Crystal structure of vinculin in complex with vinculin binding site 50 (VBS50), the integrin binding site 2 (IBS2) of talin

The cytoskeletal protein talin activates integrin receptors by binding of its FERM domain to the cytoplasmic tail of β‐integrin. Talin also couples integrins to the actin cytoskeleton, largely by binding to and activating the cytoskeletal protein vinculin, which binds to F‐actin through the agency of its five‐helix bundle tail (Vt) domain. Talin activates vinculin by means of buried amphipathic α‐helices coined vinculin binding sites (VBSs) that reside within numerous four‐ and five‐helix bundle domains that comprise the central talin rod, which are released from their buried locales by means of mechanical tension on the integrin:talin complex. In turn, these VBSs bind to the N‐terminal seven‐helix bundle (Vh1) domain of vinculin, creating an entirely new helix bundle that severs its head‐tail interactions. Interestingly, talin harbors a second integrin binding site coined IBS2 that consists of two five‐helix bundle domains that also contain a VBS (VBS50). Here we report the crystal structure of VBS50 in complex with vinculin at 2.3 Å resolution and show that intramolecular interactions of VBS50 within IBS2 are much more extensive versus its interactions with vinculin. Indeed, the IBS2‐vinculin interaction only occurs at physiological temperature and the affinity of VBS50 for vinculin is about 30 times less than other VBSs. The data support a model where integrin binding destabilizes IBS2 to allow it to bind to vinculin.     

Structure and functional implications of a complex containing a segment of U6 RNA bound by a domain of Prp24

U6 RNA plays a critical role in pre-mRNA splicing. Assembly of U6 into the spliceosome requires a significant structural rearrangement and base-pairing with U4 RNA. In the yeast Saccharomyces cerevisiae, this process requires the essential splicing factor Prp24. We present the characterization and structure of a complex containing one of Prp24''s four RNA recognition motif (RRM) domains, RRM2, and a fragment of U6 RNA. NMR methods were used to identify the preferred U6 binding sequence of RRM2 (5′-GAGA-3′), measure the affinity of the interaction, and solve the structure of RRM2 bound to the hexaribonucleotide AGAGAU. Interdomain contacts observed between RRM2 and RRM3 in a crystal structure of the free protein are not detectable in solution. A structural model of RRM1 and RRM2 bound to a longer segment of U6 RNA is presented, and a partial mechanism for Prp24''s annealing activity is proposed.     

Intermolecular versus intramolecular interactions of the vinculin binding site 33 of talin

The cytoskeletal proteins talin and vinculin are localized at cell‐matrix junctions and are key regulators of cell signaling, adhesion, and migration. Talin couples integrins via its FERM domain to F‐actin and is an important regulator of integrin activation and clustering. The 220 kDa talin rod domain comprises several four‐ and five‐helix bundles that harbor amphipathic α‐helical vinculin binding sites (VBSs). In its inactive state, the hydrophobic VBS residues involved in binding to vinculin are buried within these helix bundles, and the mechanical force emanating from bound integrin receptors is thought necessary for their release and binding to vinculin. The crystal structure of a four‐helix bundle of talin that harbors one of these VBSs, coined VBS33, was recently determined. Here we report the crystal structure of VBS33 in complex with vinculin at 2 Å resolution. Notably, comparison of the apo and vinculin bound structures shows that intermolecular interactions of the VBS33 α‐helix with vinculin are more extensive than the intramolecular interactions of the VBS33 within the talin four‐helix bundle.     

Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins.

By screening a yeast two-hybrid library with COOH-terminal fragments of vinculin/metavinculin as the bait, we identified a new protein termed raver1. Raver1 is an 80-kD multidomain protein and widely expressed but to varying amounts in different cell lines. In situ and in vitro, raver1 forms complexes with the microfilament-associated proteins vinculin, metavinculin, and alpha-actinin and colocalizes with vinculin/metavinculin and alpha-actinin at microfilament attachment sites, such as cell-cell and cell matrix contacts of epithelial cells and fibroblasts, respectively, and in costameres of skeletal muscle. The NH2-terminal part of raver1 contains three RNA recognition motifs with homology to members of the heterogeneous nuclear RNP (hnRNP) family. Raver1 colocalizes with polypyrimidine tract binding protein (PTB)/hnRNPI, a protein involved in RNA splicing of microfilament proteins, in the perinucleolar compartment and forms complexes with PTB/hnRNPI. Hence, raver1 is a dual compartment protein, which is consistent with the presence of nuclear location signal and nuclear export sequence motifs in its sequence. During muscle differentiation, raver1 migrates from the nucleus to the costamere. We propose that raver1 may coordinate RNA processing and targeting as required for microfilament anchoring in specific adhesion sites.     

