Multivalent Angiomotin-like 1 and Yes-associated protein form a dynamic complex.

Multivalent complexes formed between the cancer-promoting transcriptional co-activator, Yes-associated protein (YAP), and proteins containing short linear motifs of type PPxY modulate cell proliferation and are attractive therapeutic targets. However, challenges producing PPxY polypeptides containing the full binding domain has limited understanding of the assembly process. Here, we successfully produced a polypeptide containing the complete set of three PPxY binding sites of Angiomotin-like 1 (AMOTL1), a scaffolding protein that regulates the nucleo-cytoplasmic shuttling of YAP via WW-PPxY interactions. Using an array of biophysical techniques including isothermal titration calorimetry, size-exclusion chromatography coupled to multi-angle light scattering, and solution nuclear magnetic resonance spectroscopy, we show that the AMOTL1 polypeptide is partially disordered, and binds the YAP WW domains to form an ensemble of complexes of varying stabilities. The binding process is initiated by the binding of one YAP WW domain to one AMOTL1 PPxY motif and is completed by transient interactions of the second YAP WW domain with a second AMOTL1 PPxY motif to form an equilibrating mixture composed of various species having two YAP sites bound to two conjugate AMOTL1 sites. We rationalize that the transient interactions fine-tune the stability of the complex for rapid assembly and disassembly in response to changes in the local cellular environment.

Yorkie-Warts Complexes are an Ensemble of Interconverting Conformers Formed by Multivalent Interactions.

Multiple copies of WW domains and PPXY motif sequences are often reciprocally presented by regulatory proteins that interact at crucial regulatory steps in the cell life cycle. While biophysical studies of single WW domain-single PPXY motif complexes abound in the literature, the molecular mechanisms of multivalent WW domain-PPXY assemblies are still poorly understood. By way of investigating such assemblies, we characterized the multivalent association of the entire cognate binding domains, two WW sequences and five PPXY motifs respectively, of the Yorkie transcription coactivator and the Warts tumor suppressor. Isothermal titration calorimetry, sedimentation velocity, size-exclusion chromatography coupled to multi-angle light scattering and native-state mass spectrometry of Yorkie WW domains interactions with the full-length Warts PPXY domain, and numerous PPXY motif variants of Warts show that the two proteins assemble via binding of tandem WW domains to adjacent PPXY pairs to produce an ensemble of interconverting complexes of variable stoichiometries, binding energetics and WW domain occupancy. Apparently, the Yorkie tandem WW domains first target the two adjacent PPXY motifs at the C-terminus of the Warts polypeptide and additional WW domains bind unoccupied motifs. Similar ensembles of interconverting conformers may be common in multivalent WW domain-PPXY interactions to promote the adaptability and versatility of WW domain-PPXY mediated cellular processes.

Intrinsic disorder and amino acid specificity modulate binding of the WW2 domain in kidney and brain protein (KIBRA) to synaptopodin.

The second WW domain (WW2) of the kidney and brain scaffolding protein, KIBRA, has an isoleucine (Ile-81) rather than a second conserved tryptophan and is primarily unstructured. However, it adopts the canonical triple-stranded antiparallel ß-sheet structure of WW domains when bound to a two-PPXY motif peptide of the synaptic protein Dendrin. Here, using a series of biophysical experiments, we demonstrate that the WW2 domain remains largely disordered when bound to a 69-residue two-PPXY motif polypeptide of the synaptic and podocyte protein synaptopodin (SYNPO). Isothermal titration calorimetry and CD experiments revealed that the interactions of the disordered WW2 domain with SYNPO are significantly weaker than SYNPO's interactions with the well-folded WW1 domain and that an I81W substitution in the WW2 domain neither enhances binding affinity nor induces substantial WW2 domain folding. In the tandem polypeptide, the two WW domains synergized, enhancing the overall binding affinity with the I81W variant tandem polypeptide 2-fold compared with the WT polypeptide. Solution NMR results showed that SYNPO binding induces small but definite chemical shift perturbations in the WW2 domain, confirming the disordered state of the WW2 domain in this complex. These analyses also disclosed that SYNPO binds the tandem WW domain polypeptide in an antiparallel manner, that is, the WW1 domain binds the second PPXY motif of SYNPO. We propose a binding model consisting of a bipartite interaction mode in which the largely disordered WW2 forms a "fuzzy" complex with SYNPO. This binding mode may be important for specific cellular functions.

