Search

Intrinsically disordered proteins exist in highly flexible conformational states linked to different protein functions. In this work, we have presented evidence that HLA class-I- and class-II-binding T-cell epitopes, experimentally verified in several tumor-associated antigens and nuclear systemic autoantigens, are predominantly located in ordered protein regions or at disorder/order borderlines, defined by the majority of analyzed publicly available disorder predictors. We have also observed the overlapping of secondary structural elements and prevalently hydrophobic regions with T-cell epitopes in Epstein Barr Virus (EBV) nuclear antigen 1 (EBNA-1), cancer/testis antigen MAGE-A4, and Sm-B/B', U1 snRNPA (U1A) and U1-70kDa autoantigens. The results are in accordance with the clustering of the predicted HLA class-I and class-II epitopes in protein parts which encompass the consensus of ordered regions, determined by individual disorder predictors. Some HLA class-II epitopes and linear B-cell epitopes were located near the segments predicted to have elevated crystallographic B factor in EBNA-1, Sm-B/B' and U1 snRNP A proteins, suggesting that protein flexibility could influence the structural availability of epitopes. Naturally processed T-cell epitopes and linear B-cell epitopes could also be found within putative disordered binding sites, determined by "dips" in the prevalently disordered parts of prediction profiles of the majority of disorder predictors, and peaks in ANCHOR-prediction profile. Two minor antigenic regions within EBNA-1, mapped to the residues 58-85 and 398-458, encompassing putative disordered binding sites, contain epitopes connected with anti-Ro 60kDa and anti-Sm B/B' autoimmunity in systemic lupus erythematosus. One of these regions overlaps residues 395-450, identified as the binding site of USP7 (HAUSP), which regulates the EBNA-1 replication function. In Sm-B/B', one of the putative disordered binding sites (residues 114-165) encompasses the T-cell epitope 136-153, while another, residues 200-216, flanks two proline-rich B-cell epitopes (residues 190-198 and 216-222), overlapping the preferred CD2BP2-GYF-binding motif (R/K/G)XXPPGX(R/K), characteristic of splicosomal proteins. We have noticed that the same motif (residues 397-403) is mimicked in EBNA-1 and overlaps epitope 398-404, involved in anti-Sm B/B' autoimmunity. The majority of recognized T- and B-cell epitopes in analyzed autoantigens or tumor-associated antigens appertain to the ordered or transient protein structures. The congruence between certain B- and T-cell epitopes and predicted disordered binding sites or protein-binding eukaryotic motifs in the antigens participating in molecular complexes might influence the capture of antigens, their processing and subsequent presentation and immunodominance., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The U1A/U2B″/SNF family of proteins found in the U1 and U2 spliceosomal small nuclear ribonucleoproteins is highly conserved. In spite of the high degree of sequence and structural conservation, modern members of this protein family have unique RNA binding properties. These differences have necessarily resulted from evolutionary processes, and therefore, we reconstructed the protein phylogeny in order to understand how and when divergence occurred and how protein function has been modulated. Contrary to the conventional understanding of an ancient human U1A/U2B″ gene duplication, we show that the last common ancestor of bilaterians contained a single ancestral protein (URB). The gene for URB was synthesized, the protein was overexpressed and purified, and we assessed RNA binding to modern snRNA sequences. We find that URB binds human and Drosophila U1 snRNA SLII and U2 snRNA SLIV with higher affinity than do modern homologs, suggesting that both Drosophila SNF and human U1A/U2B″ have evolved into weaker binders of one RNA or both RNAs., (Copyright © 2013. Published by Elsevier Ltd.)

