Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis

: During muscle cell differentiation, the alternatively spliced, acidic β-domain potentiates transcription of Myocyte-specific Enhancer Factor 2 (Mef2D). Sequence analysis by the FuzDrop method indicates that the β-domain can serve as an interaction element for Mef2D higher-order assembly. In accord, we observed Mef2D mobile nuclear condensates in C2C12 cells, similar to those formed through liquid-liquid phase separation. In addition, we found Mef2D solid-like aggregates in the cytosol, the presence of which correlated with higher transcriptional activity. In parallel, we observed a progress in the early phase of myotube development, and higher MyoD and desmin expression. In accord with our predictions, the formation of aggregates was promoted by rigid β-domain variants, as well as by a disordered β-domain variant, capable of switching between liquid-like and solid-like higher-order states. Along these lines, NMR and molecular dynamics simulations corroborated that the β-domain can sample both ordered and disordered interactions leading to compact and extended conformations. These results suggest that β-domain fine-tunes Mef2D higher-order assembly to the cellular context, which provides a platform for myogenic regulatory factors and the transcriptional apparatus during the developmental process

Characterization of a Novel Interaction between Bcl-2 Members Diva and Harakiri

Interactions within proteins of the Bcl-2 family are key in the regulation of apoptosis. The death-inducing members control apoptotic mechanisms partly by antagonizing the prosurvival proteins through heterodimer formation. Structural and biophysical studies on these complexes are providing important clues to understand their function. To help improve our knowledge on protein-protein interactions within the Bcl-2 family we have studied the binding between two of its members: mouse Diva and human Harakiri. Diva has been shown to perform both prosurvival and killing activity. In contrast, Harakiri induces cell death by interacting with antiapoptotic Bcl-2 members. Here we show using ELISA and NMR that Diva and Harakiri can interact in vitro. Combining the NMR data with the previously reported three-dimensional structure of Diva we find that Harakiri binds to a specific region in Diva. This interacting surface is equivalent to the known binding area of prosurvival Bcl-2 members from the reported structures of the complexes, suggesting that Diva could function at the structural level similarly to the antiapoptotic proteins of the Bcl-2 family. We illustrate this result by building a structural model of the heterodimer using molecular docking and the NMR data as restraints. Moreover, combining circular dichroism and NMR we also show that Harakiri is largely unstructured with residual (13%) α-helical conformation. This result agrees with intrinsic disorder previously observed in other Bcl-2 members. In addition, Harakiri constructs of different length were studied to identify the region critical for the interaction. Differential affinity for Diva of these constructs suggests that the amino acid sequence flanking the interacting region could play an important role in binding

Intrinsic Order and Disorder in the Bcl-2 Member Harakiri: Insights into Its Proapoptotic Activity

Harakiri is a BH3-only member of the Bcl-2 family that localizes in membranes and induces cell death by binding to prosurvival Bcl-xL and Bcl-2. The cytosolic domain of Harakiri is largely disorder with residual α-helical conformation according to previous structural studies. As these helical structures could play an important role in Harakiri's function, we have used NMR and circular dichroism to fully characterize them at the residue-atomic level. In addition, we report structural studies on a peptide fragment spanning Harakiri's C-terminal hydrophobic sequence, which potentially operates as a transmembrane domain. We initially checked by enzyme immunoassays and NMR that peptides encompassing different lengths of the cytosolic domain are functional as they bind Bcl-xL and Bcl-2. The structural data in water indicate that the α-helical conformation is restricted to a 25-residue segment comprising the BH3 domain. However, structure calculation was precluded because of insufficient NMR restraints. To bypass this problem we used alcohol-water mixture to increase structure population and confirmed by NMR that the conformation in both milieus is equivalent. The resulting three-dimensional structure closely resembles that of peptides encompassing the BH3 domain of BH3-only members in complex with their prosurvival partners, suggesting that preformed structural elements in the disordered protein are central to binding. In contrast, the transmembrane domain forms in micelles a monomeric α-helix with a population close to 100%. Its three-dimensional structure here reported reveals features that explain its function as membrane anchor. Altogether these results are used to propose a tentative structural model of how Harakiri works

