Cheon-Gil Park

A structural pathway for activation of the kinesin motor ATPase.

Molecular motors move along actin or microtubules by rapidly hydrolyzing ATP and undergoing changes in filament‐binding affinity with steps of the nucleotide hydrolysis cycle. It is generally accepted that motor binding to its filament greatly increases the rate of ATP hydrolysis, but the structural changes in the motor associated with ATPase activation are not known. To identify the conformational changes underlying motor movement on its filament, we solved the crystal structures of three kinesin mutants that decouple nucleotide and microtubule binding by the motor, and block microtubule‐activated, but not basal, ATPase activity. Conformational changes in the structures include a disordered loop and helices in the switch I region and a visible switch II loop, which is disordered in wild‐type structures. Switch I moved closer to the bound nucleotide in two mutant structures, perturbing water‐mediated interactions with the Mg2+. This could weaken Mg2+ binding and accelerate ADP release to activate the motor ATPase. The structural changes we observe define a signaling pathway within the motor for ATPase activation that is likely to be essential for motor movement on microtubules.

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 Å crystal structure of the minus‐end kinesin, Ncd, with the coiled‐coil stalk/neck and one head rotated by ∼75° 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.

Structural polymorphism in the N-terminal oligomerization domain of NPM1

Significance Nucleophosmin (NPM1) is a multifunctional protein with critical roles in ribosome biogenesis, centrosome duplication, and tumor suppression. Despite the established importance of NPM1 as a tumor marker and potential drug target, little is currently known about the molecular mechanisms that govern its various functions. Our manuscript describes that the N-terminal domain of NPM1 (Npm-N) exhibits phosphorylation-dependent structural polymorphism along a broad conformational landscape between two extreme states: a stable, folded pentamer and a globally disordered monomer. We propose that phosphorylation-induced “regulated unfolding” of Npm-N provides a means to modulate NPM1 function and subcellular localization. Our findings will drive future structure-based studies on the roles of regulated unfolding in NPM1 biology and will provide a foundation for NPM1-targeted anticancer drug development. 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.

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.

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.The nucleolus is a membrane-less organelle formed through liquid–liquid phase separation (LLPS). Here the authors use biophysical methods and show that the nucleolar protein nucleophosmin (NPM1) also undergoes LLPS through homotypic, inter-NPM1 interactions and discuss implications for the ribosome biogenesis process.

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

AbstractIntrinsically 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.Post-translational modification of residues in an intrinsically disordered region of Bcl-XL promotes interactions with its folded core and allosterically reduces affinity for proapoptotic BH3-domain-containing proteins, resulting in apoptosis.

Large-scale Analysis of Thermo-stable, 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 spectromet...