The U1A-SL2 RNA complex is a model system for studying interactions between RNA and the RNA recognition motif (RRM), which is one of the most common RNA binding domains. We report here kinetic studies of dissociation of the U1A-SL2 RNA complex, using laser temperature jump and stopped-flow fluorescence methods with U1A proteins labeled with the intrinsic chromophore tryptophan. An analysis of the kinetic data suggests three phases of dissociation with time scales of ∼100 μs, ∼50 ms, and ∼2 s. We propose that the first step of dissociation is a fast rearrangement of the complex to form a loosely bound complex. The intermediate step is assigned to be the dissociation of the U1A-SL2 RNA complex, and the final step is assigned to a reorganization of the U1A protein structure into the conformation of the free protein. These assignments are consistent with previous proposals based on thermodynamic, NMR, and surface plasmon resonance experiments and molecular dynamics simulations. Together, these results begin to build a comprehensive model of the complex dynamic processes involved in the formation and dissociation of an RRM-RNA complex., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The investigation of spliceosomal processes is currently a topic of intense research in molecular biology. In the molecular mechanism of alternative splicing, a multi-protein-RNA complex - the spliceosome - plays a crucial role. To understand the biological processes of alternative splicing, it is essential to comprehend the biogenesis of the spliceosome. In this paper, we propose the first abstract model of the regulatory assembly pathway of the human spliceosomal subunit U1. Using Petri nets, we describe its highly ordered assembly that takes place in a stepwise manner. Petri net theory represents a mathematical formalism to model and analyze systems with concurrent processes at different abstraction levels with the possibility to combine them into a uniform description language. There exist many approaches to determine static and dynamic properties of Petri nets, which can be applied to analyze biochemical systems. In addition, Petri net tools usually provide intuitively understandable graphical network representations, which facilitate the dialog between experimentalists and theoreticians. Our Petri net model covers binding, transport, signaling, and covalent modification processes. Through the computation of structural and behavioral Petri net properties and their interpretation in biological terms, we validate our model and use it to get a better understanding of the complex processes of the assembly pathway. We can explain the basic network behavior, using minimal T-invariants which represent special pathways through the network. We find linear as well as cyclic pathways. We determine the P-invariants that represent conserved moieties in a network. The simulation of the net demonstrates the importance of the stability of complexes during the maturation pathway. We can show that complexes that dissociate too fast, hinder the formation of the complete U1 snRNP.

An MM-GBSA computational protocol was used to investigate wild-type U1A-RNA and F56 U1A mutant experimental binding free energies. The trend in mutant binding free energies compared to wild-type is well-reproduced. Following application of a linear-response-like equation to scale the various energy components, the binding free energies agree quantitatively with observed experimental values. Conformational adaptation contributes to the binding free energy for both the protein and the RNA in these systems. Small differences in DeltaGs are the result of different and sometimes quite large relative contributions from various energetic components. Residual free energy decomposition indicates differences not only at the site of mutation, but throughout the entire protein. MM-GBSA and ab initio calculations performed on model systems suggest that stacking interactions may nearly, but not completely, account for observed differences in mutant binding affinities. This study indicates that there may be different underlying causes of ostensibly similar experimentally observed binding affinities of different mutants, and thus recommends caution in the interpretation of binding affinities and specificities purely by inspection.

The widespread importance of induced fit and order-disorder transition in RNA recognition by proteins and small molecules makes it imperative that RNA motional properties are characterized quantitatively. Until now, however, very few studies have been dedicated to the systematic characterization of RNA motion and to their changes upon protein or small-molecule binding. The U1A protein-RNA complexes provide some of the best-studied examples of the role of RNA motional changes upon protein binding. Here, we report (13)C NMR relaxation studies of base and ribose dynamics for the RNA internal loop target of human U1A protein located within the 3'-untranslated region (3'-UTR) of the mRNA coding for U1A itself. We also report the semi-quantitative analysis of both fast (nano- to picosecond) and intermediate (micro- to millisecond) motions for this paradigmatic RNA system. We measure (13)C T(1), T(1rho) and heteronuclear nuclear Overhauser effects (NOEs) for sugar and base nuclei, as well as the power dependence of T(1rho) at 500 MHz and 750 MHz, and analyze these results using the model-free formalism. The results provide a much clearer picture of the type of motions experienced by this RNA in the absence of the protein than was provided by the analysis of the structure based solely on NOEs and scalar couplings. They define a model where the RNA internal loop region "breathes" on a micro- to millisecond timescale with respect to the double-helical regions. Superimposed on this slower motion, the residues at the very tip of the loop undergo faster (nano- to picosecond) motions. We hypothesize that these motions allow the RNA to sample multiple conformations so that the protein can select a structure within the ensemble that optimizes intermolecular contacts.