Differential Binding Affinities and Allosteric Conformational Changes Underlie Interactions of Yorkie and a Multivalent PPxY Partner.

Tondu domain-containing growth inhibitor (Tgi) is one of a growing number of multivalent PPxY proteins that regulate cell growth via interactions with the tandem WW domains of the transcription coactivator protein, Yorkie (Yki). These proteins are attractive candidates for targeted drug design, but the substantial amount of disorder predicted from their primary sequences makes structural studies that are foundational to drug design challenging. We have successfully overexpressed full length recombinant Tgi and Yki, experimentally confirmed that intrinsic structural disorder is common to both proteins, and assessed binding of the Yki WW domains to the three Tgi PPxY motifs using nuclear magnetic resonance and isothermal titration calorimetry. We find that the tandem WW domains positively cooperate to engage all three PPxY sites with a broad range of affinities. The first PPxY motif that is quite distant from the other two serves as the "binding initiation" site and is essential for high-affinity interactions. Importantly, by monitoring binding to the full length or larger protein domains, we obtain more physiologically relevant affinity information and identify "long-range" residues that could be targeted to fine-tune binding. This expansion of protein functionality through modulation of residues outside the recognition sequences offers potential alternative targets for drug design.

The Anchored Flexibility Model in LC8 Motif Recognition: Insights from the Chica Complex.

LC8 is a dimeric hub protein involved in a large number of interactions central to cell function. It binds short linear motifs--usually containing a Thr-Gln-Thr (TQT) triplet--in intrinsically disordered regions of its binding partners, some of which have several LC8 recognition motifs in tandem. Hallmarks of the 7-10 amino acid motif are a high variability of LC8 binding affinity and extensive sequence permutation outside the TQT triplet. To elucidate the molecular basis of motif recognition, we use a 69-residue segment of the human Chica spindle adaptor protein that contains four putative TQT recognition motifs in tandem. NMR-derived secondary chemical shifts and relaxation properties show that the Chica LC8 binding domain is essentially disordered with a dynamically restricted segment in one linker between motifs. Calorimetry of LC8 binding to synthetic motif-mimicking peptides shows that the first motif dominates LC8 recruitment. Crystal structures of the complexes of LC8 bound to each of two motif peptides show highly ordered and invariant TQT-LC8 interactions and more flexible and conformationally variable non-TQT-LC8 interactions. These data highlight rigidity in both LC8 residues that bind TQT and in the TQT portion of the motif as an important new characteristic of LC8 recognition. On the basis of these data and others in the literature, we propose that LC8 recognition is based on rigidly fixed interactions between LC8 and TQT residues that act as an anchor, coupled with inherently flexible interactions between LC8 and non-TQT residues. The "anchored flexibility" model explains the requirement for the TQT triplet and the ability of LC8 to accommodate a large variety of motif sequences and affinities.

Polybivalency and disordered proteins in ordering macromolecular assemblies.

Intrinsically disordered proteins (IDPs) are prevalent in macromolecular assemblies and are thought to mediate protein recognition in complex regulatory processes and signaling pathways. The formation of a polybivalent scaffold is a key process by which IDPs drive early steps in macromolecular assemblies. Three intrinsically disordered proteins, IC, Swallow and Nup159, are core components, respectively, of cytoplasmic dynein, bicoid mRNA localization apparatus, and nuclear pore complexes. In all three systems, the hub protein LC8 recognizes on the IDP, short linear motifs that are fully disordered in the apo form, but adopt a ß-strand when bound to LC8. The IDP/LC8 complex forms a bivalent scaffold primed to bind additional bivalent ligands. Scaffold formation also promotes self-association and/or higher order organization of the IDP components at a site distant from LC8 binding. Rigorous thermodynamic analyses imply that association of additional bivalent ligands is driven by entropic effects where the first binding event is weak but subsequent binding of additional ligands occurs with higher affinity. Here, we review specific examples of macromolecular assemblies in which polybivalency of aligned IDP duplexes not only enhances binding affinity and results in formation of a stable complex but also compensates unfavorable steric and enthalpic interactions. We propose that polybivalent scaffold assembly involving IDPs and LC8-like proteins is a general process in the cell biology of a class of multi-protein structures that are stable yet fine-tuned for diverse cellular requirements.

