Expanding the Paradigm: Intrinsically Disordered Proteins and Allosteric Regulation

Allosteric regulatory processes are implicated at all levels of biological function. Recent advances in our understanding of the diverse and functionally significant class of intrinsically disordered proteins have identified a multitude of ways in which disordered proteins function within the confines of the allosteric paradigm. Allostery within or mediated by intrinsically disordered proteins ensures robust and efficient signal integration through mechanisms that would be extremely unfavorable or even impossible for globular protein interaction partners. Here, we highlight recent examples that indicate the breadth of biological outcomes that can be achieved through allosteric regulation by intrinsically disordered proteins. Ongoing and future work in this rapidly evolving area of research will expand our appreciation of the central role of intrinsically disordered proteins in ensuring the fidelity and efficiency of cellular regulation.     

Packing, specificity, and mutability at the binding interface between the p160 coactivator and CREB-binding protein

Among the most common interaction motifs between nuclear proteins is the recognition of one or more amphipathic helices. In an effort to determine principles behind this recognition, we have investigated the interaction between the p160 coactivator protein ACTR and the ACTR-binding domain of the CREB-binding protein, CBP. The two proteins use relatively small portions of their primary sequences to form a single synergistically folded domain consisting of six intertwined alpha-helices, three from each protein. Neither of the component polypeptides forms a cooperatively folded domain in isolation. However, a considerable amount of residual secondary structure remains in the isolated CBP domain according to CD spectroscopy. Chemical denaturation, differential scanning calorimetry, and ANS binding experiments demonstrate that the isolated CBP domain is not entirely unfolded but forms a helical state with the characteristics of a molten globule. Mutations probing the functional and energetic significance of a buried intermolecular Arg-Asp salt bridge in the interface of the protein complex suggest that these residues are tuned for functional discrimination and not strictly for binding affinity or stability. These results suggest a mechanism for formation of the complex where the unfolded ACTR domain interacts with the partly folded CBP domain in a rapid and specific manner to form the final stable complex.     

Identification of Cys255 in HIF‐1α as a novel site for development of covalent inhibitors of HIF‐1α/ARNT PasB domain protein–protein interaction

The heterodimer HIF‐1α (hypoxia inducible factor)/HIF‐β (also known as ARNT‐aryl hydrocarbon nuclear translocator) is a key mediator of cellular response to hypoxia. The interaction between these monomer units can be modified by the action of small molecules in the binding interface between their C‐terminal heterodimerization (PasB) domains. Taking advantage of the presence of several cysteine residues located in the allosteric cavity of HIF‐1α PasB domain, we applied a cysteine‐based reactomics “hotspot identification” strategy to locate regions of HIF‐1α PasB domain critical for its interaction with ARNT. COMPOUND 5 was identified using a mass spectrometry‐based primary screening strategy and was shown to react specifically with Cys255 of the HIF‐1α PasB domain. Biophysical characterization of the interaction between PasB domains of HIF‐1α and ARNT revealed that covalent binding of COMPOUND 5 to Cys255 reduced binding affinity between HIF‐1α and ARNT PasB domains approximately 10‐fold. Detailed NMR structural analysis of HIF‐1α‐PasB‐COMPOUND 5 conjugate showed significant local conformation changes in the HIF‐1α associated with key residues involved in the HIF‐1α/ARNT PasB domain interaction as revealed by the crystal structure of the HIF‐1α/ARNT PasB heterodimer. Our screening strategy could be applied to other targets to identify pockets surrounding reactive cysteines suitable for development of small molecule modulators of protein function.     

Isolation of zoosporogenous actinomycetes from desert soils

We have undertaken a study to estimate the species diversity of zoosporogenous actinomycetes that can be isolated from an arid environment. The study site encompassed an area of approximately 22 000 square kilometers of the Mojave Desert along the California-Nevada border. A series of 29 soil samples was collected along two intersecting transects of approximately 190 and 240 km which traversed a number of distinct ecosystems. A₀ horizon soils were collected from the rhizosphere of the predominant vegetation at each sampling site and screened for the target genera using selective isolation techniques: chemoattraction (xylose and -collidine) and baiting with hair. Following incubation of primary isolation plates for 28 days at 28°C, all colonies that exhibited filamentous growth, presence of sporangia and/or motile spores upon direct microscopic observation (450 and 1000×) were further characterized by fatty acid analysis (FAME). Most of the isolates fell into three broad clusters that roughly correlated with presumptive genus assignments. Individual isolates could be assigned to 226 FAME biotypes based on chromatographic similarity (85%). The dominant species (514/826 isolates) belong to a previously undescribed taxon that morphologically resemblesGeodermatophilus but possesses unique FAME profiles that include at least three novel lipids. The remainder of the isolates were species ofActinoplanes, indeterminate species or vagrant isolates ofStreptomyces.     