The yeast splicing factor pre-mRNA processing protein 40 (Prp40) comprises two N-terminal WW domains, separated by a ten-residue linker, and six consecutive FF domains. In the spliceosome, the Prp40 WW domains participate in cross-intron bridging by interacting with proline-rich regions present in the branch-point binding protein (BBP) and the U5 small nuclear ribonucleoprotein component Prp8. Furthermore, binding of Prp40 to the phosphorylated C-terminal domain (CTD) of the largest subunit of RNA polymerase II is thought to link splicing to transcription. To gain insight into this complex interaction network we have determined the solution structure of the tandem Prp40 WW domains by NMR spectroscopy and performed chemical shift mapping experiments with different proline-rich peptides. The WW domains each adopt the characteristic triple-stranded beta-sheet structure and are connected by a stable alpha-helical linker. On the basis of a detailed analysis of residual dipolar couplings (RDC) and 15N relaxation data we show that the tandem Prp40 WW domains behave in solution as a single folded unit with unique alignment and diffusion tensor, respectively. Using [1H-15N]-RDCs, we were able to accurately define the relative orientation of the WW domains revealing that the binding pockets of each domain face opposite sides of the structure. Furthermore, we found that both Prp40 WW domains interact with PPxY motifs (where x is any residue) present in peptides derived from the splicing factors BBP and Prp8. Moreover, the Prp40 WW domains are shown to bind proline-rich peptides devoid of aromatic residues, which are also recognised by the Abl-SH3 domain and the WW domain of the mammalian Prp40 orthologue formin binding protein 11. In contrast, no interaction was observed between the Prp40 WW domains and the CTD repeats used in this work.

The N-terminal RNA-binding domain of the human U1A protein (RBD1) undergoes local conformational changes upon binding to its target RNA. Here, the wild-type RBD1 and two mutants are examined with molecular dynamics simulations that are analyzed using the reorientational eigenmode dynamics (RED) formalism. The results reveal changes in the magnitude and extent of coupled intra-domain motions resulting from single amino acid substitutions. Interpretation of the novel RED results and corresponding NMR relaxation data suggests that the loss of collective motions in the mutants could account for their weak RNA-binding.

We perform folding simulations on 18 small proteins with using a simple Go-like protein model and analyze the folding rate constants, characteristics of the transition state ensemble, and those of the denatured states in terms of native topology and chain length. Near the folding transition temperature, the folding rate k(F) scales as k(F) approximately exp(-c RCO N(0.6)) where RCO and N are the relative contact order and number of residues, respectively. Here the topology RCO dependence of the rates is close to that found experimentally (k(F) approximately exp(-c RCO)), while the chain length N dependence is in harmony with the predicted scaling property (k(F) approximately exp(-c N(2/3))). Thus, this may provides a unified scaling law in folding rates at the transition temperature, k(F) approximately exp(-c RCO N(2/3)). The degree of residual structure in the denatured state is highly correlated with RCO, namely, proteins with smaller RCO tend to have more ordered structure in the denatured state. This is consistent with the observation that many helical proteins such as myoglobin and protein A, have partial helices, in the denatured states. The characteristics of the transition state ensemble calculated by the current model, which uses native topology but not sequence specific information, are consistent with experimental phi-value data for about half of proteins., (Copyright 2001 Academic Press.)

Significant advances have been made in elucidating the biogenesis pathway and three-dimensional structure of the UsnRNPs, the building blocks of the spliceosome. U2 and U4/U6*U5 tri-snRNPs functionally associate with the pre-mRNA at an earlier stage of spliceosome assembly than previously thought, and additional evidence supporting UsnRNA-mediated catalysis of pre-mRNA splicing has been presented.

An adaptive binding mechanism, requiring large conformational rearrangements, occurs commonly with many RNA-protein associations. To explore this process of reorganization, we have investigated the conformational change upon spliceosomal U1A-RNA binding with molecular dynamics (MD) simulations and free energy analyses. We computed the energetic cost of conformational change in U1A-hairpin and U1A-internal loop binding using a hybrid of molecular mechanics and continuum solvent methods. Encouragingly, in all four free energy comparisons (two slightly different proteins, two different RNAs), the free macromolecule was more stable than the bound form by the physically reasonable value of approximately 10 kcal/mol. We calculated the absolute binding free energies for both complexes to be in the same range as that found experimentally., (Copyright 2000 Academic Press.)

Well-ordered crystals of a genomic hepatitis delta virus (HDV) ribozyme, a large, globular RNA, were obtained employing a new crystallization method. A high-affinity binding site for the spliceosomal protein U1A was engineered into a segment of the catalytic RNA that is dispensable for catalysis. Because molecular surfaces of proteins are more chemically varied than those of RNA, the presence of the protein moiety was expected to facilitate crystallization and improve crystal order. The HDV ribozyme-U1A complex crystallized readily, and its structure was solved using standard techniques for heavy-atom derivatization of protein crystals. Over 1200 A(2) of the solvent-accessible surface area of the complex are involved in crystal contacts. As protein-protein interactions comprise 85% of this buried area, these crystals appear to be held together predominantly by the protein component of the complex. Our crystallization method should be useful for the structure determination of other biochemically important RNAs for which protein partners do not exist or are experimentally intractable. The refined model of the complex (R-free=27.9% for all reflections between 20.0 and 2.3 A) reveals an RNA with a deep active site cleft. Well-ordered metal ions are not observed crystallographically in this cavity. Biochemical results of previous workers had suggested an important role in catalysis for cytosine 75. The pyrimidine base of this residue is buried at the bottom of the active site in an environment that could raise its pK(a) value. We propose that this highly conserved cytosine may be the general base that catalyzes the transesterification., (Copyright 2000 Academic Press.)