A network of assembly factors is involved in remodeling rRNA elements during preribosome maturation.

Eukaryotic ribosome biogenesis involves ∼200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a "distribution box," transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation.

Solution NMR structures of Pyrenophora tritici-repentis ToxB and its inactive homolog reveal potential determinants of toxin activity.

Pyrenophora tritici-repentis Ptr ToxB (ToxB) is a proteinaceous host-selective toxin produced by Pyrenophora tritici-repentis (P. tritici-repentis), a plant pathogenic fungus that causes the disease tan spot of wheat. One feature that distinguishes ToxB from other host-selective toxins is that it has naturally occurring homologs in non-pathogenic P. tritici-repentis isolates that lack toxic activity. There are no high-resolution structures for any of the ToxB homologs, or for any protein with >30% sequence identity, and therefore what underlies activity remains an open question. Here, we present the NMR structures of ToxB and its inactive homolog Ptr toxb. Both proteins adopt a ß-sandwich fold comprising three strands in each half that are bridged together by two disulfide bonds. The inactive toxb, however, shows higher flexibility localized to the sequence-divergent ß-sandwich half. The absence of toxic activity is attributed to a more open structure in the vicinity of one disulfide bond, higher flexibility, and residue differences in an exposed loop that likely impacts interaction with putative targets. We propose that activity is regulated by perturbations in a putative active site loop and changes in dynamics distant from the site of activity. Interestingly, the new structures identify AvrPiz-t, a secreted avirulence protein produced by the rice blast fungus, as a structural homolog to ToxB. This homology suggests that fungal proteins involved in either disease susceptibility such as ToxB or resistance such as AvrPiz-t may have a common evolutionary origin.

NMR Characterization of Self-Association Domains Promoted by Interactions with LC8 Hub Protein.

Most proteins in interaction networks have a small number of partners, while a few, called hubs, participate in a large number of interactions and play a central role in cell homeostasis. One highly conserved hub is a protein called LC8 that was originally identified as an essential component of the multi-subunit complex dynein but later shown to be also critical in multiple protein complexes in diverse systems. What is intriguing about this hub protein is that it does not passively bind its various partners but emerging evidence suggests that LC8 acts as a dimerization engine that promotes self-association and/or higher order organization of its primarily disordered monomeric partners. This structural organization process does not require ATP but is triggered by long-range allosteric regulation initiated by LC8 binding a pair of disordered chains forming a bivalent or polybivalent scaffold. This review focuses on the role of LC8 in promoting self-association of two of its binding partners, a dynein intermediate chain and a non dynein protein called Swallow.

Structural features of LC8-induced self-association of swallow.

Cell functions depend on the collective activity of protein networks within which a few proteins, called hubs, participate in a large number of interactions. Dynein light chain LC8, first discovered as a subunit of the motor protein dynein, is considered to have a role broader than that of dynein, and its participation in diverse systems fits the description of a hub. Among its partners is Swallow with which LC8 is essential for proper localization of bicoid mRNA at the anterior cortex of Drosophila oocytes. Why LC8 is essential in this process is not clear, but emerging evidence suggests that LC8 functions by promoting self-association and/or structural organization of its diverse binding partners. This work addresses the energetics and structural features of LC8-induced Swallow self-association distant from LC8 binding. Mutational design based on a hypothetical helical wheel, intermonomer nuclear Overhauser effects assigned to residues expected at interface positions, and circular dichroism spectral characteristics indicate that the LC8-promoted dimer of Swallow is a coiled coil. Secondary chemical shifts and (15)N backbone relaxation identify the boundaries and distinguishing structural features of the coiled coil. Thermodynamic analysis of Swallow polypeptides designed to decouple self-association from LC8 binding reveals that the higher binding affinity of the engineered bivalent Swallow is of purely entropic origin and that the linker separating the coiled coil from the LC8 binding site remains disordered. We speculate that the LC8-promoted coiled coil is critical for bicoid mRNA localization because it favors structural organization of Swallow, which except for the central LC8-promoted coiled coil is primarily disordered.

