Regulation of apoptosis by an intrinsically disordered region of Bcl-xL.

Intrinsically disordered regions (IDRs) of proteins often regulate function upon post-translational modification (PTM) through interactions with folded domains. An IDR linking two α-helices (α1-α2) of the antiapoptotic protein Bcl-xL experiences several PTMs that reduce antiapoptotic activity. Here, we report that PTMs within the α1-α2 IDR promote its interaction with the folded core of Bcl-xL that inhibits the proapoptotic activity of two types of regulatory targets, BH3-only proteins and p53. This autoregulation utilizes an allosteric pathway whereby, in one direction, the IDR induces a direct displacement of p53 from Bcl-xL coupled to allosteric displacement of simultaneously bound BH3-only partners. This pathway operates in the opposite direction when the BH3-only protein PUMA binds to the BH3 binding groove of Bcl-xL, directly displacing other bound BH3-only proteins, and allosterically remodels the distal site, displacing p53. Our findings show how an IDR enhances functional versatility through PTM-dependent allosteric regulation of a folded protein domain.

Structural polymorphism in the N-terminal oligomerization domain of NPM1.

Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer-pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions.

Real-Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP†NMR Spectroscopy.

The kinase inhibitory domain of the cell cycle regulatory protein p27Kip1 (p27) was nuclear spin hyperpolarized using dissolution dynamic nuclear polarization (D-DNP). While intrinsically disordered in isolation, p27 adopts secondary structural motifs, including an α-helical structure, upon binding to cyclin-dependent kinase 2 (Cdk2)/cyclin A. The sensitivity gains obtained with hyperpolarization enable the real-time observation of 13 C NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds. Time-dependent intensity changes are dependent on the extent of folding and binding, as manifested in differential spin relaxation. The analysis of signal decay rates suggests the existence of a partially folded p27 intermediate during the timescale of the D-DNP NMR experiment.

Defining the condensate landscape of fusion oncoproteins.

Fusion oncoproteins (FOs) arise from chromosomal translocations in ~17% of cancers and are often oncogenic drivers. Although some FOs can promote oncogenesis by undergoing liquid-liquid phase separation (LLPS) to form aberrant biomolecular condensates, the generality of this phenomenon is unknown. We explored this question by testing 166 FOs in HeLa cells and found that 58% formed condensates. The condensate-forming FOs displayed physicochemical features distinct from those of condensate-negative FOs and segregated into distinct feature-based groups that aligned with their sub-cellular localization and biological function. Using Machine Learning, we developed a predictor of FO condensation behavior, and discovered that 67% of ~3000 additional FOs likely form condensates, with 35% of those predicted to function by altering gene expression. 47% of the predicted condensate-negative FOs were associated with cell signaling functions, suggesting a functional dichotomy between condensate-positive and -negative FOs. Our Datasets and reagents are rich resources to interrogate FO condensation in the future.

Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation.

NUP98 fusion oncoproteins (FO) are drivers in pediatric leukemias and many transform hematopoietic cells. Most NUP98 FOs harbor an intrinsically disordered region from NUP98 that is prone to liquid-liquid phase separation (LLPS) in vitro. A predominant class of NUP98 FOs, including NUP98-HOXA9 (NHA9), retains a DNA-binding homeodomain, whereas others harbor other types of DNA- or chromatin-binding domains. NUP98 FOs have long been known to form puncta, but long-standing questions are how nuclear puncta form and how they drive leukemogenesis. Here we studied NHA9 condensates and show that homotypic interactions and different types of heterotypic interactions are required to form nuclear puncta, which are associated with aberrant transcriptional activity and transformation of hematopoietic stem and progenitor cells. We also show that three additional leukemia-associated NUP98 FOs (NUP98-PRRX1, NUP98-KDM5A, and NUP98-LNP1) form nuclear puncta and transform hematopoietic cells. These findings indicate that LLPS is critical for leukemogenesis by NUP98 FOs. SIGNIFICANCE: We show that homotypic and heterotypic mechanisms of LLPS control NUP98-HOXA9 puncta formation, modulating transcriptional activity and transforming hematopoietic cells. Importantly, these mechanisms are generalizable to other NUP98 FOs that share similar domain structures. These findings address long-standing questions on how nuclear puncta form and their link to leukemogenesis. This article is highlighted in the In This Issue feature, p. 873.