The mammalian spliceosomal protein U1A binds a hairpin RNA with picomolar affinity. To examine the origin of this binding specificity, we carried out computational mutagenesis on protein and RNA residues in the U1A-RNA binding interface. Our computational mutagenesis methods calculate the relative binding affinity between mutant and wild-type as the sum of molecular mechanical energies and solvation free energies estimated with a continuum solvent model. We obtained good agreement with experimental studies and we verified mutations that abolish and improve binding. Therefore, we offer these methods as computationally inexpensive tools for investigating and predicting the effects of site-specific mutagenesis., (Copyright 2000 Academic Press.)

The ribonucleoprotein (RNP) domain is one of the most common eukaryotic protein domains, and is found in many proteins involved in recognition of a wide variety of RNAs. Two structures of RNA complexes of human U1A protein have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning how RNP domains discriminate between different RNAs. In this work, we extend the investigation of U1A-RNA recognition by comparing the dynamics of U1A protein both free and in complex with RNA. We have also investigated the trimolecular complex between two U1A proteins and the complete polyadenylation inhibition element to study the effect of RNA-dependent protein-protein interactions on protein conformational flexibility. We report that changes in backbone dynamics upon complex formation identify regions of the protein where conformational exchange processes are quenched in the RNA-bound conformation. Furthermore, amino acids whose side-chains experience significant changes in conformational flexibility coincide with residues particularly important for the specificity of the U1A protein/RNA interaction. This study adds a new dimension to the description of the coordinated changes in structure and dynamics that are critical to define the biological specificity of U1A and other RNP proteins., (Copyright 1999 Academic Press.)

The small nuclear ribonucleoprotein particles (snRNP) U1, U2, U4, and U5 contain a common set of eight Sm proteins that bind to the conserved single-stranded 5'-PuAU3-6GPu-3' (Sm binding site) region of their constituent U snRNA (small nuclear RNA), forming the Sm core RNP. Using native and in vitro reconstituted U1 snRNPs, accessibility of the RNA within the Sm core RNP to chemical structure probes was analyzed. Hydroxyl radical footprinting of in vitro reconstituted U1 snRNP demonstrated that riboses within a large continuous RNA region, including the Sm binding site, were protected. This protection was dependent on the binding of the Sm proteins. In contrast with the riboses, the phosphate groups within the Sm core site were accessible to modifying reagents. The invariant adenosine residue at the 5' end, as well as an adenosine two nucleotides downstream of the Sm binding site, showed an unexpected reactivity with dimethyl sulfate. This novel reactivity could be attributed to N7-methylation of the adenosine and was not observed in naked RNA, indicating that it is an intrinsic property of the RNA- protein interactions within the Sm core RNP. Further, this reactivity was observed concomitantly with formation of the Sm subcore intermediate during Sm core RNP assembly. As the Sm subcore can be viewed as the commitment complex in this assembly pathway, these results suggest that the peculiar reactivity of the Sm site adenosine bases may be diagnostic for proper assembly of the Sm core RNP. Consistent with this idea, a strong correlation was found between the unusual N7-A methylation sensitivity of the Sm core RNP and its ability to be imported into the nucleus of Xenopus laevis oocytes., (Copyright 1999 Academic Press.)

Pairwise coupling theory is applied here to determine the energetic interactions between two elements of the N-terminal RNA binding domain (RBD) of the human U1A protein. The novel application of the theory to this system incorporates both measurements of protein stability and RNA binding to define thermodynamic cycles. In this first example of the application, two regions of the protein are selected for study: tyrosine 13, one of the conserved aromatic residues on the surface of the beta-sheet, and the C-terminal tail of the RBD. The six initial pairwise coupling free energies derived from this system describe the communication between these positions, both in the free and RNA-bound states of the protein. The results show that in the absence of RNA, these two elements of the protein act independently. However, when RNA is bound, there is indirect coupling between Tyr13 and the tail, mediated through the RNA. Subsequent thermodynamic cycles involving additional perturbations to the C-terminal tail further define the communication between the C terminus and the beta-sheet. This work demonstrates the general applicability of the pairwise coupling theory to protein:nucleic acid interactions, and illustrates the necessity of such analyses to describe the network of energetic interactions that comprise RNA recognition by this RBD.