Multiple recognition motifs in nucleoporin Nup159 provide a stable and rigid Nup159-Dyn2 assembly.

Dyn2 is the yeast ortholog of the molecular hub LC8, which binds disordered proteins and promotes their self-association and higher order assembly. Dyn2 is proposed to dimerize and stabilize the Nup82-Nsp1-Nup159 complex of the nuclear pore assembly through its interaction with nucleoporin Nup159. Nup159 has six LC8 recognition motifs separated by short linkers. NMR experiments reported here show that the Dyn2 binding domain of Nup159 is intrinsically disordered and that binding of one equivalent of Dyn2 dimer aligns two Nup159 chains along the full Dyn2 binding domain to form a bivalent scaffold that promotes binding of other Dyn2 dimers. Isothermal titration calorimetry of Dyn2 binding to Nup constructs of increasing lengths determine that the third LC8 recognition motifs does not bind Dyn2. A new approach to identifying active LC8 recognition motifs based on NMR-detected ß-sheet propensities is presented. Isothermal titration calorimetry experiments also show that, due to unfavorable entropy changes, a Nup-Dyn2 complex with three Dyn2 dimers is more stable than the wild-type complex with five Dyn2 dimers. The calorimetric results argue that, from a thermodynamics perspective, only three Dyn2 dimers are needed for optimal stability and suggest that the evolutionary adaptation of multiple tandem LC8 recognition motifs imparts to the complex other properties such as rigidity and a kink in the rod-like structure. These findings extend the repertoire of functions of intrinsically disordered protein to fine-tuning and versatile assembly of higher order macromolecular complexes.

Intrinsic disorder in dynein intermediate chain modulates its interactions with NudE and dynactin.

The functional diversity of cytoplasmic dynein is in part attributed to multiple interactions between noncatalytic dynein subunits and an array of regulatory proteins. This study focuses on the interaction between the dynein intermediate chain subunit (IC) and a dynein regulator protein (NudE). We use isothermal titration calorimetry and NMR spectroscopy to map their interacting sections to their respective N-terminal domains, which are predicted to form dimeric coiled-coils. Interestingly, the specific residues within IC that interact with NudE are a subset of the bi-segmental binding region reported for p150(Glued), a subunit of the dynein activator protein dynactin. Although the IC binding domains of both NudE and p150(Glued) form dimeric coiled-coils and bind IC at a common site, we observe distinct binding modes for each regulatory protein: 1) NudE binds region 1 of the bi-segmental binding footprint of p150(Glued), whereas p150(Glued) requires regions 1 and 2 to match the binding affinity of NudE with region 1 alone. 2) Compared with unbound IC, NudE-bound IC shows a slight increase in flexibility in region 2, in contrast to the increase in ordered structure observed for p150(Glued)-bound IC (Morgan, J. L., Song, Y., and Barbar, E. (2011) J. Biol. Chem. 286, 39349-39359). 3) Although NudE has a higher affinity for the common binding segment on IC, when all three proteins are in solution, IC preferentially binds p150(Glued). These results underscore the importance of a bi-segmental binding region of IC and disorder in region 2 and flanking linkers in selecting which regulatory protein binds IC.

Conformational dynamics promote binding diversity of dynein light chain LC8.

A highly conserved and ubiquitous protein known as LC8 binds over twenty different partners, characteristic of a molecular hub (Barbar, 2008 Biochemistry, 47, 503-508). Structural studies of LC8 complexes with binding partners having diverse recognition sequences show that the same binding groove of LC8 accommodates the various binding motifs. Here we use thermodynamics and dynamics measurements of LC8 complexes to group LC8 binding partners in two categories: those whose binding is enthalpically driven and those that are entropically favored. Peptides that are enthalpically driven completely silence the millisecond-microsecond relaxation signal, suggesting a significant rigidifying of the binding groove, while peptides in the entropically favored group exhibit the same conformational dynamics as the free protein, suggesting that the peptide sits loosely in the binding groove and so retains flexibility of the groove, and presumably of the bound peptide. The inherent disorder in the LC8 binding groove and in LC8 binding partners allows both types of binding, accounts for the lack of a conserved recognition consensus motif and underlies the binding specificity and broad selectivity observed in LC8 binding.

