Protein aggregation profile of the bacterial cytosol

Background: protein misfolding is usually deleterious for the cell, either as a consequence of the loss of protein function or the buildup of insoluble and toxic aggregates. The aggregation behavior of a given polypeptide is strongly influenced by the intrinsic properties encoded in its sequence. This has allowed the development of effective computational methods to predict protein aggregation propensity. Methodology/Principal Findings: here, we use the AGGRESCAN algorithm to approximate the aggregation profile of an experimental cytosolic Escherichia coli proteome. The analysis indicates that the aggregation propensity of bacterial proteins is associated with their length, conformation, location, function, and abundance. The data are consistent with the predictions of other algorithms on different theoretical proteomes. Conclusions/Significance: overall, the study suggests that the avoidance of protein aggregation in functional environments acts as a strong evolutionary constraint on polypeptide sequences in both prokaryotic and eukaryotic organisms

Origin of a folded repeat protein from an intrinsically disordered ancestor.

Repetitive proteins are thought to have arisen through the amplification of subdomain-sized peptides. Many of these originated in a non-repetitive context as cofactors of RNA-based replication and catalysis, and required the RNA to assume their active conformation. In search of the origins of one of the most widespread repeat protein families, the tetratricopeptide repeat (TPR), we identified several potential homologs of its repeated helical hairpin in non-repetitive proteins, including the putatively ancient ribosomal protein S20 (RPS20), which only becomes structured in the context of the ribosome. We evaluated the ability of the RPS20 hairpin to form a TPR fold by amplification and obtained structures identical to natural TPRs for variants with 2-5 point mutations per repeat. The mutations were neutral in the parent organism, suggesting that they could have been sampled in the course of evolution. TPRs could thus have plausibly arisen by amplification from an ancestral helical hairpin

Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases

Mitogen-activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less-characterized disordered regions. We used a structurally consistent model on kinase-docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under-explored part of the human proteome and applied experimental tools specifically tailored to detect low-affinity protein-protein interactions for their validation in vitro and in cell-based assays. The combined computational and experimental approach enabled the identification of many novel MAPK-docking motifs that were elusive for other large-scale protein-protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase-mediated partnerships evolved over time. The analysis suggests that most human MAPK-binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK-binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles

A novel regulatory unit in the N-terminal region of c-Src

[eng] c-Src is a central player in several cellular signaling pathways. It controls impor- tant cellular processes like cellular proliferation, survival or motility. Therefore, a number of tumoral diseases have been related to abnormal c-Src activity. Among them, colorectal cancer stands out, as c-Src deregulation correlates with tumor progression and clinical outcome. This tyrosine kinase is part of a larger group of functionally and structurally related proteins termed Src Family Kinases. These proteins share the same domain architecture: a cassette formed by a catalytic domain (SH1), two reg- ulatory domains, SH2 and SH3, and a variable intrinsically disordered region (the Unique domain) that ultimately anchors to the inner face of the cellular membrane via the N-terminal SH4 domain, also disordered. The sequence and structure of the cassette are highly conserved, and thus unsurprisingly Src Family Kinases perform closely related and often overlapping functions. However, the role of intrinsically disordered regions has remained unclear, although they are known to be functionally relevant. In this work, the structural and functional relationship between the intrinsically disordered SH4 and Unique domains with the neighboring folded SH3 domain in c-Src is explored. Interactions between disordered and ordered proteins are often characterized by the formation of complexes that are specific and functional but structurally heterogeneous. Moreover, conformational plasticity is a fundamental feature for function. These assemblies are known as fuzzy complexes. Here this theoretical framework, usually applied to isolated partners, is extended to the intramolecular interface between covalently bound domains instead of isolated pairs. The concept of fuzzy binding is also used in order to describe interactions based on sets of dynamic, transient, and promiscuous contacts between ill-defined sets of interactors. In order to characterize the system, an integrative strategy using short and long range Nuclear Magnetic Resonance techniques and Small Angle X-ray Scattering is applied to several constructs containing different combinations of bound or isolated domains. It is demonstrated that the folded SH3 domain acts as a scaffold for the disordered region, which interacts in a specific manner with its partner. Both disordered domains, SH4 and Unique, are involved in the process albeit they contribute differently. Additionally, it is shown that the Unique domain is not a random coil, but contains a significant degree of pre-arrangement that is independent of the scaffold. Sequence determinants are then searched by comparison of the sequences of different Src Family Kinases. Four conserved phenylalanine residues are found and their implication in Unique domain pre-organization and Unique:SH3 domain interaction tested. All these amino acids are found to favor compaction of the intrinsically disordered region, and at the same time to perturb close contact with the scaffold. In addition, mutations in the interacting zones of the SH3 domain are also studied to test reciprocity. In all, the fuzzy complex model is proven for the SH4:Unique:SH3 system. Then, the results are extrapolated to the full-length c-Src to test its biological relevance. A co evolutionary analysis suggests that the fuzzy model may be a general feature for the whole Src Family, so the closest member of the family, Yes, is also tested experimentally. The initial results on long-range contacts suggests a similar arrangement between the scaffold and the disordered region. In all, it is suggested that plastic, fuzzy interfaces between ordered and disordered domains may be a relevant mode for the transmission of functional information within multidomain proteins. Finally, a first approach for a structural study of the c-Src fuzzy complex in a native-like lipid environment, including natural co-translational modifications, is presented. A protocol for sample preparation is developed and Dynamic Nuclear Polarization solid state NMR is shown to be an adequate tool for further analysis.[spa] c-Src es una tirosina quinasa clave en múltiples rutas de señalización celulares. Su desregulación ha sido asociada a diversos procesos tumorales, entre los que destaca el cáncer de cólon. Una actividad anómala de c-Src se correlaciona con el desarrollo tumoral y pronóstico clínico desfavorable. c-Src forma parte de un grupo de proteínas relacionadas estructural y funcional- mente, la Familia de Quinasas Src. Todas ellas comparten la misma arquitectura modular, que incluye un dominio catalítico (SH1), dos dominios regulatorios, SH2 y SH3, y a continuación una región variable intrínsecamente desordenada que incluye los dominios Único y SH4. Mientras que el segmento ordenado está bien caracterizado, el papel de la región desordenada no está claro, aunque es funcionalmente relevante. En este trabajo se explora la relación estructural y funcional entre la región desordenada y el dominio ordenado adyacente SH3. Dado que este tipo de interacciones implican un grado significativo de heterogeneidad estructural, se ha aplicado el concepto de unión difusa para caracterizar este sistema. Este marco teórico permite modelar interacciones basadas en contactos dinámicos y transitorios entre múltiples interactores vagamente definidos, que sin embargo son específicos y funcionales. Para ello, se ha usado una estrategia que implica el uso combinado de técnicas de Resonancia Magnética Nuclear de largo y corto alcance, así como Dispersión de rayos X a Bajo Ángulo. Se demuestra así que el dominio plegado SH3 actúa como armazón para la región desordenada, que a su vez contiene un grado significativo de pre-organización estructural. Se han identificado cuatro fenilalaninas en el dominio Único responsables de esta pre-formación que también afectan a la interacción entre la región desordenada y el armazón. Los resultados demuestran que el conjunto de dominios SH4, Único y SH3 forman una unidad funcional que puede ser definida como un complejo difuso. Además, datos teóricos y experimentales de otros miembros de la familia sugieren que el modelo difuso es una característica común de todos ellos. Finalmente, se ha demostrado que la Resonancia Magnética de estado sólido con Polarización Dinámica Nuclear es una técnica adecuada para el estudio estructural de c-Src unida a una matriz lipídica similar a la natural