The solution structure of a fragment of the human U1A spliceosomal protein containing residues 2 to 117 (U1A117) determined using multi-dimensional heteronuclear NMR is presented. The C-terminal region of the molecule is considerably more ordered in the free protein than thought previously and its conformation is different from that seen in the crystal structure of the complex with U1 RNA hairpin II. The residues between Asp90 and Lys98 form an alpha-helix that lies across the beta-sheet, with residues IIe93, IIe94 and Met97 making contacts with Leu44, Phe56 and IIe58. This interaction prevents solvent exposure of hydrophobic residues on the surface of the beta-sheet, thereby stabilising the protein. Upon RNA binding, helix C moves away from this position, changing its orientation by 135 degrees to allow Tyr13, Phe56 and Gln54 to stack with bases of the RNA, and also allowing Leu44 to contact the RNA. The new position of helix C in the complex with RNA is stabilised by hydrophobic interactions from IIe93 and IIe94 to IIe58, Leu 41, Val62 and His 10, as well as a hydrogen bond between Ser91 and Thr11. The movement of helix C mainly involves changes in the main-chain torsion angles of Thr89, Asp90 and Ser91, the helix thereby acting as a "lid" over the RNA binding surface.

Initiation of translation in prokaryotes requires the formation of a complex between the messenger RNA, the 30 S ribosomal subunit and the initiator tRNA(fMet). Initiation factor IF3 binds to the 30 S ribosomal subunit and proof-reads the initiation complex, thereby ensuring the accuracy of this step. IF3 also plays a pleiotropic role in the regulation of translation, as a result of differential influences exerted on the levels of the initiation of translation of genes or groups of genes. IF3 is composed of two independent domain or roughly identical sizes. We have expressed and purified the C-terminal domain of E. coli IF3 and shown that it retains both the 30 S particle binding and 70 S ribosome dissociating activities of the native protein. We have obtained 1H and 15N NMR resonance assignments and its 3D solution structure was calculated using 551 restraints. It is composed of a mixed beta-sheet backed by two alpha-helices. It shows a striking resemblance to the U1A small nuclear ribonucleoprotein structure, which binds to the U1 snRNA in the eukaryotic spliceosome. This suggests a convergent evolution process for these two proteins that are associated with ribonucleoproteic complexes.

The hairpin is one of the most commonly found structural motifs of RNA and is often a binding site for proteins. Crystallisation of U1A spliceosomal protein bound to a RNA hairpin, its natural binding site on U1snRNA, is described. RNA oligonucleotides were synthesised either chemically or by in vitro transcription using T7 RNA polymerase and purified to homogeneity by gel electrophoresis. Crystallisation trials with the wild-type protein sequence and RNA hairpins containing various stem sequences and overhanging nucleotides only resulted in a cubic crystal form which diffracted to 7-8 A resolution. A new crystal form was grown by using a protein variant containing mutations of two surface residues. The N-terminal sequence of the protein was also varied to reduce heterogeneity which was detected by protein mass spectrometry. A further crystallisation search using the double mutant protein and varying the RNA hairpins resulted in crystals diffracting to beyond 1.7 A. The methods and strategy described in this paper may be applicable to crystallisation of other RNA-protein complexes.

Research on the interplay between chromatin and transcription has progressed along three lines during the past year. Evidence has been reported for disruption of nucleosomes by transcriptional regulatory proteins in cell-free systems; displacement of the histone octamer during transcription has been conclusively demonstrated; and insights into transcriptional repression by heterochromatin have been gained from studies of silent mating loci and telomeres in yeast.

We have obtained backbone 1H, 15N, and 13C assignments and determined the secondary structure and folding topology of the C-terminal RNA-binding domain (RBD) of the human U1A protein. The secondary structure derived from NOE data is in excellent agreement with the predicted structure from the 1H and 13C chemical shift indices. This 88 amino acid domain exhibits a beta alpha beta-beta alpha beta folding pattern, with conserved RNP1 and RNP2 sequences located in two adjacent strands of a four-strand antiparallel beta-sheet. This global folding pattern is typical of this class of RNA binding proteins. Although this domain contains residues that are conserved in all RBDs, its RNA binding properties are very unusual. RNA binding studies show that this domain does not bind U1, U2 or U5 snRNA, an RNA hairpin, rA16, rU16, rC16 or rA3U3GUA4, nor does it show significant association to populations of random sequence RNAs.