Evaluating conformational changes in protein structures binding RNA

Many protein-RNA recognition events are known to exhibit conformational changes from qualitative observations of individual complexes. However, a quantitative estimation of conformational changes is required if protein-RNA docking and template-based methods for RNA binding site prediction are to be developed. This study presents the first quantitative evaluation of conformational changes that occur when proteins bind RNA. The analysis of twelve RNA-binding proteins in the bound and unbound states using error-scaled difference distance matrices is presented. The binding site residues are mapped to each structure, and the conformational changes that affect these residues are evaluated. Of the twelve proteins four exhibit greater movements in nonbinding site residues, and a further four show the greatest movements in binding site residues. The remaining four proteins display no significant conformational change. When interface residues are found to be in conformationally variable regions of the protein they are typically seen to move less than 2 A between the bound and unbound conformations. The current data indicate that conformational changes in the binding site residues of RNA binding proteins may not be as significant as previously suggested, but a larger data set is required before wider conclusions may be drawn. The implications of the observed conformational changes for protein function prediction are discussed.     

Annexin B12 is a sensor of membrane curvature and undergoes major curvature-dependent structural changes

The regulation of membrane curvature plays an important role in many membrane trafficking and fusion events. Recent studies have begun to identify some of the proteins involved in controlling and sensing the curvature of cellular membranes. A mechanistic understanding of these processes is limited, however, as structural information for the membrane-bound forms of these proteins is scarce. Here, we employed a combination of biochemical and biophysical approaches to study the interaction of annexin B12 with membranes of different curvatures. We observed selective and Ca(2+)-independent binding of annexin B12 to negatively charged vesicles that were either highly curved or that contained lipids with negative intrinsic curvature. This novel curvature-dependent membrane interaction induced major structural rearrangements in the protein and resulted in a backbone fold that was different from that of the well characterized Ca(2+)-dependent membrane-bound form of annexin B12. Following curvature-dependent membrane interaction, the protein retained a predominantly alpha-helical structure but EPR spectroscopy studies of nitroxide side chains placed at selected sites on annexin B12 showed that the protein underwent inside-out refolding that brought previously buried hydrophobic residues into contact with the membrane. These structural changes were reminiscent of those previously observed following Ca(2+)-independent interaction of annexins with membranes at mildly acidic pH, yet they occurred at neutral pH in the presence of curved membranes. The present data demonstrate that annexin B12 is a sensor of membrane curvature and that membrane curvature can trigger large scale conformational changes. We speculate that membrane curvature could be a physiological signal that induces the previously reported Ca(2+)-independent membrane interaction of annexins in vivo.     

The Mad2 spindle checkpoint protein undergoes similar major conformational changes upon binding to either Mad1 or Cdc20.

Mad2 participates in spindle checkpoint inhibition of APC(Cdc20). We show that RNAi-mediated suppression of Mad1 function in mammalian cells causes loss of Mad2 kinetochore localization and impairment of the spindle checkpoint. Mad1 and Cdc20 contain Mad2 binding motifs that share a common consensus. We have identified a class of Mad2 binding peptides with a similar consensus. Binding of one of these ligands, MBP1, triggers an extensive rearrangement of the tertiary structure of Mad2. Mad2 also undergoes a similar striking structural change upon binding to a Mad1 or Cdc20 binding motif peptide. Our data suggest that, upon checkpoint activation, Mad1 recruits Mad2 to unattached kinetochores and may promote binding of Mad2 to Cdc20.     

PTEN phosphatase selectively binds phosphoinositides and undergoes structural changes

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor that is mutated or deleted in a variety of human tumors, and even loss of only one PTEN gene profoundly affects carcinogenesis. PTEN encodes a phosphatidylinositol phosphate phosphatase specific for the 3-position of the inositol ring. Despite its importance, we are just beginning to understand the regulatory circuits that maintain the correct levels of PTEN phosphatase activity. Several independent studies reported that PI(4,5)P2 enhances PTEN phosphatase activity, but the reasons for this enhancement are currently being debated. In this study, PTEN bound to PI(4,5)P2-bearing vesicles has increased alpha-helicity, providing direct spectroscopic proof of a conformational change. Neither PI(3,5)P2 nor PI(3,4,5)P3 induced this conformational change. On the basis of experiments with two mutant PTEN proteins, it is shown that PI(4,5)P2 induces this conformational change by binding to the PTEN N-terminal domain. Using PTEN protein and a 21-amino acid peptide based on the PTEN N-terminus, we tested all natural phosphatidylinositol phosphates and found preferential binding of PI(4,5)P2. PTEN also binds to phosphatidylserine-bearing vesicles, resulting in a slight increase in beta-sheet content. In addition, PTEN binds synergistically to PI(4,5)P2 and phosphatidylserine, and hence, these anionic lipids do not compete for PTEN binding sites. Collectively, these results demonstrate that PTEN binds to membranes through multiple sites, but only PI(4,5)P2 binding to the N-terminal domain triggers a conformational change with increased alpha-helicity.     