Dynamic anticipation by Cdk2/Cyclin A-bound p27 mediates signal integration in cell cycle regulation.

p27Kip1 is an intrinsically disordered protein (IDP) that inhibits cyclin-dependent kinase (Cdk)/cyclin complexes (e.g., Cdk2/cyclin A), causing cell cycle arrest. Cell division progresses when stably Cdk2/cyclin A-bound p27 is phosphorylated on one or two structurally occluded tyrosine residues and a distal threonine residue (T187), triggering degradation of p27. Here, using an integrated biophysical approach, we show that Cdk2/cyclin A-bound p27 samples lowly-populated conformations that provide access to the non-receptor tyrosine kinases, BCR-ABL and Src, which phosphorylate Y88 or Y88 and Y74, respectively, thereby promoting intra-assembly phosphorylation (of p27) on distal T187. Even when tightly bound to Cdk2/cyclin A, intrinsic flexibility enables p27 to integrate and process signaling inputs, and generate outputs including altered Cdk2 activity, p27 stability, and, ultimately, cell cycle progression. Intrinsic dynamics within multi-component assemblies may be a general mechanism of signaling by regulatory IDPs, which can be subverted in human disease.

Self-interaction of NPM1 modulates multiple mechanisms of liquid-liquid phase separation.

Nucleophosmin (NPM1) is an abundant, oligomeric protein in the granular component of the nucleolus with roles in ribosome biogenesis. Pentameric NPM1 undergoes liquid-liquid phase separation (LLPS) via heterotypic interactions with nucleolar components, including ribosomal RNA (rRNA) and proteins which display multivalent arginine-rich linear motifs (R-motifs), and is integral to the liquid-like nucleolar matrix. Here we show that NPM1 can also undergo LLPS via homotypic interactions between its polyampholytic intrinsically disordered regions, a mechanism that opposes LLPS via heterotypic interactions. Using a combination of biophysical techniques, including confocal microscopy, SAXS, analytical ultracentrifugation, and single-molecule fluorescence, we describe how conformational changes within NPM1 control valency and switching between the different LLPS mechanisms. We propose that this newly discovered interplay between multiple LLPS mechanisms may influence the direction of vectorial pre-ribosomal particle assembly within, and exit from the nucleolus as part of the ribosome biogenesis process.

Large-scale analysis of thermostable, mammalian proteins provides insights into the intrinsically disordered proteome.

Intrinsically disordered proteins are predicted to be highly abundant and play broad biological roles in eukaryotic cells. In particular, by virtue of their structural malleability and propensity to interact with multiple binding partners, disordered proteins are thought to be specialized for roles in signaling and regulation. However, these concepts are based on in silico analyses of translated whole genome sequences, not on large-scale analyses of proteins expressed in living cells. Therefore, whether these concepts broadly apply to expressed proteins is currently unknown. Previous studies have shown that heat-treatment of cell extracts lead to partial enrichment of soluble, disordered proteins. On the basis of this observation, we sought to address the current dearth of knowledge about expressed, disordered proteins by performing a large-scale proteomics study of thermostable proteins isolated from mouse fibroblast cells. With the use of novel multidimensional chromatography methods and mass spectrometry, we identified a total of 1320 thermostable proteins from these cells. Further, we used a variety of bioinformatics methods to analyze the structural and biological properties of these proteins. Interestingly, more than 900 of these expressed proteins were predicted to be substantially disordered. These were divided into two categories, with 514 predicted to be predominantly disordered and 395 predicted to exhibit both disordered and ordered/folded features. In addition, 411 of the thermostable proteins were predicted to be folded. Despite the use of heat treatment (60 min at 98 degrees C) to partially enrich for disordered proteins, which might have been expected to select for small proteins, the sequences of these proteins exhibited a wide range of lengths (622 +/- 555 residues (average length +/- standard deviation) for disordered proteins and 569 +/- 598 residues for folded proteins). Computational structural analyses revealed several unexpected features of the thermostable proteins: (1) disordered domains and coiled-coil domains occurred together in a large number of disordered proteins, suggesting functional interplay between these domains; and (2) more than 170 proteins contained lengthy domains (>300 residues) known to be folded. Reference to Gene Ontology Consortium functional annotations revealed that, while disordered proteins play diverse biological roles in mouse fibroblasts, they do exhibit heightened involvement in several functional categories, including, cytoskeletal structure and cell movement, metabolic and biosynthetic processes, organelle structure, cell division, gene transcription, and ribonucleoprotein complexes. We believe that these results reflect the general properties of the mouse intrinsically disordered proteome (IDP-ome) although they also reflect the specialized physiology of fibroblast cells. Large-scale identification of expressed, thermostable proteins from other cell types in the future, grown under varied physiological conditions, will dramatically expand our understanding of the structural and biological properties of disordered eukaryotic proteins.