Crystal structure of human vinculin

Alterations in the actin cytoskeleton following the formation of cell-matrix and cell-cell junctions are orchestrated by vinculin. Vinculin associates with a large number of cytoskeletal and signaling proteins, and this flexibility is thought to contribute to rapid dissociation and reassociations of adhesion complexes. Intramolecular interactions between vinculin's head (Vh) and tail (Vt) domains limit access of its binding sites for other adhesion proteins. While the crystal structures of the Vh and Vt domains are known, these domains represent less than half of the entire protein and are separated by a large central region of unknown structure and function. Here we report the crystal structure of human full-length vinculin to 2.85 A resolution. In its resting state, vinculin is a loosely packed collection of alpha-helical bundles held together by Vh-Vt interactions. The three new well ordered alpha-helical bundle domains are similar in their structure to either Vh (Vh2 and Vh3) or to Vt (Vt2) and their loose packing provides the necessary flexibility that allows vinculin to interact with its various protein partners at sites of cell adhesion.     

Solution structure of the two RNA recognition motifs of hnRNP A1 using segmental isotope labeling: how the relative orientation between RRMs influences the nucleic acid binding topology

Human hnRNP A1 is a multi-functional protein involved in many aspects of nucleic-acid processing such as alternative splicing, micro-RNA biogenesis, nucleo-cytoplasmic mRNA transport and telomere biogenesis and maintenance. The N-terminal region of hnRNP A1, also named unwinding protein 1 (UP1), is composed of two closely related RNA recognition motifs (RRM), and is followed by a C-terminal glycine rich region. Although crystal structures of UP1 revealed inter-domain interactions between RRM1 and RRM2 in both the free and bound form of UP1, these interactions have never been established in solution. Moreover, the relative orientation of hnRNP A1 RRMs is different in the free and bound crystal structures of UP1, raising the question of the biological significance of this domain movement. In the present study, we have used NMR spectroscopy in combination with segmental isotope labeling techniques to carefully analyze the inter-RRM contacts present in solution and subsequently determine the structure of UP1 in solution. Our data unambiguously demonstrate that hnRNP A1 RRMs interact in solution, and surprisingly, the relative orientation of the two RRMs observed in solution is different from the one found in the crystal structure of free UP1 and rather resembles the one observed in the nucleic-acid bound form of the protein. This strongly supports the idea that the two RRMs of hnRNP A1 have a single defined relative orientation which is the conformation previously observed in the bound form and now observed in solution using NMR. It is likely that the conformation in the crystal structure of the free form is a less stable form induced by crystal contacts. Importantly, the relative orientation of the RRMs in proteins containing multiple-RRMs strongly influences the RNA binding topologies that are practically accessible to these proteins. Indeed, RRM domains are asymmetric binding platforms contacting single-stranded nucleic acids in a single defined orientation. Therefore, the path of the nucleic acid molecule on the multiple RRM domains is strongly dependent on whether the RRMs are interacting with each other. The different nucleic acid recognition modes by multiple-RRM domains are briefly reviewed and analyzed on the basis of the current structural information.     

The Interaction of Vinculin with Actin

Vinculin can interact with F-actin both in recruitment of actin filaments to the growing focal adhesions and also in capping of actin filaments to regulate actin dynamics. Using molecular dynamics, both interactions are simulated using different vinculin conformations. Vinculin is simulated either with only its vinculin tail domain (Vt), with all residues in its closed conformation, with all residues in an open I conformation, and with all residues in an open II conformation. The open I conformation results from movement of domain 1 away from Vt; the open II conformation results from complete dissociation of Vt from the vinculin head domains. Simulation of vinculin binding along the actin filament showed that Vt alone can bind along the actin filaments, that vinculin in its closed conformation cannot bind along the actin filaments, and that vinculin in its open I conformation can bind along the actin filaments. The simulations confirm that movement of domain 1 away from Vt in formation of vinculin 1 is sufficient for allowing Vt to bind along the actin filament. Simulation of Vt capping actin filaments probe six possible bound structures and suggest that vinculin would cap actin filaments by interacting with both S1 and S3 of the barbed-end, using the surface of Vt normally occluded by D4 and nearby vinculin head domain residues. Simulation of D4 separation from Vt after D1 separation formed the open II conformation. Binding of open II vinculin to the barbed-end suggests this conformation allows for vinculin capping. Three binding sites on F-actin are suggested as regions that could link to vinculin. Vinculin is suggested to function as a variable switch at the focal adhesions. The conformation of vinculin and the precise F-actin binding conformation is dependent on the level of mechanical load on the focal adhesion.     