Transposition of two amino acids changes a promiscuous RNA binding protein into a sequence-specific RNA binding protein

In yeast (Saccharomyces cerevisiae), the branchpoint binding protein (BBP) recognizes the conserved yeast branchpoint sequence (UACUAAC) with a high level of specificity and affinity, while the human branchpoint binding protein (SF1) binds the less-conserved consensus branchpoint sequence (CURAY) in human introns with a lower level of specificity and affinity. To determine which amino acids in BBP provide the additional specificity and affinity absent in SF1, a panel of chimeric SF1 proteins was tested in RNA binding assays with wild-type and mutant RNA substrates. This approach revealed that the QUA2 domain of BBP is responsible for the enhanced RNA binding affinity and specificity displayed by BBP compared with SF1. Within the QUA2 domain, a transposition of adjacent arginine and lysine residues is primarily responsible for the switch in RNA binding between BBP and SF1. Alignment of multiple branchpoint binding proteins and the related STAR/GSG proteins suggests that the identity of these two amino acids and the RNA target sequences of all of these proteins are correlated.     

A major determinant of hnRNP C protein binding to RNA is a novel bZIP-like RNA binding domain.

The C protein tetramer of hnRNP complexes binds approximately 150-230 nt of RNA with high cooperativity (McAfee J et al., 1996, Biochemistry 35:1212-1222). Three contiguously bound tetramers fold 700-nt lengths of RNA into a 19S triangular intermediate that nucleates 40S hnRNP assembly in vitro (Huang M et al., 1994, Mol Cell Biol 14:518-533). Although it has been assumed that the consensus RNA recognition motif (RRM) of C protein (residues 8-87) is the primary determinant of RNA binding, we report here that a recombinant construct containing residues 1-115 has very low affinity for RNA at physiological ionic strength (100 mM NaCl). Moreover, we demonstrate that an N-terminal deletion construct lacking the consensus RRM but containing residues 140-290 binds RNA with an affinity sufficient to account for the total free energy change observed for the binding of intact protein. Like native C protein, the 140-290 construct is a tetramer in solution and binds RNA stoichiometrically in a salt-resistant manner in 100-300 mM NaCl. Residues 140-179 of the N-terminal truncated variant contain 11 basic and 2 acidic residues, whereas residues 180-207 specify a leucine zipper motif that directs dimer assembly. Elements within the 50-residue carboxy terminus of C protein are required for tetramer assembly. A basic region followed by a leucine zipper is identical to the domain organization of the basic-leucine zipper (bZIP) class of DNA binding proteins. Sequence homologies with other proteins containing RRMs and the bZIP motif suggest that residues 140-207 represent a conserved bZIP-like RNA binding motif (designated bZLM). The steric orientation of four high-affinity RNA binding sites about rigid leucine zipper domains may explain in part C protein''s asymmetry, its large occluded site size, and its RNA folding activity.     

Structure of tandem RNA recognition motifs from polypyrimidine tract binding protein reveals novel features of the RRM fold

Polypyrimidine tract binding protein (PTB), an RNA binding protein containing four RNA recognition motifs (RRMs), is involved in both pre-mRNA splicing and translation initiation directed by picornaviral internal ribosome entry sites. Sequence comparisons previously indicated that PTB is a non-canonical RRM protein. The solution structure of a PTB fragment containing RRMs 3 and 4 shows that the protein consists of two domains connected by a long, flexible linker. The two domains tumble independently in solution, having no fixed relative orientation. In addition to the betaalphabetabetaalphabeta topology, which is characteristic of RRM domains, the C-terminal extension of PTB RRM-3 incorporates an unanticipated fifth beta-strand, which extends the RNA binding surface. The long, disordered polypeptide connecting beta4 and beta5 in RRM-3 is poised above the RNA binding surface and is likely to contribute to RNA recognition. Mutational analyses show that both RRM-3 and RRM-4 contribute to RNA binding specificity and that, despite its unusual sequence, PTB binds RNA in a manner akin to that of other RRM proteins.     

