WW or WoW: the WW domains in a union of bliss

WW domains are small protein modules that recognize proline-rich peptide motifs or phosphorylated-serine/threonine proline sites in cognate proteins. Within host proteins these modules are joined to other protein domains or to a variety of catalytic domains acting together as adaptors or targeting anchors of enzymes. An important aspect of signaling by WW domains is their ability to recognize their cognate ligands in tandem. Tandem WW domains not only act in a synergistic manner but also appear to chaperone the function of each other. In this review, we focus on structure, function, and mechanism of the tandem WW domains co-operativity as well as independent actions. We emphasize here the implications of tandem arrangement and cooperative function of the domains for signaling pathways.     

Structural analysis of WW domains and design of a WW prototype

Two new NMR structures of WW domains, the mouse formin binding protein and a putative 84.5 kDa protein from Saccharomyces cerevisiae, show that this domain, only 35 amino acids in length, defines the smallest monomeric triple-stranded antiparallel beta-sheet protein domain that is stable in the absence of disulfide bonds, tightly bound ions or ligands. The structural roles of conserved residues have been studied using site-directed mutagenesis of both wild type domains. Crucial interactions responsible for the stability of the WW structure have been identified. Based on a network of highly conserved long range interactions across the beta-sheet structure that supports the WW fold and on a systematic analysis of conserved residues in the WW family, we have designed a folded prototype WW sequence.     

Molecular basis for chromatin assembly and modification by multiprotein complexes

Abstract: Epigenetic regulation of the chromatin landscape is often orchestrated through modulation of nucleosomes. Nucleosomes are composed of two copies each of the four core histones, H2A, H2B, H3, and H4, wrapped in ~150 bp of DNA. We focus this review on recent structural studies that further elucidate the mechanisms used by macromolecular complexes to mediate histone modification and nucleosome assembly. Nucleosome assembly, spacing, and variant histone incorporation are coordinated by chromatin remodeler and histone chaperone complexes. Several recent structural studies highlight how disparate families of histone chaperones and chromatin remodelers share similar features that underlie how they interact with their respective histone or nucleosome substrates. Post‐translational modification of histone residues is mediated by enzymatic subunits within large complexes. Until recently, relatively little was known about how association with auxiliary subunits serves to modulate the activity and specificity of the enzymatic subunit. Analysis of several recent structures highlights the different modes that auxiliary subunits use to influence enzymatic activity or direct specificity toward individual histone residues.     

Theoretical investigation of IL-6 multiprotein receptor assembly

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates cell growth, differentiation, and cellular functions in many cell lineages. Recently, evidences for the formation of an active hexameric complex with an IL-6:IL-6Rα:gp130 stoichiometry of 2:2:2 have been obtained by different experimental approaches. Analysis of the electrostatic potential complementarity between IL-6 and its receptors has been used, in this study, to guide the assembly of homology-based 3D models of the components. The results strongly support a mechanism whereby the active cytokine (IL-6:IL-6Rα) associates with the signal transducing gp130 protein, and the trimeric complex formed further dimerizes to form the hexameric species. Furthermore, computational simulations of the multiprotein complexes provide a rationalization of data from mutation experiments and highlight some key protein–protein interactions which have not yet been the subject of mutagenesis studies. Proteins 29:528–544, 1997. © 1997 Wiley-Liss, Inc.     

Structural basis for phosphoserine-proline recognition by group IV WW domains

Pin1 contains an N-terminal WW domain and a C-terminal peptidyl-prolyl cis-trans isomerase (PPIase) domain connected by a flexible linker. To address the energetic and structural basis for WW domain recognition of phosphoserine (P.Ser)/phosphothreonine (P. Thr)- proline containing proteins, we report the energetic and structural analysis of a Pin1-phosphopeptide complex. The X-ray crystal structure of Pin1 bound to a doubly phosphorylated peptide (Tyr-P.Ser-Pro-Thr-P.Ser-Pro-Ser) representing a heptad repeat of the RNA polymerase II large subunit's C-terminal domain (CTD), reveals the residues involved in the recognition of a single P.Ser side chain, the rings of two prolines, and the backbone of the CTD peptide. The side chains of neighboring Arg and Ser residues along with a backbone amide contribute to recognition of P.Ser. The lack of widespread conservation of the Arg and Ser residues responsible for P.Ser recognition in the WW domain family suggests that only a subset of WW domains can bind P.Ser-Pro in a similar fashion to that of Pin1.     