Actividad de la proteina intrínsecamente desordenada p15(PAF) en el replisoma o cómo el desorden orquesta la replicación celular

[spa] La abrazadera deslizante eucariota (PCNA), juega un papel esencial como componente del replisoma. PCNA, de forma toroidal, rodea el DNA y ata las polimerasas y otros factores a la plantilla genómica para una síntesis rápida y procesiva. PCNA puede deslizar bidireccionalmente a lo largo del dúplex de DNA rastreando su columna vertebral mediante un mecanismo de «rueda dentada» basado en interacciones polares efímeras que mantienen la orientación de la pinza invariante en relación con la doble hélice. La mutación de residuos en esta interfaz de interacción PCNA-DNA hace desfavorable el inicio de la síntesis de DNA por Pol δ, por lo tanto, es necesaria una pinza orientada correctamente en el DNA para el ensamblaje de una holoenzima pol δ-PCNA competente en replicación. La cara del interior del anillo de PCNA, además de ser crucial para la función de PCNA como factor de procesividad durante la replicación, está altamente regulada para controlar la resistencia al daño en el DNA. Se puede modular (i) a través de la acetilación de su lisina 20, lo que estimula la reparación por recombinación homóloga, o (ii) mediante la unión de p15PAF, lo que desactiva el baipás de lesión en el DNA. p15PAF es una proteína intrínsecamente desordenada que atraviesa el canal del anillo de PCNA, uniendo su dominio PIP-box al bolsillo hidrofóbico de la cara frontal de la pinza y estableciendo contactos también con la superficie deslizante de la abrazadera para asomar su cola N-terminal por la cara trasera. Cuando dos moléculas de p15PAF ocupan dos subunidades del homotrímero de PCNA, el DNA dentro del canal del anillo se une a la subunidad que queda desocupada y no desplaza a p15PAF de la pared del anillo interno de PCNA. Cuando p15PAF está unida a PCNA, se reduce la superficie deslizante disponible de la abrazadera, así que p15PAF puede estar funcionando como un cinturón que abrocha el DNA a PCNA durante la síntesis por la polimerasa replicativa Pol δ. Esta restricción de la superficie deslizante, sin embargo, necesita ser eliminada para un baipás eficaz de la lesión del DNA por parte de la polimerasa de síntesis translesión Pol η. PCNA es estable en forma de anillo cerrado y, por lo tanto, debe cargarse activamente en las uniones cebador/plantilla del DNA, colocándose exactamente en el lugar y posición correctas para una replicación procesiva. La apertura y carga de PCNA la lleva a cabo el cargador de la pinza RFC. Una vez en el DNA, PCNA se vuelve a sellar alrededor del DNA y entonces el cargador de la pinza es expulsado. Cuando PCNA ya no es necesaria anclada en el DNA, el complejo RFC es el encargado de retirarla abriéndola y soltándola fuera de la doble hebra. Pero la flexibilidad intrínseca de PCNA hace que tenga cierta predisposición a estar en estado abierto separando dos de sus subunidades a través de su interfaz. Esto, que favorece la apertura del anillo por parte de RFC para lograr el ensamblado alrededor del DNA, puede ser un problema para mantenerla cerrada en la unión cebador/plantilla. De hecho, la estabilidad de las interfaces entre subunidades de PCNA disminuye cuando se une al DNA después de ser cargada por RFC, y dicha estabilidad solo la ve recuperada cuando p15PAF se ancla por su dominio PIP-box a sus bolsillos hidrofóbicos, grapando así las subunidades de la pinza e impidiendo su salida prematura del complejo con el DNA. Además de estabilizar la forma cerrada del anillo de PCNA, cuando p15PAF está anclada a su cara frontal, impide que RFC se aproxime, se una a ella y la desenganche de la unión cebador/plantilla.