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p27$^{Kip1}$ (p27) is an intrinsically disordered protein (IDP) that folds upon binding to cyclin-dependent kinase (Cdk)$/$cyclin complexes (e.g., Cdk2$/$cyclin A), inhibiting their catalytic activity and causing cell cycle arrest. However, cell division progresses when stably Cdk2$/$cyclin A-bound p27 is phosphorylated on one or two structurally occluded tyrosine residues $[$tyrosines 88 (Y88) and 74 (Y74)$]$ and a distal threonine residue $[$threonine 187 (T187)$]$. These events trigger ubiquitination and degradation of p27, fully activating Cdk2$/$cyclin A to drive cell division. Using an integrated approach comprising structural, biochemical, biophysical and single-molecule fluorescence methods, we show that Cdk2$/$cyclin A-bound p27 samples lowly-populated conformations that dynamically anticipate the sequential steps of this signaling cascade. "Dynamic anticipation" provides access to the non-receptor tyrosine kinases, BCR-ABL and Src, which sequentially phosphorylate Y88 and Y74 and promote intra-assembly phosphorylation (of p27) on distal T187. Tyrosine phosphorylation also allosterically relieves p27-dependent inhibition of substrate binding to Cdk2$/$cyclin A, a phenomenon we term "cross-complex allostery". 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, as exemplified by hyper-active BCR-ABL and Src in certain cancers., 35 pages, 5 figures, supporting information 37 pages

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 13C 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.

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.

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 Mg 2+ . This could weaken Mg 2+ 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.

The crystal structures of gyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Escherichia coli have been determined in three different enzymatic states, NAD(+)-free, NAD(+)-bound, and hemiacetal intermediate. The NAD(+)-free structure reported here has been determined from monoclinic and tetragonal crystal forms. The conformational changes in GAPDH induced by cofactor binding are limited to the residues that bind the adenine moiety of NAD(+). Glyceraldehyde 3-phosphate (GAP), the substrate of GAPDH, binds to the enzyme with its C3 phosphate in a hydrophilic pocket, called the "new P(i)" site, which is different from the originally proposed binding site for inorganic phosphate. This observed location of the C3 phosphate is consistent with the flip-flop model proposed for the enzyme mechanism [Skarzynski, T., Moody, P. C., and Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187]. Via incorporation of the new P(i) site in this model, it is now proposed that the C3 phosphate of GAP initially binds at the new P(i) site and then flips to the P(s) site before hydride transfer. A superposition of NAD(+)-bound and hemiacetal intermediate structures reveals an interaction between the hydroxyl oxygen at the hemiacetal C1 of GAP and the nicotinamide ring. This finding suggests that the cofactor NAD(+) may stabilize the transition state oxyanion of the hemiacetal intermediate in support of the flip-flop model for GAP binding.

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.

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.

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.

Pantothenate kinase (PanK) is a key regulatory enzyme in the coenzyme A (CoA) biosynthetic pathway and catalyzes the phosphorylation of pantothenic acid to form phosphopantothenate. CoA is a feedback inhibitor of PanK activity by competitive binding to the ATP site. The structures of the Escherichia coli enzyme, in complex with a nonhydrolyzable analogue of ATP, 5'-adenylimido-diphosphate (AMPPNP), or with CoA, were determined at 2.6 and 2.5 A, respectively. Both structures show that two dimers occupy an asymmetric unit; each subunit has a alpha/beta mononucleotide-binding fold with an extensive antiparallel coiled coil formed by two long helices along the dimerization interface. The two ligands, AMPPNP and CoA, associate with PanK in very different ways, but their phosphate binding sites overlap, explaining the kinetic competition between CoA and ATP. Residues Asp(127), His(177), and Arg(243) are proposed to be involved in catalysis, based on modeling of the pentacoordinate transition state. The more potent inhibition by CoA, compared with the CoA thioesters, is explained by a tight interaction of the CoA thiol group with the side chains of aromatic residues, which is predicted to discriminate against the CoA thioesters. The PanK structure provides the framework for a more detailed understanding of the mechanism of catalysis and feedback regulation of PanK.

Proteins that are fully or partially unstructured are named intrinsically disordered proteins (IDP). The function of IDP's, in context of the structure-function paradigm, is challenged by their inherent flexibility. The current hypothesis of induced-folding mechanism of IDP's suggests many advantages over well-ordered and structured proteins because of it ability to recognize and regulate multiple pathways by adopting numerous conformations. One of the most prominent examples is the disordered polypeptide p27. p27 is a small IDP that inhibits cyclin-dependent kinases (Cdk) regulating cell proliferation. Furthermore, phosphorylation of p27 leads to its degradation by the ubiquitin-protease pathway. By applying single-molecule fluorescence spectroscopy in combination with biochemical assays, we tested the hypothesis that bound disordered regions of p27 are essential for its regulatory functions. However, the existence of regulatory modification sites within bound, folded regions of disordered proteins raises a key question regarding how these sites become accessible for enzymatic modification. Our results show that p27 is dynamic even when bound to the Cdk2/CyclinA complex. Moreover, sequential phosphorylation at positions Y88 and Y74 of p27 relieves Cdk2 inhibition with rheostat-like precision. These observations explain the mechanism of how intra and inter-molecular phosphorylation of partially activated Cdk2/CyclinA are regulated during the cell cycle and provide the basis for understanding how dynamics within Cdk2/CyclinA-bound p27 mediate signal transmission.