Alternative conformations at the RNA-binding surface of the N-terminal U2AF(65) RNA recognition motif

The essential pre-mRNA splicing factor, U2 auxiliary factor 65KD (U2AF(65)) recognizes the polypyrimidine tract (Py-tract) consensus sequence of the pre-mRNA using two RNA recognition motifs (RRMs), the most prevalent class of eukaryotic RNA-binding domain. The Py-tracts of higher eukaryotic pre-mRNAs are often interrupted with purines, yet U2AF(65) must identify these degenerate Py-tracts for accurate pre-mRNA splicing. Previously, the structure of a U2AF(65) variant in complex with poly(U) RNA suggested that rearrangement of flexible side-chains or bound water molecules may contribute to degenerate Py-tract recognition by U2AF(65). Here, the X-ray structure of the N-terminal RRM domain of U2AF(65) (RRM1) is described at 1.47 A resolution in the absence of RNA. Notably, RNA-binding by U2AF(65) selectively stabilizes pre-existing alternative conformations of three side-chains located at the RNA interface (Arg150, Lys225, and Arg227). Additionally, a flexible loop connecting the beta2/beta3 strands undergoes a conformational change to interact with the RNA. These pre-existing alternative conformations may contribute to the ability of U2AF(65) to recognize a variety of Py-tract sequences. This rare, high-resolution view of an important member of the RRM class of RNA-binding domains highlights the role of alternative side-chain conformations in RNA recognition.     

Structural basis for RNA recognition by the N-terminal tandem RRM domains of human RBM45

RBM45 is an RNA-binding protein involved in neural development, whose aggregation is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). However, the mechanisms of RNA-binding and aggregation of RBM45 remain unelucidated. Here, we report the crystal structure of the N-terminal tandem RRM domains of human RBM45 in complex with single-stranded DNA (ssDNA). Our structural and biochemical results revealed that both the RRM1 and RRM2 of RBM45 recognized the GAC sequence of RNA/ssDNA. Two aromatic residues and an arginine residue in each RRM were critical for RNA-binding, and the interdomain linker was also involved in RNA-binding. Two RRMs formed a pair of antiparallel RNA-binding sites, indicating that the N-terminal tandem RRM domains of RBM45 bound separate GAC motifs in one RNA strand or GAC motifs in different RNA strands. Our findings will be helpful in the identification of physiologic targets of RBM45 and provide evidence for understanding the physiologic and pathologic functions of RBM45.     

Novel RNA recognition motif domain in the cytoplasmic polyadenylation element binding protein 3

The family of cytoplasmic polyadenylation element binding proteins CPEB1, CPEB2, CPEB3, and CPEB4 binds to the 3′‐untranslated region (3′‐UTR) of mRNA, and plays significant roles in mRNA metabolism and translation regulation. They have a common domain organization, involving two consecutive RNA recognition motif (RRM) domains followed by a zinc finger domain in the C‐terminal region. We solved the solution structure of the first RRM domain (RRM1) of human CPEB3, which revealed that CPEB3 RRM1 exhibits structural features distinct from those of the canonical RRM domain. Our structural data provide important information about the RNA binding ability of CPEB3 RRM1. Proteins 2014; 82:2879–2886. © 2014 Wiley Periodicals, Inc.     

Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases

Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B‐cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand‐bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS‐354825) at 1.9 Å resolution or to 4‐amino‐5‐(4‐phenoxyphenyl)‐7H‐pyrrolospyrimidin‐ 7‐yl‐cyclopentane at 1.6 Å resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp‐Glu‐Ile motif in the N‐terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.     