Studies on the inference of protein binding regions across fold space based on structural similarities

The emerging picture of a continuous protein fold space highlights the existence of non obvious structural similarities between proteins with apparent different topologies. The identification of structure resemblances across fold space and the analysis of similar recognition regions may be a valuable source of information towards protein structure-based functional characterization. In this work, we use non-sequential structural alignment methods (ns-SAs) to identify structural similarities between protein pairs independently of their SCOP hierarchy, and we calculate the significance of binding region conservation using the interacting residues overlap in the ns-SA. We cluster the binding inferences for each family to distinguish already known family binding regions from putative new ones. Our methodology exploits the enormous amount of data available in the PDB to identify binding region similarities within protein families and to propose putative binding regions. Our results indicate that there is a plethora of structurally common binding regions among proteins, independently of current fold classifications. We obtain a 6- to 8-fold enrichment of novel binding regions, and identify binding inferences for 728 protein families that so far lack binding information in the PDB. We explore binding mode analogies between ligands from commonly clustered binding regions to investigate the utility of our methodology. A comprehensive analysis of the obtained binding inferences may help in the functional characterization of protein recognition and assist rational engineering. The data obtained in this work is available in the download link at www.scowlp.org.     

Two distinct binding modes of a protein cofactor with its target RNA

Like most cellular RNA enzymes, the bI5 group I intron requires binding by a protein cofactor to fold correctly. Here, we use single-molecule approaches to monitor the structural dynamics of the bI5 RNA in real time as it assembles with its CBP2 protein cofactor. These experiments show that CBP2 binds to the target RNA in two distinct modes with apparently opposite effects: a "non-specific" mode that forms rapidly and induces large conformational fluctuations in the RNA, and a "specific" mode that forms slowly and stabilizes the native RNA structure. The bI5 RNA folds though multiple pathways toward the native state, typically traversing dynamic intermediate states induced by non-specific binding of CBP2. These results suggest that the protein cofactor-assisted RNA folding involves sequential non-specific and specific protein-RNA interactions. The non-specific interaction potentially increases the local concentration of CBP2 and the number of conformational states accessible to the RNA, which may promote the formation of specific RNA-protein interactions.     

Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions

Contradictory reports in the literature have emphasised either the sequence of small interfering RNAs (siRNA) or the structure of their target molecules to be the major determinant of the efficiency of RNA interference (RNAi) approaches. In the present study, we analyse systematically the contributions of these parameters to siRNA activity by using deliberately designed mRNA constructs. The siRNA target sites were included in well-defined structural elements rendering them either highly accessible or completely involved in stable base-pairing. Furthermore, complementary sequence elements and various hairpins with different stem lengths and designs were used as target sites. Only one of the strands of the siRNA duplex was found to be capable of silencing via its respective target site, indicating that thermodynamic characteristics intrinsic to the siRNA strands are a basic determinant of siRNA activity. A significant obstruction of gene silencing by the same siRNA, however, was observed to be caused by structural features of the substrate RNA. Bioinformatic analysis of the mRNA structures suggests a direct correlation between the extent of gene-knockdown and the local free energy in the target region. Our findings indicate that, although a favourable siRNA sequence is a necessary prerequisite for efficient RNAi, complex target structures may limit the applicability even of carefully chosen siRNAs.     

Demonstration that the insulin receptor undergoes an early structural modification following insulin binding

Processing of the insulin receptor by hepatocytes was studied using a 125I-labelled photoreactive insulin derivative which could be covalently attached to the receptor and facilitate the analysis of receptor structure in isolated subcellular fractions by SDS-polyacrylamide gel electrophoresis. Following binding at the cell surface, the label was rapidly internalised and located in a low-density subcellular fraction ('endosomes'). The intact receptor (350 000 molecular weight) and binding (alpha) subunit (135 000), produced by in vitro disulphide reduction of the samples, were found in the plasma membrane fraction but not in endosomes. In endosomes, the label was concentrated in a band at 140 000 (non-reduced) which on reduction generated species of 100 000 and 68 000 predominantly. The insulin receptor therefore undergoes an early structural change during endocytosis. This modification does not involve complete disulphide reduction and may be due to a proteolytic event.     