Proteomic studies of the intrinsically unstructured mammalian proteome.

Intrinsically unstructured proteins (IUPs) represent an important class of proteins primarily involved in cellular signaling and regulation. The aim of this study was to develop methodology for the enrichment and identification of IUPs. We show that heat treatment of NIH3T3 mouse fibroblast cell extracts at 98 degrees C selects for IUPs. The majority of these IUPs were cytosolic or nuclear proteins involved in cell signaling or regulation. These studies represent the first large-scale experimental investigation of the intrinsically unstructured mammalian proteome.

Rotation of the stalk/neck and one head in a new crystal structure of the kinesin motor protein, Ncd.

Molecular motors undergo conformational changes to produce force and move along cytoskeletal filaments. Structural changes have been detected in kinesin motors; however, further changes are expected because previous crystal structures are in the same or closely related conformations. We report here a 2.5 A crystal structure of the minus-end kinesin, Ncd, with the coiled-coil stalk/neck and one head rotated by approximately 75 degrees relative to the other head. The two heads are asymmetrically positioned with respect to the stalk and show asymmetry of nucleotide state: one head is fully occupied, but the other is unstably bound to ADP. Unlike previous structures, our new atomic model can be fit into cryoelectron microscopy density maps of the motor attached to microtubules, where it appears to resemble a one-head-bound motor with the stalk rotated towards the minus end. Interactions between neck and motor core residues, observed in the head that moves with the stalk, are disrupted in the other head, permitting rotation of the stalk/neck. The rotation could represent a force-producing stroke that directs the motor to the minus end.

Crystal structures of the Dab homology domains of mouse disabled 1 and 2.

Disabled (Dab) 1 and 2 are mammalian homologues of Drosophila DAB. Dab1 is a key cytoplasmic mediator in Reelin signaling that controls cell positioning in the developing central nervous system, whereas Dab2 is an adapter protein that plays a role in endocytosis. DAB family proteins possess an amino-terminal DAB homology (DH) domain that is similar to the phosphotyrosine binding/phosphotyrosine interaction (PTB/PI) domain. We have solved the structures of the DH domains of Dab2 (Dab2-DH) and Dab1 (Dab1-DH) in three different ligand forms, ligand-free Dab2-DH, the binary complex of Dab2-DH with the Asn-Pro-X-Tyr (NPXY) peptide of amyloid precursor protein (APP), and the ternary complex of Dab1-DH with the APP peptide and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3, the head group of phosphatidylinositol-4,5-diphosphate (PtdIns-4,5-P2)). The similarity of these structures suggests that the rigid Dab DH domain maintains two independent pockets for binding of the APP/lipoprotein receptors and phosphoinositides. Mutagenesis confirmed the structural determinants specific for the NPXY sequence and PtdIns-4,5-P2 binding. NMR spectroscopy confirmed that the DH domain binds to Ins-1,4,5-P3 independent of the NPXY peptides. These findings suggest that simultaneous interaction of the rigid DH domain with the NPXY sequence and PtdIns-4,5-P2 plays a role in the attachment of Dab proteins to the APP/lipoprotein receptors and phosphoinositide-rich membranes.