WW domain sequence activity relationships identified using ligand recognition propensities of 42 WW domains

WW domains mediate protein-protein interactions in a number of different cellular functions by recognizing proline-containing peptide sequences. We determined peptide recognition propensities for 42 WW domains using NMR spectroscopy and peptide library screens. As potential ligands, we studied both model peptides and peptides based on naturally occurring sequences, including phosphorylated residues. Thirty-two WW domains were classified into six groups according to detected ligand recognition preferences for binding the motifs PPx(Y/poY), (p/phi)P(p,g)PPpR, (p/phi)PPRgpPp, PPLPp, (p/xi)PPPPP, and (poS/poT)P (motifs according to modified Seefeld Convention 2001). In addition to these distinct binding motifs, group-specific WW domain consensus sequences were identified. For PPxY-recognizing domains, phospho-tyrosine binding was also observed. Based on the sequences of the PPx(Y/poY)-specific group, a profile hidden Markov model was calculated and used to predict PPx(Y/poY)-recognition activity for WW domains, which were not assayed. PPx(Y/poY)-binding was found to be a common property of NEDD4-like ubiquitin ligases.     

CENP-C is a structural platform for kinetochore assembly

Centromeres provide a region of chromatin upon which kinetochores are assembled in mitosis. Centromeric protein C (CENP-C) is a core component of this centromeric chromatin that, when depleted, prevents the proper formation of both centromeres and kinetochores. CENP-C localizes to centromeres throughout the cell cycle via its C-terminal part, whereas its N-terminal part appears necessary for recruitment of some but not all components of the Mis12 complex of the kinetochore. We now find that all kinetochore proteins belonging to the KMN (KNL1/Spc105, the Mis12 complex, and the Ndc80 complex) network bind to the N-terminal part of Drosophila CENP-C. Moreover, we show that the Mis12 complex component Nnf1 interacts directly with CENP-C in vitro. To test whether CENP-C's N-terminal part was sufficient to recruit KMN proteins, we targeted it to the centrosome by fusing it to a domain of Plk4 kinase. The Mis12 and Ndc80 complexes and Spc105 protein were then all recruited to centrosomes at the expense of centromeres, leading to mitotic abnormalities typical of cells with defective kinetochores. Thus, the N-terminal part of Drosophila CENP-C is sufficient to recruit core kinetochore components and acts as the principal linkage between centromere and kinetochore during mitosis.     

Conformational rearrangements of RIG-I receptor on formation of a multiprotein:dsRNA assembly

The retinoic acid inducible gene-I (RIG-I)-like family of receptors is positioned at the front line of our innate cellular defence system. RIG-I detects and binds to foreign duplex RNA in the cytoplasm of both immune and non-immune cells, and initiates the induction of type I interferons and pro-inflammatory cytokines. The mechanism of RIG-I activation by double-stranded RNA (dsRNA) involves a molecular rearrangement proposed to expose the N-terminal pair of caspase activation recruitment domains, enabling an interaction with interferon-beta promoter stimulator 1 (IPS-1) and thereby initiating downstream signalling. dsRNA is particularly stimulatory when longer than 20 bp, potentially through allowing binding of more than one RIG-I molecule. Here, we characterize full-length RIG-I and RIG-I subdomains combined with a stimulatory 29mer dsRNA using multi-angle light scattering and size-exclusion chromatography–coupled small-angle X-ray scattering, to build up a molecular model of RIG-I before and after the formation of a 2:1 protein:dsRNA assembly. We report the small-angle X-ray scattering–derived solution structure of the human apo-RIG-I and observe that on binding of RIG-I to dsRNA in a 2:1 ratio, the complex becomes highly extended and flexible. Hence, here we present the first model of the fully activated oligomeric RIG-I.     

A unified representation of multiprotein complex data for modeling interaction networks

The protein interaction network presents one perspective for understanding cellular processes. Recent experiments employing high-throughput mass spectrometric characterizations have resulted in large data sets of physiologically relevant multiprotein complexes. We present a unified representation of such data sets based on an underlying bipartite graph model that is an advance over existing models of the network. Our unified representation allows for weighting of connections between proteins shared in more than one complex, as well as addressing the higher level organization that occurs when the network is viewed as consisting of protein complexes that share components. This representation also allows for the application of the rigorous MinMaxCut graph clustering algorithm for the determination of relevant protein modules in the networks. Statistically significant annotations of clusters in the protein-protein and complex-complex networks using terms from the Gene Ontology indicate that this method will be useful for posing hypotheses about uncharacterized components of protein complexes or uncharacterized relationships between protein complexes.     

Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

The second WW domain in mammalian Salvador protein (SAV1 WW2) is quite atypical, as it forms a beta-clam-like homodimer. The second WW domain in human MAGI1 (membrane associated guanylate kinase, WW and PDZ domain containing 1) (MAGI1 WW2) shares high sequence similarity with SAV1 WW2, suggesting comparable dimerization. However, an analytical ultracentrifugation study revealed that MAGI1 WW2 (Leu355-Pro390) chiefly exists as a monomer at low protein concentrations, with an association constant of 1.3 x 10(2) M(-1). We determined its solution structure, and a structural comparison with the dimeric SAV1 WW2 suggested that an Asp residue is crucial for the inhibition of the dimerization. The substitution of this acidic residue with Ser resulted in the dimerization of MAGI1 WW2. The spin-relaxation data suggested that the MAGI1 WW2 undergoes a dynamic process of transient dimerization that is limited by the charge repulsion. Additionally, we characterized a longer construct of this WW domain with a C-terminal extension (Leu355-Glu401), as the formation of an extra alpha-helix was predicted. An NMR structural determination confirmed the formation of an alpha-helix in the extended C-terminal region, which appears to be independent from the dimerization regulation. A thermal denaturation study revealed that the dimerized MAGI1 WW2 with the Asp-to-Ser mutation gained apparent stability in a protein concentration-dependent manner. A structural comparison between the two constructs with different lengths suggested that the formation of the C-terminal alpha-helix stabilized the global fold by facilitating contacts between the N-terminal linker region and the main body of the WW domain.     

Structural characterisation of PinA WW domain and a comparison with other group IV WW domains, Pin1 and Ess1

The NMR solution structure of the PinA WW domain from Aspergillus nidulans is presented. The backbone of the PinA WW domain is composed of a triple-stranded anti-parallel beta-sheet and an alpha-helix similar to Ess1 and Pin1 without the alpha-helix linker. Large RMS deviations in Loop I were observed both from the NMR structures and molecular dynamics simulation suggest that the Loop I of PinA WW domain is flexible and solvent accessible, thus enabling it to bind the pS/pT-P motif. The WW domain in this structure are stabilised by a hydrophobic core. It is shown that the linker flexibility of PinA is restricted because of an alpha-helical structure in the linker region. The combination of NMR structural data and detailed Molecular Dynamics simulations enables a comprehensive structural and dynamic understanding of this protein.     

A single point mutation in a group I WW domain shifts its specificity to that of group II WW domains.

WW domains can be divided into three groups based on their binding specificity. By random mutagenesis, we switched the specificity of the Yes-associated protein (YAP) WW1 domain, a Group I WW domain, to that of the FE65 WW domain, which belongs to Group II. We showed that a single mutation, leucine 190 (betaB5) to tryptophan, is required to switch from Group I to Group II. Although this single substitution in YAP WW1 domain is sufficient to precipitate the two protein isoforms of Mena, an in vivo ligand of FE65, we showed that an additional substitution, histidine 192 (betaB7) to glycine, significantly increased the ability of YAP to mimic FE65. This double mutant (L190W/H192G) precipitates eight of the nine protein bands that FE65 pulls down from rat brain protein lysates. Based on both our data and a sequence comparison between Group I and Group II WW domains, we propose that a block of three consecutive aromatic amino acids within the second beta-sheet of the domain is required, but not always sufficient, for a WW domain to belong to Group II. These data deepen our understanding of WW domain binding specificity and provide a basis for the rational design of modified WW domains with potential therapeutic applications.     

A method for multiprotein assembly in cells reveals independent action of kinesins in complex

Teams of processive molecular motors are critical for intracellular transport and organization, yet coordination between motors remains poorly understood. Here, we develop a system using protein components to generate assemblies of defined spacing and composition inside cells. This system is applicable to studying macromolecular complexes in the context of cell signaling, motility, and intracellular trafficking. We use the system to study the emergent behavior of kinesin motors in teams. We find that two kinesin motors in complex act independently (do not help or hinder each other) and can alternate their activities. For complexes containing a slow kinesin-1 and fast kinesin-3 motor, the slow motor dominates motility in vitro but the fast motor can dominate on certain subpopulations of microtubules in cells. Both motors showed dynamic interactions with the complex, suggesting that motor–cargo linkages are sensitive to forces applied by the motors. We conclude that kinesin motors in complex act independently in a manner regulated by the microtubule track.     

Yeast KRE genes provide evidence for a pathway of cell wall beta-glucan assembly

The Saccharomyces cerevisiae KRE1 gene encodes a Ser/Thr-rich protein, that is directed into the yeast secretory pathway, where it is highly modified, probably through addition of O-linked mannose residues. Gene disruption of the KRE1 locus leads to a 40% reduced level of cell wall (1----6)-beta-glucan. Structural analysis of the (1----6)-beta-glucan fraction, isolated from a strain with a krel disruption mutation, showed that it had an altered structure with a smaller average polymer size. Mutations in two other loci, KRE5 and KRE6 also lead to a defect in cell wall (1----6)-beta-glucan production and appear to be epistatic to KRE1. These findings outline a possible pathway of assembly of yeast cell wall (1----6)-beta-glucan.     