Light chain-dependent self-association of dynein intermediate chain.

Dynein light chains are bivalent dimers that bind two copies of dynein intermediate chain IC to form a cargo attachment subcomplex. The interaction of light chain LC8 with the natively disordered N-terminal domain of IC induces helix formation at distant IC sites in or near a region predicted to form a coiled-coil. This fostered the hypothesis that LC8 binding promotes IC self-association to form a coiled-coil or other interchain helical structure. However, recent studies show that the predicted coiled-coil sequence partially overlaps the light chain LC7 recognition sequence on IC, raising questions about the apparently contradictory effects of LC8 and LC7. Here, we use NMR and fluorescence quenching to localize IC self-association to residues within the predicted coiled-coil that also correspond to helix 1 of the LC7 recognition sequence. LC8 binding promotes IC self-association of helix 1 from each of two IC chains, whereas LC7 binding reverses self-association by incorporating the same residues into two symmetrical, but distant, helices of the LC7-IC complex. Isothermal titration experiments confirm the distinction of LC8 enhancement of IC self-association and LC7 binding effects. When all three light chains are bound, IC self-association is shifted to another region. Such flexibility in association modes may function in maintaining a stable and versatile light chain-intermediate chain assembly under changing cellular conditions.

TPR-Mediated self-association of plant SGT1.

The tetratricopeptide repeat (TPR) domain mediates inter-protein associations in a number of systems. The domain is also thought to mediate oligomerization of some proteins, but this has remained controversial, with conflicting data appearing in the literature. By way of investigating such TPR-mediated self-associations we used a variety of biophysical techniques to characterize purified recombinant Sgt1, a TPR-containing protein found in all eukaryotes that is involved in a broad range of biological processes, including kinetochore assembly in humans and yeast and disease resistance in plants. We show that recombinant Sgt1 from Arabidopsis, barley, and yeast self-associates in vitro while recombinant human Sgt1 does not. Further experiments on barley Sgt1 demonstrate unambiguously a TPR-mediated dimerization, which is concentration- and ionic-strength-dependent and results in a global increase in helical structure and stability of the protein. Dimerization is also redox sensitive, being completely abolished by the formation of an intramolecular disulfide bond where the contributing cysteines are conserved in plant Sgt1s. The dimer interface was mapped through cross-linking and mass spectrometry to the C-terminal region of the TPR domain. Our study, which provides the first biophysical characterization of plant Sgt1, highlights how TPR domains can mediate self-association in solution and that sequence variation in the regions involved in oligomerization affects the propensity of TPR-containing proteins to dimerize.

Potential role for phosphorylation in differential regulation of the assembly of dynein light chains.

The homodimeric light chains LC8 and Tctex-1 are integral parts of the microtubule motor cytoplasmic dynein, as they directly associate with dynein intermediate chain IC and various cellular cargoes. These light chains appear to regulate assembly of the dynein complex by binding to and promoting dimerization of IC. In addition, both LC8 and Tctex-1 play roles in signaling, apoptosis, and neuronal development that are independent of their function in dynein, but it is unclear how these various activities are modulated. Both light chains undergo specific phosphorylation, and here we present biochemical and NMR analyses of phosphomimetic mutants that indicate how phosphorylation may regulate light chain function. For both LC8 and Tctex-1, phosphorylation promotes dissociation from IC while retaining their binding activity with other non-dynein proteins. Although LC8 and Tctex-1 are homologs having a common fold, their reduced affinity for IC upon phosphorylation arises by different mechanisms. In the case of Tctex-1, phosphorylation directly masks the IC binding site at the dimer interface, whereas for LC8, phosphorylation dissociates the dimer and indirectly eliminates the binding site. This modulation of the monomer-dimer equilibrium by phosphorylation provides a novel mechanism for discrimination among LC8 binding partners.

