted and later identified as novel in vitro substrates for TG2. By studying the sequence of TG2 substrate proteins lacking available crystal structure, the strong favorable influence on substrate selection of the presence of substrate glutamine and lysine residues in intrinsically disordered regions could also be revealed. The collected structural data have provided novel understanding of how this versatile enzyme selects its substrates in various cell compartments and tissues.     

Computational analyses,molecular dynamics,and mutagenesis studies of unprocessed form of [NiFe] hydrogenase reveal the role of disorder for efficient enzyme maturation

Biological production and oxidation of hydrogen is mediated by hydrogenases, key enzymes for these energy-relevant reactions. Synthesis of [NiFe] hydrogenases involves a complex series of biochemical reactions to assemble protein subunits and metallic cofactors required for enzyme function. A final step in this biosynthetic pathway is the processing of a C-terminal tail (CTT) from its large subunit, thus allowing proper insertion of nickel in the unique NiFe(CN)₂CO cofactor present in these enzymes. In silico modelling and Molecular Dynamics (MD) analyses of processed vs. unprocessed forms of Rhizobium leguminosarum bv. viciae (Rlv) hydrogenase large subunit HupL showed that its CTT (residues 582-596) is an intrinsically disordered region (IDR) that likely provides the required flexibility to the protein for the final steps of proteolytic maturation. Prediction of pKa values of ionizable side chains in both forms of the enzyme's large subunit also revealed that the presence of the CTT strongly modify the protonation state of some key residues around the active site. Furthermore, MD simulations and mutant analyses revealed that two glutamate residues (E27 in the N-terminal region and E589 inside the CTT) likely contribute to the process of nickel incorporation into the enzyme. Computational analysis also revealed structural details on the interaction of Rlv hydrogenase LSU with the endoprotease HupD responsible for the removal of CTT.