Distinct functions of the closely related tandem RNA-recognition motifs of hnRNP A1.

hnRNP A1 regulates alternative splicing by antagonizing SR proteins. It consists of two closely related, tandem RNA-recognition motifs (RRMs), followed by a glycine-rich domain. Analysis of variant proteins with duplications, deletions, or swaps of the RRMs showed that although both RRMs are required for alternative splicing function, each RRM plays distinct roles, and their relative position is important. Surprisingly, RRM2 but not RRM1 could support this function when duplicated, despite their very similar structure. Specific RNA binding and annealing are not sufficient for hnRNP A1 alternative splicing function. These observations, together with phylogenetic and structural data, suggest that the two RRMs are quasi-symmetric but functionally nonequivalent modules that evolved as components of a single bipartite domain.     

The PTB interacting protein raver1 regulates alpha-tropomyosin alternative splicing

Regulated switching of the mutually exclusive exons 2 and 3 of alpha-tropomyosin (TM) involves repression of exon 3 in smooth muscle cells. Polypyrimidine tract-binding protein (PTB) is necessary but not sufficient for regulation of TM splicing. Raver1 was identified in two-hybrid screens by its interactions with the cytoskeletal proteins actinin and vinculin, and was also found to interact with PTB. Consistent with these interactions raver1 can be localized in either the nucleus or cytoplasm. Here we show that raver1 is able to promote the smooth muscle-specific alternative splicing of TM by enhancing PTB-mediated repression of exon 3. This activity of raver1 is dependent upon characterized PTB-binding regulatory elements and upon a region of raver1 necessary for interaction with PTB. Heterologous recruitment of raver1, or just its C-terminus, induced very high levels of exon 3 skipping, bypassing the usual need for PTB binding sites downstream of exon 3. This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor.     

Nucleotide recognition by CopA,a Cu+‐transporting P‐type ATPase

Heavy metal pumps constitute a large subgroup in P‐type ion‐transporting ATPases. One of the outstanding features is that the nucleotide binding N‐domain lacks residues critical for ATP binding in other well‐studied P‐type ATPases. Instead, they possess an HP‐motif and a Gly‐rich sequence in the N‐domain, and their mutations impair ATP binding. Here, we describe 1.85 Å resolution crystal structures of the P‐ and N‐domains of CopA, an archaeal Cu⁺‐transporting ATPase, with bound nucleotides. These crystal structures show that CopA recognises the adenine ring completely differently from other P‐type ATPases. The crystal structure of the His462Gln mutant, in the HP‐motif, a disease‐causing mutation in human Cu⁺‐ATPases, shows that the Gln side chain mimics the imidazole ring, but only partially, explaining the reduction in ATPase activity. These crystal structures lead us to propose a role of the His and a mechanism for removing Mg²⁺ from ATP before phosphoryl transfer.     

ETR-3 and CELF4 protein domains required for RNA binding and splicing activity in vivo

Members of the CUG-BP and ETR-3 like factor (CELF) protein family bind within conserved intronic elements (called MSEs) flanking the cardiac troponin T (cTNT) alternative exon 5 and promote exon inclusion in vivo and in vitro. Here we use a comparative deletion analysis of two family members (ETR-3 and CELF4) to identify separate domains required for RNA binding and splicing activity in vivo. CELF proteins contain two adjacent RNA binding domains (RRM1 and RRM2) near the N-terminus and one RRM (RRM3) near the C-terminus, which are separated by a 160–230 residue divergent domain of unknown function. Either RRM1 or RRM2 of CELF4 are necessary and sufficient for binding MSE RNA and RRM2 plus an additional 66 amino acids of the divergent domain are as effective as full-length protein in activating MSE-dependent splicing in vivo. Non-overlapping N- and C-terminal regions of ETR-3 containing either RRM1 and RRM2 or RRM3 plus segments of the adjacent divergent domain activate MSE-dependent exon inclusion demonstrating an unusual functional redundancy of the N- and C-termini of the protein. These results identify specific regions of ETR-3 and CELF4 that are likely targets of protein–protein interactions required for splicing activation.     