Heteronuclear NMR identifies a nascent helix in intrinsically disordered dynein intermediate chain: implications for folding and dimerization.

The intermediate chain of dynein forms a tight subcomplex with dimeric light chains LC8 and Tctex-1, and together they constitute the cargo attachment complex. There is considerable interest in identifying the role of these light chains in the assembly of the two copies of the intermediate chain. The N-terminal domain of the intermediate chain, IC1-289, contains the binding sites for the light chains, and is a highly disordered monomer but gains helical structure upon binding to light chains LC8 and Tctex-1. To provide insights into the structural and dynamic changes that occur in the intermediate chain upon light chains binding, we have used NMR spectroscopy to compare the properties of two distinct sub-domains of IC1-289: IC84-143 which is the light chains binding domain, and IC198-237, which contains a predicted coiled coil necessary for the increase in ordered structure upon light chain binding. Neither construct has stable secondary structure when probed by circular dichroism and amide chemical shift dispersion. Specific residues of IC84-143 involved in binding to the light chains were identified by their increase in resonance line broadening and the corresponding large intensity reduction in 1H-15N HSQC spectra. Interestingly, IC84-143 shows no sign of structure formation after binding to either LC8 or Tctex-1 or to both. IC198-237, on the other hand, contains a population of a nascent helix at low temperature as identified by heteronuclear NMR relaxation measurements, secondary chemical shifts, and sequential amide-amide connectivities. These data are consistent with a model for light chain binding coupled to intermediate chain dimerization through forming a coiled coil distant from the binding site.

Folding is coupled to dimerization of Tctex-1 dynein light chain.

Equilibrium analyses have been performed to elucidate the role of dimerization in folding and stability of dynein light chain Tctex-1. The equilibrium unfolding transition was monitored by intrinsic fluorescence intensity, fluorescence anisotropy, and circular dichroism and was modeled as a two-state mechanism where a folded dimer dissociates to two unfolded monomers without populating thermodynamically stable monomeric or dimeric intermediates. Sedimentation equilibrium and chemical cross-linking experiments performed at increasing concentrations of denaturants show no change in the association state before the unfolding transition and are consistent with the two-state model of dissociation coupled to unfolding. A linear dependence on denaturant concentration is observed by fluorescence intensity and anisotropy before unfolding in the 0-2 M GdnCl, and 0-4 M urea concentration range. This change is not protein concentration-dependent and possibly reflects relief of quenching associated with premelting conformational disorder in the vicinity of Trp 83. The data clearly show that the dissociation-coupled unfolding mechanism of Tctex-1 is different from the three-state denaturation mechanism of its structural homologue light chain LC8. The absence of a stable monomer in Tctex-1 offers insight into its functional differences from LC8.

Ionization of His 55 at the dimer interface of dynein light-chain LC8 is coupled to dimer dissociation.

LC8 is a highly conserved light-chain subunit of cytoplasmic dynein that interacts with a wide variety of cellular proteins and is presumed to play a fundamental role in dynein assembly and cargo recruitment and in the assembly of protein complexes unrelated to dynein. LC8 is a dimer at physiological pH but dissociates to a folded monomer at pH < 4.8. We have suggested that acid-induced dimer dissociation is due to protonation of His 55, which is stacked against His 55' and completely buried in the dimer interface. In this work, we show that the pH-induced dissociation is reversible and indeed governed by the ionization state of His 55. Mutagenesis of His 55 to Lys results in a monomer in the pH range of 3-8, while the mutation to Ala results in a dimer in the same pH range. Mutations that disrupt intermolecular hydrogen bonds between Tyr 65 and Lys 44' and His 55 and Thr 67' do not change the association state of the dimer. Titration curves for His 55 and the two other histidines, His 72 and 68, were determined by (13)C-(1)H NMR for H55K and for WT-LC8 in the monomeric and dimeric states. The pK(a) values of His 72 and His 68 are 6 in the WT dimer and 6.2-6.5 in monomeric H55K, while the pK(a) of His 55 is about 4.5 in the WT dimer. These results indicate that deprotonation of His 55 is linked to dimer formation and that mutation of His 55 to a small neutral residue or to a positively charged residue uncouples the protonation and dissociation processes.