141 DNA origami as a platform for assembly of nanophotonic elements

Achieving DNA-functionalized semiconductor quantum dots (QDs) that are robust enough to be compatible with the DNA nanotechnology that withstand precipitation at high temperature and ionic strength is a challenge. Here we report a method that facilitates the synthesis of stable core/shell (1–20 monolayers) QD-DNA conjugates in which the end part (5–10 nucleotides) of the phosphorothiolated oligonucleotides is embedded within the shell of the QD. These reliable QD-DNA conjugates exhibit excellent chemical, colloidal and photonic stability over a wide pH range (4–12) and at high salt concentrations (>100?mM Na⁺ or Mg²⁺), bright fluorescence emission with quantum yields of upto 70%, and broad spectral tunability with emission ranging from UV to NIR (360–800?nm). The assembly of these different QDs into DNA origami in a well-controlled pattern was demonstrated (Deng, Samanta, Nangreave, Yan, & Liu, 2012). We also used DNA origami as a platform to co-assemble a gold nanoparticle with 20?nm diameter (AuNP) and an organic fluorophore (TAMRA) and studied the distance dependent plasmonic interactions between the particle and the dye using steady state fluorescence and lifetime measurements. Greater fluorescence quenching was found at smaller inter-particle distances, which was accompanied by an enhancement of the decay rate. We further fabricated 20?nm and 30?nm AuNP homodimers with different inter-particle distances using DNA origami scaffolds and positioned a Cy3 fluorophore between the AuNPs in both the assemblies. Up to 50% enhancement of the Cy3 fluorescence quantum efficiency was observed for the dye between the 30?nm AuNPs. These results are in good agreement with the theoretical simulations (Pal et al., 2013).     

HSP90 as a platform for the assembly of more effective cancer chemotherapy

Since initial discovery of the first HSP90 inhibitor over a decade and a half ago, tremendous progress has been made in developing potent and selective compounds with which to target this chaperone in the treatment of cancers. These compounds have been invaluable in dissecting how HSP90 supports the dramatic alterations in cellular physiology that constitute the malignant phenotype and give rise to the clinical manifestations of diverse cancers. Unfortunately, single agent activity for HSP90 inhibitors has been disappointingly modest against recurrent, refractory cancers in most of the clinical trials that have been reported to date. This problem could be due to pharmacological limitations of the first-generation inhibitors that have been most extensively studied. But we suggest it may well be intrinsic to the target itself. This review will focus on how the utilization of HSP90 by cancer cells might be targeted to enhance the activity of other anticancer drugs while at the same time limiting the ability of advanced cancers to adapt and evolve drug resistance; the net result being more durable disease control. A better understanding of these fundamental issues will surely make the ongoing clinical development of HSP90 inhibitors as anticancer drugs less empiric, more efficient and hopefully more successful. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Human SAP18 mediates assembly of a splicing regulatory multiprotein complex via its ubiquitin-like fold

RNPS1, Acinus, and SAP18 form the apoptosis- and splicing-associated protein (ASAP) complex, which is also part of the exon junction complex. Whereas RNPS1 was originally identified as a general activator of mRNA processing, all three proteins have been found within functional spliceosomes. Both RNPS1 and Acinus contain typical motifs of splicing regulatory proteins including arginine/serine-rich domains. Due to the absence of such structural features, however, a function of SAP18 in splicing regulation is completely unknown. Here we have investigated splicing regulatory activities of the ASAP components. Whereas a full-length Acinus isoform displayed only limited splicing regulatory activity, both RNPS1 and, surprisingly, SAP18 strongly modulated splicing regulation. Detailed mutational analysis and three-dimensional modeling data revealed that the ubiquitin-like fold of SAP18 was required for efficient splicing regulatory activity. Coimmunoprecipitation and immunofluorescence experiments demonstrated that SAP18 assembles a nuclear speckle-localized splicing regulatory multiprotein complex including RNPS1 and Acinus via its ubiquitin-like fold. Our results therefore suggest a novel function of SAP18 in splicing regulation.     

Comparative analysis of Saccharomyces cerevisiae WW domains and their interacting proteins

Background  

The WW domain is found in a large number of eukaryotic proteins implicated in a variety of cellular processes. WW domains bind proline-rich protein and peptide ligands, but the protein interaction partners of many WW domain-containing proteins in Saccharomyces cerevisiae are largely unknown.