1H, 13C and 15N chemical shift referencing in biomolecular NMR

Summary A considerable degree of variability exists in the way that ¹H, ¹³C and ¹⁵N chemical shifts are reported and referenced for biomolecules. In this article we explore some of the reasons for this situation and propose guidelines for future chemical shift referencing and for conversion from many common ¹H, ¹³C and ¹⁵N chemical shift standards, now used in biomolecular NMR, to those proposed here.Abbreviations TMS tetramethylsilane - TSP 3-(trimethylsilyl)-propionate, sodium salt - DSS 2,2-dimethyl-2-silapentane-5-sulfonate, sodium salt - TFE 2,2,2-trifluoroethanol - DMSO dimethyl sulfoxide     

Condensin minimizes topoisomerase II‐mediated entanglements of DNA in vivo

The juxtaposition of intracellular DNA segments, together with the DNA‐passage activity of topoisomerase II, leads to the formation of DNA knots and interlinks, which jeopardize chromatin structure and gene expression. Recent studies in budding yeast have shown that some mechanism minimizes the knotting probability of intracellular DNA. Here, we tested whether this is achieved via the intrinsic capacity of topoisomerase II for simplifying the equilibrium topology of DNA; or whether it is mediated by SMC (structural maintenance of chromosomes) protein complexes like condensin or cohesin, whose capacity to extrude DNA loops could enforce dissolution of DNA knots by topoisomerase II. We show that the low knotting probability of DNA does not depend on the simplification capacity of topoisomerase II nor on the activities of cohesin or Smc5/6 complexes. However, inactivation of condensin increases the occurrence of DNA knots throughout the cell cycle. These results suggest an in vivo role for the DNA loop extrusion activity of condensin and may explain why condensin disruption produces a variety of alterations in interphase chromatin, in addition to persistent sister chromatid interlinks in mitotic chromatin.     

PP2A-B56γ is required for an efficient spindle assembly checkpoint

The Spindle Assembly Checkpoint (SAC) is part of a complex feedback system designed to ensure that cells do not proceed through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. The formation of the kinetochore complex and the implementation of the SAC are regulated by multiple kinases and phosphatases. BubR1 is a phosphoprotein that is part of the Cdc20 containing mitotic checkpoint complex that inhibits the APC/C so that Cyclin B1 and Securin are not degraded, thus preventing cells going into anaphase. In this study, we found that PP2A in association with its B56γ regulatory subunit, are needed for the stability of BubR1 during nocodazole induced cell cycle arrest. In primary cells that lack B56γ, BubR1 is prematurely degraded and the cells proceed through mitosis. The reduced SAC efficiency results in cells with abnormal chromosomal segregation, a hallmark of transformed cells. Previous studies on PP2A's role in the SAC and kinetochore formation were done using siRNAs to all 5 of the B56 family members. In our study we show that inactivation of only the PP2A-B56γ subunit can affect the efficiency of the SAC. We also provide data that show the intracellular locations of the B56 subunits varies between family members, which is consistent with the hypothesis that they are not completely functionally redundant.     

Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin

Flower color is mainly determined by anthocyanins. Rosa hybrida lacks violet to blue flower varieties due to the absence of delphinidin-based anthocyanins, usually the major constituents of violet and blue flowers, because roses do not possess flavonoid 3',5'-hydoxylase (F3'5'H), a key enzyme for delphinidin biosynthesis. Other factors such as the presence of co-pigments and the vacuolar pH also affect flower color. We analyzed the flavonoid composition of hundreds of rose cultivars and measured the pH of their petal juice in order to select hosts of genetic transformation that would be suitable for the exclusive accumulation of delphinidin and the resulting color change toward blue. Expression of the viola F3'5'H gene in some of the selected cultivars resulted in the accumulation of a high percentage of delphinidin (up to 95%) and a novel bluish flower color. For more exclusive and dominant accumulation of delphinidin irrespective of the hosts, we down-regulated the endogenous dihydroflavonol 4-reductase (DFR) gene and overexpressed the Irisxhollandica DFR gene in addition to the viola F3'5'H gene in a rose cultivar. The resultant roses exclusively accumulated delphinidin in the petals, and the flowers had blue hues not achieved by hybridization breeding. Moreover, the ability for exclusive accumulation of delphinidin was inherited by the next generations.