[eng] The eukaryotic sliding clamp (PCNA) is an essential replisome's component. PCNA, with a toroidal shape, surrounds DNA and binds polymerases and other factors to the genomic template for rapid and processive synthesis. PCNA can slide bi-directionally along the DNA duplex using a "cogwheel" mechanism based on ephemeral polar interactions that maintain the orientation of the clamp invariant relative to the double helix. However, mutations in the PCNA-DNA interaction interface render unfavourable the initiation of DNA synthesis by Pol δ. Therefore, a correctly oriented clamp on the DNA is necessary to assemble a competent pol δ-PCNA holoenzyme. Tight regulation of the inner face of PCNA, which is crucial for PCNA function as a processivity factor during replication, controls the DNA damage resistance. The inner PCNA ring face can be regulated (i) through acetylation of its lysine 20, which stimulates repair by homologous recombination, or (ii) by p15PAF binding, which deactivates the bypass of DNA damage. p15PAF is an intrinsically disordered protein that crosses the channel of the PCNA ring, attaching its PIP-box domain to the hydrophobic pocket on the front face of the clamp and establishing contacts with the sliding surface to show its N-terminal tail through the rear face. When two p15PAF molecules occupy two subunits of the PCNA homotrimer, the DNA within the ring channel binds to the unoccupied subunit and does not displace p15PAF from the inner ring wall of PCNA. When p15PAF is bound to PCNA, the available slip surface of the clamp is reduced, so p15PAF may be functioning as a belt that binds DNA to PCNA during synthesis by the replicative polymerase Pol δ. This sliding surface restriction, however, needs to be removed for efficient bypass of DNA damage by the translesion synthesis polymerase Pol η. PCNA is a stable closed ring and must be actively loaded onto the primer/template junctions of DNA, getting precisely in the right place and position for processive replication. RFC clamp loader opens and loads PCNA onto the DNA. Once in the DNA, PCNA reseals around the DNA and RFC is then ejected. When PCNA is no longer needed around the DNA, the RFC complex opens and unloads it. But the local flexibility of PCNA's subunits interfaces makes it have a certain predisposition to be in the open state. This local flexibility, which favours the RFC opening of the ring to achieve assembly around the DNA, can be a problem in keeping it closed at the primer/template junction. Furthermore, the stability of the interfaces between PCNA subunits decreases further when it binds to DNA. Interestingly, the PCNA homotrimer interfaces stability recovers when p15PAF is anchored by its PIP-box domain to PCNA's hydrophobic pockets, thus stapling the clamp subunits and preventing their premature exit from the complex with DNA. In addition, when p15PAF is anchored to PCNA front face, it prevents RFC from approaching, binding to, and unloading PCNA from the primer/template junction