Crystal structure of Lon protease: molecular architecture of gated entry to a sequestered degradation chamber

Lon proteases are distributed in all kingdoms of life and are required for survival of cells under stress. Lon is a tandem fusion of an AAA+ molecular chaperone and a protease with a serine‐lysine catalytic dyad. We report the 2.0‐Å resolution crystal structure of Thermococcus onnurineus NA1 Lon (TonLon). The structure is a three‐tiered hexagonal cylinder with a large sequestered chamber accessible through an axial channel. Conserved loops extending from the AAA+ domain combine with an insertion domain containing the membrane anchor to form an apical domain that serves as a gate governing substrate access to an internal unfolding and degradation chamber. Alternating AAA+ domains are in tight‐ and weak‐binding nucleotide states with different domain orientations and intersubunit contacts, reflecting intramolecular dynamics during ATP‐driven protein unfolding and translocation. The bowl‐shaped proteolytic chamber is contiguous with the chaperone chamber allowing internalized proteins direct access to the proteolytic sites without further gating restrictions.     

Mutations in RRM4 uncouple the splicing repression and RNA-binding activities of polypyrimidine tract binding protein

The polypyrimidine tract binding protein (PTB, or hnRNP I) contains four RNA-binding domains of the ribonucleoprotein fold type (RRMs 1, 2, 3, and 4), and mediates the negative regulation of alternative splicing through sequence-specific binding to intronic splicing repressor elements. To assess the roles of individual RRM domains in splicing repression, a neural-specific splicing extract was used to screen for loss-of-function mutations that fail to switch splicing from the neural to nonneural pathway. These results show that three RRMs are sufficient for wild-type RNA binding and splicing repression activity, provided that RRM4 is intact. Surprisingly, the deletion of RRM4, or as few as 12 RRM4 residues, effectively uncouples these functions. Such an uncoupling phenotype is unique to RRM4, and suggests a possible regulatory role for this domain either in mediating specific RNA contacts, and/or contacts with putative splicing corepressors. Evidence of a role for RRM4 in anchoring PTB binding adjacent to the branch site is shown by mobility shift and RNA footprinting assays.     

Crystal structure of Cwc2 reveals a novel architecture of a multipartite RNA-binding protein

The yeast splicing factor Cwc2 contacts several catalytically important RNA elements in the active spliceosome, suggesting that Cwc2 is involved in determining their spatial arrangement at the spliceosome's catalytic centre. We have determined the crystal structure of the Cwc2 functional core, revealing how a previously uncharacterized Torus domain, an RNA recognition motif (RRM) and a zinc finger (ZnF) are tightly integrated in a compact folding unit. The ZnF plays a pivotal role in the architecture of the whole assembly. UV-induced crosslinking of Cwc2-U6 snRNA allowed the identification by mass spectrometry of six RNA-contacting sites: four in or close to the RRM domain, one in the ZnF and one on a protruding element connecting the Torus and RRM domains. The three distinct regions contacting RNA are connected by a contiguous and conserved positively charged surface, suggesting an expanded interface for RNA accommodation. Cwc2 mutations confirmed that the connector element plays a crucial role in splicing. We conclude that Cwc2 acts as a multipartite RNA-binding platform to bring RNA elements of the spliceosome's catalytic centre into an active conformation.     

Proteome-Level Analysis Indicates Global Mechanisms for Post-Translational Regulation of RRM Domains

RRM, or RNA-recognition motif, domains are the largest class of single-stranded RNA binding domains in the human proteome and play important roles in RNA processing, splicing, export, stability, packaging, and degradation. Using a current database of post-translational modifications (PTMs), ProteomeScout, we found that RRM domains are also one of the most heavily modified domains in the human proteome. Here, we present two interesting findings about RRM domain modifications, found by mapping known PTMs onto RRM domain alignments and structures. First, we find significant overlap of ubiquitination and acetylation within RRM domains, suggesting the possibility for ubiquitination-acetylation crosstalk. Additionally, an analysis of quantitative study of ubiquitination changes in response to proteasome inhibition highlights the uniqueness of RRM domain ubiquitination – RRM domain ubiquitination decreases in response to proteasome inhibition, whereas the majority of sites increase. Second, we found conservation of tyrosine phosphorylation within the RNP1 and RNP2 consensus sequences, which coordinate RNA binding – suggesting a possible role for regulation of RNA binding by tyrosine kinase signaling. These observations suggest there are unique regulatory mechanisms of RRM function that have yet to be uncovered and that the RRM domain represents a model system for further studies on understanding PTM crosstalk.