Mi-Kyung Yun

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.

PUMA binding induces partial unfolding within BCL-xL to disrupt p53 binding and promote apoptosis

Following DNA damage, nuclear p53 induces the expression of PUMA, a BH3-only protein that binds and inhibits the anti-apoptotic BCL-2 repertoire, including BCL-xL. PUMA, unique amongst BH3-only proteins, disrupts the interaction between cytosolic p53 and BCL-xL, allowing p53 to promote apoptosis via direct activation of the BCL-2 effector molecules, BAX and BAK. Structural investigations using nuclear magnetic resonance spectroscopy and X-ray crystallography revealed that PUMA binding induced partial unfolding of two α-helices within BCL-xL. Wild-type PUMA or a PUMA mutant incapable of causing binding-induced unfolding of BCL-xL equivalently inhibited the anti-apoptotic BCL-2 repertoire to sensitize for death receptor (DR)-activated apoptosis, but only wild-type PUMA promoted p53-dependent, DNA damage-induced apoptosis. Our data suggest that PUMA-induced partial unfolding of BCL-xL disrupts interactions between cytosolic p53 and BCL-xL, releasing the bound p53 to initiate apoptosis. We propose that regulated unfolding of BCL-xL provides a mechanism to promote PUMA-dependent signaling within the apoptotic pathways.

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.

Catalysis and sulfa drug resistance in dihydropteroate synthase.

Sulfas Crystal View The sulfonamide antibiotics (sulfa drugs) have been used to treat infections for over 70 years; however, emerging resistance has eroded their clinical utility. Sulfa drugs target dihydropteroate synthase, a key enzyme in the bacterial folate pathway. By performing the reaction in the crystalline form of the enzyme, Yun et al. (p. 1110) have characterized the key structural intermediates. In combining structural data with theoretical and mutagenesis studies, they propose a detailed mechanism for dihydropteroate synthase catalysis. By resolving this structure with a sulfa drug bound to the enzyme, they showed how inhibition occurred and indicated how resistance could emerge. Structures of a target enzyme in the bacteria that cause anthrax and bubonic plague may lead to effective drugs. The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an SN1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.

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.

Fused Oxacycle Synthesis via Radical Cyclization of β- Alkoxyacrylates.

Abstract β-Alkoxyacrylates are efficient radical acceptors in intramolecular addition of O-stannyl ketyls. This cyclization reaction can be applied reiteratively to form fused oxacycles.

Kar3 interaction with Cik1 alters motor structure and function

Kar3, a kinesin‐14 motor of Saccharomyces cerevisiae required for mitosis and karyogamy, reportedly interacts with Cik1, a nonmotor protein, via its central, predicted coiled coil. Despite this, neither Kar3 nor Cik1 homodimers have been observed in vivo. Here we show that Kar3 is a dimer in vitro by analytical ultracentrifugation. The motor domains appear as paired particles by rotary‐shadow electron microscopy (EM) and circular dichroism (CD) spectroscopy of the nonmotor region shows characteristics of helical structure, typical of coiled coils. Remarkably, the Kar3/Cik1 nonmotor region shows greater helicity by CD analysis and rotary‐shadow EM reveals a stalk joined to one large or two smaller particles. The highly helical Kar3/Cik1 nonmotor region and visible stalk indicate that dimerization with Cik1 causes structural changes in Kar3. The Cik1 and Kar3 stalk regions preferentially associate with one another rather than forming homodimers. Kar3/Cik1 moves on microtubules at 2–2.4 μm min−1, 2–5‐fold faster than Kar3, and destabilizes microtubules at the lagging ends. Thus, structural changes in Kar3 upon dimerization with Cik1 alter the motor velocity and likely regulate Kar3 activity in vivo.

Discovery of LB30057, a benzamidrazone-based selective oral thrombin inhibitor

Systematic variation of the so-called P-pocket moiety of benzamidrazone-based selective thrombin inhibitors led to the discovery of LB30057. It is potent (Ki = 0.38 nM for human thrombin), selective (Ki = 3290 nM for bovine trypsin), and orally bioavailable (58% oral bioavailability in dogs). LB30057 was efficacious in thrombosis animal models.

Modulation of Pantothenate Kinase 3 Activity by Small Molecules that Interact with the Substrate/Allosteric Regulatory Domain

Pantothenate kinase (PanK) catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis. PanK3 is stringently regulated by acetyl-CoA and uses an ordered kinetic mechanism with ATP as the leading substrate. Biochemical analysis of site-directed mutants indicates that pantothenate binds in a tunnel adjacent to the active site that is occupied by the pantothenate moiety of the acetyl-CoA regulator in the PanK3acetyl-CoA binary complex. A high-throughput screen for PanK3 inhibitors and activators was applied to a bioactive compound library. Thiazolidinediones, sulfonylureas and steroids were inhibitors, and fatty acyl-amides and tamoxifen were activators. The PanK3 activators and inhibitors either stimulated or repressed CoA biosynthesis in HepG2/C3A cells. The flexible allosteric acetyl-CoA regulatory domain of PanK3 also binds the substrates, pantothenate and pantetheine, and small molecule inhibitors and activators to modulate PanK3 activity.

STRUCTURAL MODIFICATION OF AN ORALLY ACTIVE THROMBIN INHIBITOR, LB30057: REPLACEMENT OF THE D-POCKET-BINDING NAPHTHYL MOIETY

An amidrazonophenylalanine derivative LB30057 (2) was identified as a potent (Ki = 0.38 nM), selective, and orally active thrombin inhibitor. As a continuation of studies into benzamidrazone-based thrombin inhibitors, we have structurally modified compound 2 by replacing the naphthyl group with a variety of hydrophobic moieties. This study led to discovery of several compounds with significantly enhanced potency in thrombin inhibition without sacrificing selectivity against trypsin and oral absorption. The highest activity was obtained with compound 23 (Ki = 0.045 nM).