The Elephant Evolved p53 Isoforms that Escape MDM2-Mediated Repression and Cancer

The p53 tumor suppressor is a transcription factor with roles in cell development, apoptosis, oncogenesis, aging, and homeostasis in response to stresses and infections. p53 is tightly regulated by the MDM2 E3 ubiquitin ligase. The p53-MDM2 pathway has coevolved, with MDM2 remaining largely conserved, whereas the TP53 gene morphed into various isoforms. Studies on prevertebrate ancestral homologs revealed the transition from an environmentally induced mechanism activating p53 to a tightly regulated system involving cell signaling. The evolution of this mechanism depends on structural changes in the interacting protein motifs. Elephants such as Loxodonta africana constitute ideal models to investigate this coevolution as they are large and long-living as well as having 20 copies of TP53 isoformic sequences expressing a variety of BOX-I MDM2-binding motifs. Collectively, these isoforms would enhance sensitivity to cellular stresses, such as DNA damage, presumably accounting for strong cancer defenses and other adaptations favoring healthy aging. Here we investigate the molecular evolution of the p53-MDM2 system by combining in silico modeling and in vitro assays to explore structural and functional aspects of p53 isoforms retaining the MDM2 interaction, whereas forming distinct pools of cell signaling. The methodology used demonstrates, for the first time that in silico docking simulations can be used to explore functional aspects of elephant p53 isoforms. Our observations elucidate structural and mechanistic aspects of p53 regulation, facilitate understanding of complex cell signaling, and suggest testable hypotheses of p53 evolution referencing Peto's Paradox

Structural insight into how the human helicase subunit MCM2 may act as a histone chaperone together with ASF1 at the replication fork

International audienceMCM2 is a subunit of the replicative helicase machinery shown to interact with histones H3 and H4 during the replication process through its N-terminal domain. During replication, this interaction has been proposed to assist disassembly and assembly of nu-cleosomes on DNA. However, how this interaction participates in crosstalk with histone chaperones at the replication fork remains to be elucidated. Here, we solved the crystal structure of the ternary complex between the histone-binding domain of Mcm2 and the histones H3-H4 at 2.9 ˚ A resolution. Histones H3 and H4 assemble as a tetramer in the crystal structure , but MCM2 interacts only with a single molecule of H3-H4. The latter interaction exploits binding surfaces that contact either DNA or H2B when H3-H4 dimers are incorporated in the nucleosome core particle. Upon binding of the ternary complex with the histone chaperone ASF1, the histone tetramer dissociates and both MCM2 and ASF1 interact simultaneously with the histones forming a 1:1:1:1 het-eromeric complex. Thermodynamic analysis of the quaternary complex together with structural model-ing support that ASF1 and MCM2 could form a chaperoning module for histones H3 and H4 protecting them from promiscuous interactions. This suggests an additional function for MCM2 outside its helicase function as a proper histone chaperone connected to the replication pathway