1,2,6-thiadiazinones as novel narrow spectrum calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) inhibitors

We demonstrate for the first time that 4H-1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4H-1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors

Optimal design of thermally stable proteins

Motivation: For many biotechnological purposes, it is desirable to redesign proteins to be more structurally and functionally stable at higher temperatures. For example, chemical reactions are intrinsically faster at higher temperatures, so using enzymes that are stable at higher temperatures would lead to more efficient industrial processes. We describe an innovative and computationally efficient method called Improved Configurational Entropy (ICE), which can be used to redesign a protein to be more thermally stable (i.e. stable at high temperatures). This can be accomplished by systematically modifying the amino acid sequence via local structural entropy (LSE) minimization. The minimization problem is modeled as a shortest path problem in an acyclic graph with nonnegative weights and is solved efficiently using Dijkstra's method

Characterizing the conformational dynamics of metal-free PsaA using molecular dynamics simulations and electron paramagnetic resonance spectroscopy

Prokaryotic metal-ion receptor proteins, or solute-binding proteins, facilitate the acquisition of metal ions from the extracellular environment. Pneumococcal surface antigen A (PsaA) is the primary Mn2+-recruiting protein of the human pathogen Streptococcus pneumoniae and is essential for its in vivo colonization and virulence. The recently reported high-resolution structures of metal- free and metal-bound PsaA have provided the first insights into the mechanism of PsaA-facilitated metal binding. However, the conformational dynamics of metal-free PsaA in solution remain unknown. Here, we use continuous wave electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations to study the relative flexibility of the structural domains in metal-free PsaA and its distribution of conformations in solution. The results show that the crystal structure of the metal-free PsaA is a good representation of the dominant conformation in solution, but the protein also samples structurally distinct conformations that are not captured by the crystal structure. Further, these results suggest that the metal binding site is larger and more solvent exposed than indicated by the metal-free crystal structure. Collectively, this study provides atomic-resolution insight into the conformational dynamics of PsaA prior to metal binding and lays the groundwork for future EPR and MD based studies of PsaA in solution

WNT activates the AAK1 kinase to promote clathrin-mediated endocytosis of LRP6 and establish a negative feedback loop

beta-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity2617993FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2013/50724-5; 2016/17469-0M.B.M. acknowledges support from the NIH (RO1-CA187799 and U24-DK116204-01). M.J.A. received financial support from NIH T32 Predoctoral Training Grants in Pharmacology (T32-GM007040-43 and T32-GM007040-42), an Initiative for Maximizing Student Diversity Grant (R25-GM055336-16), and the NIH National Cancer Institute (NCI) NRSA Predoctoral Fellowship to Promote Diversity in Health-Related Research (F31CA228289). M.P.W. received support from the Lymphoma Research Foundation (337444) and the NIH (T32-CA009156-35). Y.N. was supported by grants-in-aid from the Japan Society for the Promotion of Science (JSPS) (15KK0356 and 16K11493). T.T. was supported by the Howard Hughes Medical Institute Gilliam Fellowship for Advanced Study. M.V.G. was supported by Cancer Research UK (grants C7379/A15291 and C7379/A24639 to Mariann Bienz). The UNC Flow Cytometry Core Facility is supported in part by Cancer Center Core Support Grant P30 CA016086 to the UNC Lineberger Comprehensive Cancer Center, and research reported in this publication was supported by the Center for AIDS Research (award number 5P30AI050410), and the content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The Structural Genomics Consortium (SGC) is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, the Canada Foundation for Innovation, the Eshelman Institute for Innovation, Genome Canada, the Innovative Medicines Initiative (European Union [EU]/European Federation of Pharmaceutical Industries and Associations [EFPIA]) (ULTRA-DD grant no. 115766), Janssen, Merck & Company, Merck KGaA, Novartis Pharma AG, the Ontario Ministry of Economic Development and Innovation, Pfizer, the São Paulo Research Foundation (FAPESP) (2013/50724-5), Takeda, and the Wellcome Trust (106169/ZZ14/Z). R.R.R. received financial support from FAPESP (2016/17469-0). We would also like to thank Claire Strain-Damerell and Pavel Savitsky for cloning various mutants of AAK1 and BMP2K proteins that were used in the crystallization trials. Additionally, we thank Dr. Sean Conner for providing the AAK1 plasmids, Dr. Stephane Angers for kindly providing the HEK293T DVL TKO cells, and Dr. Mariann Bienz for providing comments and feedback. We would like to thank members of the Major laboratory for their feedback and expertise regarding experimental design and project directio

The Pneumococcal Iron Uptake Protein a (PiuA) Specifically Recognizes Tetradentate FeIIIbis- and Mono-Catechol Complexes

Streptococcus pneumoniae (Spn) is an important Gram-positive human pathogen that causes millions of infections worldwide with an increasing occurrence of antibiotic resistance. Fe acquisition is a crucial virulence determinant in Spn; further, Spn relies on exogenous FeIII-siderophore scavenging to meet nutritional Fe needs. Recent studies suggest that the human catecholamine stress hormone, norepinephrine (NE), facilitates Fe acquisition in Spn under conditions of transferrin-mediated Fe starvation. Here we show that the solute binding lipoprotein PiuA from the piu Fe acquisition ABC transporter PiuBCDA, previously described as an Fe-hemin binding protein, binds tetradentate catechol FeIII complexes, including NE and the hydrolysis products of enterobactin. Two protein-derived ligands (H238, Y300) create a coordinately-saturated FeIII complex, which parallel recent studies in the Gram-negative intestinal pathogen Campylobacter jejuni. Our in vitro studies using NMR spectroscopy and 54Fe LC-ICP-MS confirm the FeIII can move from transferrin to apo-PiuA in a NE-dependent manner. Structural analysis of PiuA FeIII-bis-catechol and GaIII-bis-catechol and GaIII-(NE)2 complexes by NMR spectroscopy reveals only localized structural perturbations in PiuA upon ligand binding, largely consistent with recent descriptions of other solute binding proteins of type II ABC transporters. We speculate that tetradentate FeIII complexes formed by mono- and bis-catechol species are important Fe sources in Gram-positive human pathogens, since PiuA functions in the same way as SstD from Staphylococcus aureus