High-throughput prediction of RNA,DNA and protein binding regions mediated by intrinsic disorder

Intrinsically disordered proteins and regions (IDPs and IDRs) lack stable 3D structure under physiological conditions in-vitro, are common in eukaryotes, and facilitate interactions with RNA, DNA and proteins. Current methods for prediction of IDPs and IDRs do not provide insights into their functions, except for a handful of methods that address predictions of protein-binding regions. We report first-of-its-kind computational method DisoRDPbind for high-throughput prediction of RNA, DNA and protein binding residues located in IDRs from protein sequences. DisoRDPbind is implemented using a runtime-efficient multi-layered design that utilizes information extracted from physiochemical properties of amino acids, sequence complexity, putative secondary structure and disorder and sequence alignment. Empirical tests demonstrate that it provides accurate predictions that are competitive with other predictors of disorder-mediated protein binding regions and complementary to the methods that predict RNA- and DNA-binding residues annotated based on crystal structures. Application in Homo sapiens, Mus musculus, Caenorhabditis elegans and Drosophila melanogaster proteomes reveals that RNA- and DNA-binding proteins predicted by DisoRDPbind complement and overlap with the corresponding known binding proteins collected from several sources. Also, the number of the putative protein-binding regions predicted with DisoRDPbind correlates with the promiscuity of proteins in the corresponding protein–protein interaction networks. Webserver: http://biomine.ece.ualberta.ca/DisoRDPbind/     

HIV-1 structural gene expression requires binding of the Rev trans-activator to its RNA target sequence

Expression of human immunodeficiency virus type 1 structural proteins requires both the viral Rev trans-activator and its cis-acting RNA target sequence, the Rev response element (RRE). The RRE has been mapped to a conserved region of the HIV-1 env gene and is predicted to form a complex, highly stable RNA stem-loop structure. Site-directed mutagenesis was used to define a small subdomain of the RRE, termed stem-loop II, that is essential for biological activity. Gel retardation assays demonstrated that the Rev trans-activator is a sequence-specific RNA binding protein. The RRE stem-loop II subdomain was found to be both necessary and sufficient for the binding of Rev by the RRE. We propose that the HIV-1 Rev trans-activator belongs to a new class of sequence-specific RNA binding proteins characterized by the presence of an arginine-rich binding motif.     

Gene overlap results in a viral protein having an RNA binding domain and a major coat protein domain

L-A double-stranded RNA (dsRNA) replicates in vivo in yeast in a conservative, asynchronous (first [+] strand then [-] strand), intraviral process. New particles are formed by packaging (+) strands. Added viral (+) single-stranded RNA (ssRNA) is specifically bound by empty virus-like particles (VLPs) and, in a reaction requiring a host factor, is converted in vitro to dsRNA. We find that the isolated binding complex replicates only if it was formed in the presence of the host factor. The VLP minor 180 kd protein, but not the major coat protein, has ssRNA binding activity on Western blots. The 180 kd protein shares a common antigenic domain with the major coat protein, the latter known to be encoded by L-A dsRNA. The 180 kd protein, but not the major coat protein, also shares an antigenic domain with a sequence encoded by the 3' end of the L-A (+) strand. Thus the 180 kd protein is also encoded by L-A dsRNA and consists of a major coat protein domain and a ssRNA binding domain.     

Evidence for major structural changes in subunit C of the vacuolar ATPase due to nucleotide binding

The ability of subunit C of eukaryotic V-ATPases to bind ADP and ATP is demonstrated by photoaffinity labeling and fluorescence correlation spectroscopy (FCS). Quantitation of the photoaffinity and the FCS data indicate that the ATP-analogues bind more weakly to subunit C than the ADP-analogues. Site-directed mutagenesis and N-terminal sequencing of subunit C from Arabidopsis (VHA-C) and yeast (Vma5p) have been used to map the C-terminal region of subunit C as the nucleotide-binding site. Tryptophan fluorescence quenching and decreased susceptibility to tryptic digestion of subunit C after binding of different nucleotides provides evidence for structural changes in this subunit caused by nucleotide-binding.