Protein interactions: anything new?

: How do proteins interact in the cellular environment? Which interactions stabilize liquid-liquid phase separated condensates? Are the concepts, which have been developed for specific protein complexes also applicable to higher-order assemblies? Recent discoveries prompt for a universal framework for protein interactions, which can be applied across the scales of protein communities. Here, we discuss how our views on protein interactions have evolved from rigid structures to conformational ensembles of proteins and discuss the open problems, in particular related to biomolecular condensates. Protein interactions have evolved to follow changes in the cellular environment, which manifests in multiple modes of interactions between the same partners. Such cellular context-dependence requires multiplicity of binding modes (MBM) by sampling multiple minima of the interaction energy landscape. We demonstrate that the energy landscape framework of protein folding can be applied to explain this phenomenon, opening a perspective toward a physics-based, universal model for cellular protein behaviors

ASC pyrin domain self-associates and binds NLRP3 protein using equivalent binding interfaces

16 p.-15 fig.Death domain superfamily members typically act as adaptors mediating in the assembly of supramolecular complexes with critical apoptosis and inflammation functions. These modular proteins consist of death domains, death effector domains, caspase recruitment domains, and pyrin domains (PYD). Despite the high structural similarity among them, only homotypic interactions participate in complex formation, suggesting that subtle factors differentiate each interaction type. It is thus critical to identify these factors as an essential step toward the understanding of the molecular basis of apoptosis and inflammation. The proteins apoptosis-associated speck-like protein containing a CARD (ASC) and NLRP3 play key roles in the regulation of apoptosis and inflammation through self-association and protein-protein interactions mediated by their PYDs. To better understand the molecular basis of their function, we have characterized ASC and NLRP3 PYD self-association and their intermolecular interaction by solution NMR spectroscopy and analytical ultracentrifugation. We found that ASC self-associates and binds NLRP3 PYD through equivalent protein regions, with higher binding affinity for the latter. These regions are located at opposite sides of the protein allowing multimeric complex formation previously shown in ASC PYD fibril assemblies. We show that NLRP3 PYD coexists in solution as a monomer and highly populated large-order oligomerized species. Despite this, we determined its monomeric three-dimensional solution structure by NMR and characterized its binding to ASC PYD. Using our novel structural data, we propose molecular models of ASC·ASC and ASC·NLRP3 PYD early supramolecular complexes, providing new insights into the molecular mechanisms of inflammasome and apoptosis signaling.This work was supported in part by Spanish Ministry of Science and Innovation Plan Nacional I+D+i Program, Grants BFU2008-03278 and BFU2011-23797, and Programa Ingenio-2010-CONSOLIDER Grant CSD2009-00088 (to E. dA.).Peer reviewe

Changes in the NMR spectra of Hrk_ΔTM resulting from the interaction with Diva.

<p>¹H_amide-¹H_aliphatic region of TOCSY spectra of Hrk_ΔTM (black) and Hrk_ΔTM/Diva mixture (0.85 mM/0.12 mM) (red). The small dispersion in ¹H amide chemical shifts, the absence of signals for methyl groups close to 0 ppm and the severe signal overlap are characteristic of disordered proteins. Changes in the chemical shifts of Hrk_ΔTM upon the addition of Diva likely result from an overall structural rearrangement toward the helical conformation in addition to the interaction with Diva. Signals in the [¹H-¹H]-TOCSY spectrum of the mixture are observed only for Hrk_ΔTM because of the low concentration of Diva relative to Hrk_ΔTM and Diva's broader signals, most likely at the noise level according to its larger molecular weight (∼18 kDa for Diva, ∼6 kDa for Hrk_ΔTM).</p

Harakiri constructs and ELISA binding.

<p>(A) One-letter code amino acid sequence of human Harakiri and constructs studied. The BH3 region, the transmembrane domain and the predicted α-helical segments are indicated. (B) Difference in ELISA absorbance relative to the control for the binding of Diva to Hrk_ΔTM, Hrk_medium and Hrk_BH3 vs. fragment concentration. Shown values are average of two measurements. Thin bars represent the standard deviation. The absorbance value for the control is 0.12±0.01.</p

3D model of the Diva/Harakiri heterodimer compared to other prosurvival/BH3_peptide complexes.

<p>(A,B,C) Surface and ribbon representation of the Diva/Hrk_ΔTM heterodimer (A), Mcl-1/Bid_BH3 (pdb ID 2KBW) (B) and Bcl-X_L/Bad_BH3 (pdb ID 2BZW) (C) complexes. The interacting surface in Diva and the other prosurvival proteins is colored in orange. The BH3-only protein fragments are shown as green ribbons and the N-termini are indicated. Residues Arg 22, Ala 54 and Ala 59 in Hrk_ΔTM are indicated to show that the N and C-terminal regions of the cytosolic domain of Harakiri are outside the critical binding region. The orientation of the three complexes is equivalent. Fig. 7 was created with MOLMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015575#pone.0015575-Koradi1" target="_blank">[42]</a>.</p

Determination of the apparent dissociation constant of the Diva/Harakiri complex.

<p>Chemical shift changes of signals in the [¹H-¹⁵N]-HSQC spectra of Diva shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015575#pone-0015575-g002" target="_blank">Fig. 2B</a> vs. the total concentration of Hrk_ΔTM (black circles). Fittings of the experimental data to equation (11) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015575#s4" target="_blank">Materials and Methods</a>) are shown with a continuous line. The corresponding residues and individually measured K<i>_dapp</i> values are indicated. The concentration of Hrk_ΔTM (mM) for each point is: 0, 0.04, 0.08, 0.12, 0.16, 0.24, 0.29, 0.37, 0.44, 0.61, 0.73, 0.81, 0.85, 1.30, 1.60, 1.80. Diva's NMR signals undergo significant broadening for the last three titration points at the largest Hrk_ΔTM concentration, and thus the error in chemical shift determination for these points is likely larger.</p