Single gene evolution: A punctuated history of chance events

In the present work I have developed and characterized an in vivo selection system to investigate molecular evolution. The essential gene that codes for adenylate kinase (AK; EC 2.4.7.3) in the gram-positive moderate thermophile Geobacillus stearothermophilus was replaced through homologous recombination with its counterpart from the mesophilic Bacillus subtilis. PCR, DNA sequencing and Southern analysis confirmed proper gene replacement and preservation of neighboring genes in the recombinant strain. Recombinant cells (NUB3621-R:ThEV) displayed a temperature sensitive phenotype, with a highest growing temperature almost 20°C lower than that of wild-type cells (56°C vs 75°C). The temperature sensitive phenotype in recombinant cells was linked to a disruption of adenylate homeostasis at high temperatures, secondary to B. subtilis AK heat inactivation, as shown by enzyme activity assays, temperature denaturation profiles and adenylate level measurements. Evolution of a single gene, B. subtilis adk, was investigated by steady-state growth of the recombinant strain from 55 to 70°C in a turbidostat. The temporal characteristics of B. subtilis adk evolution were probed by DNA sequence analysis at various temperatures. The appearance of more fit strains from pre-existing genetic variation, rapid extinctions generated by clonal interference and selection coupled to a strong dependence on historical context and chance events resulted in a punctuated pattern of evolution. The organism fitness at different temperatures could be linked to its expressed AK variant and allowed the investigation of the biochemical and structural basis of adaptation. Heat denaturation and enzyme activity studies showed that all isolated AK mutants were more stable than the wild-type protein and were responsible for the punctuated pattern of evolution detected during the population analyses. The atomic structure of one of the isolated AK variants (Q199R) revealed that its increased thermostability is due to unique electrostatic interactions absent in the wild-type structure. Taken together our population, biochemical and structural analysis suggests that natural selection observed at the molecular level is guided by the same principles that act at the organismal level

Prokaryotic substrate-binding proteins as targets for antimicrobial therapies

The rapid emergence of multidrug-resistant bacteria over the last two decades has catalyzed a shift away from traditional antibiotic development strategies and encouraged the search for unconventional drug targets. Prokaryotic substrate- binding proteins (SBPs), together with their cognate ATP-binding cassette (ABC) transporters, facilitate the unidirectional, transbilayer movement of specific extracytosolic cargoes against a concentration gradient, powered by ATP hydrolysis. In Gram-negative bacteria, SBPs are found in the periplasmic space, whereas in Gram-positive organisms these proteins are anchored to the outer cell wall by a lipid moiety. SBPs are vital components of the substrate-translocation machinery, as they determine cargo specificity and are involved in coupling the cargo uptake process with ABC transporter- mediated ATP hydrolysis. In this review, we focus on "Cluster A-1" divalent metal-binding proteins from within the SBP family. Acquisition of transition row metal ions is essential for bacterial colonization and virulence and Cluster A-1 SBPs play an integral role in this process. Cluster A-1 SBPs lack homologs in humans, bypass the need to deliver compounds into the bacterial cell, and are therefore potential drug targets against Gram-positive bacteria. Here we discuss the role SBPs play in the prokaryotic substrate-translocation machinery with emphasis in the substrate-binding mechanism of Cluster A-1 SBPs, the role of these proteins in virulence and their potential use as drug targets.M. Counago, Rafael; A. McDevitt, Christopher; P. Ween, Miranda; Kobe, Bostja

Crystal structure of rice importin-alpha and structural basis of its interaction with plant-specific nuclear localization signals

In the classical nucleocytoplasmic import pathway, nuclear localization signals (NLSs) in cargo proteins are recognized by the import receptor importin-alpha. Importin-alpha has two separate NLS binding sites (the major and the minor site), both of which recognize positively charged amino acid clusters in NLSs. Little is known about the molecular basis of the unique features of the classical nuclear import pathway in plants. We determined the crystal structure of rice (Oryza sativa) importin-alpha 1a at 2-angstrom resolution. The structure reveals that the autoinhibitory mechanism mediated by the importin-beta binding domain of importin-alpha operates in plants, with NLS-mimicking sequences binding to both minor and major NLS binding sites. Consistent with yeast and mammalian proteins, rice importin-alpha binds the prototypical NLS from simian virus 40 large T-antigen preferentially at the major NLS binding site. We show that two NLSs, previously described as plant specific, bind to and are functional with plant, mammalian, and yeast importin-alpha proteins but interact with rice importin-alpha more strongly. The crystal structures of their complexes with rice importin-alpha show that they bind to the minor NLS binding site. By contrast, the crystal structures of their complexes with mouse (Mus musculus) importin-alpha show preferential binding to the major NLS binding site. Our results reveal the molecular basis of a number of features of the classical nuclear transport pathway specific to plants