Preparation of CDK/Cyclin Inhibitor Complexes for Structural Determination.

The abundance of biochemical and structural knowledge on the Cyclin-Dependent Kinases (CDKs) has provided a comprehensive but not exhaustive insight into the molecular determinants that govern their function mechanisms. The implementation of structural and functional CDK models towards developing novel anticancer strategies that will specifically target individual or multiple CDKs remains a critical need.More than 250 CDKs crystal structures are available to-date, including truncated or whole, modified or not, active or inactive forms, co-crystallized with the cyclins and/or their respective putative inhibitors, though, to our knowledge, there is no NMR solved structure available to date. We hitherto attempt to provide a useful guide from protein production to crystallization for CDK/Inhibitors complexes based on an overview of the already elucidated CDK structures, constructs and the preferable expression vectors in each case, in order to yield the respective crystals.

Efficient soluble expression of active recombinant human cyclin A2 mediated by E. coli molecular chaperones.

Bacterial expression of human proteins continues to present a critical challenge in protein crystallography and drug design. While human cyclin A constructs have been extensively characterized in complex with cyclin dependent kinase 2 (CDK2), efforts to express the monomeric human cyclin A2 in Escherichia coli in a stable form, without the kinase subunit, have been laden with technical difficulties, including solubility, yield and purity. Here, optimized conditions are described with the aim of generating for first time, sufficient quantities of human recombinant cyclin A2 in a soluble and active form for crystallization and ligand characterization purposes. The studies involve implementation of a His-tagged heterologous expression system under conditions of auto-induction and mediated by molecular chaperone-expressing plasmids. A high yield of human cyclin A2 was obtained in natively folded and soluble form, through co-expression with groups of molecular chaperones from E. coli in various combinations. A one-step affinity chromatography method was utilized to purify the fusion protein products to homogeneity, and the biological activity confirmed through ligand-binding affinity to inhibitory peptides, representing alternatives for the key determinants of the CDK2 substrate recruitment site on the cyclin regulatory subunit. As a whole, obtaining the active cyclin A without the CDK partner (referred to as monomeric in this work) in a straightforward and facile manner will obviate protein--production issues with the CDK2/cyclin A complex and enable drug discovery efforts for non-ATP competitive CDK inhibition through the cyclin groove.

Iterative conversion of cyclin binding groove peptides into druglike CDK inhibitors with antitumor activity.

The cyclin groove is an important recognition site for substrates of the cell cycle cyclin dependent kinases and provides an opportunity for highly selective inhibition of kinase activity through a non-ATP competitive mechanism. The key peptide residues of the cyclin binding motif have been studied in order to precisely define the structure-activity relationship for CDK kinase inhibition. Through this information, new insights into the interactions of peptide CDK inhibitors with key subsites of the cyclin binding groove provide for the replacement of binding determinants with more druglike functionality through REPLACE, a strategy for the iterative conversion of peptidic blockers of protein-protein interactions into pharmaceutically relevant compounds. As a result, REPLACE is further exemplified in combining optimized peptidic sequences with effective N-terminal capping groups to generate more stable compounds possessing antitumor activity consistent with on-target inhibition of cell cycle CDKs. The compounds described here represent prototypes for a next generation of kinase therapeutics with high efficacy and kinome selectivity, thus avoiding problems observed with first generation CDK inhibitors.

Quantification of the effects of ionic strength, viscosity, and hydrophobicity on protein-ligand binding affinity.

In order to quantify the interactions between molecules of biological interest, the determination of the dissociation constant (K d) is essential. Estimation of the binding affinity in this way is routinely performed in "favorable" conditions for macromolecules. Crucial data for ligand-protein binding elucidation is mainly derived from techniques (e.g., macromolecular crystallography) that require the addition of high concentration of salts and/or other additives. In this study we have evaluated the effect of temperature, ionic strength, viscosity, and hydrophobicity on the K d of three previously characterized protein-ligand systems, based on variation in their binding sites, in order to provide insight into how these often overlooked unconventional circumstances impact binding affinity. Our conclusions are as follows: (1) increasing solvent viscosity in general is detrimental to ligand binding, (2) moderate increases in temperature have marginal effects on the dissociation constant, and (3) the degree of hydrophobicity of the ligand and the binding site determines the extent of the influence of cosolvents and salt concentration on ligand binding affinity.

Escherichia coli genome-wide promoter analysis: identification of additional AtoC binding target elements.

BACKGROUND: Studies on bacterial signal transduction systems have revealed complex networks of functional interactions, where the response regulators play a pivotal role. The AtoSC system of E. coli activates the expression of atoDAEB operon genes, and the subsequent catabolism of short-chain fatty acids, upon acetoacetate induction. Transcriptome and phenotypic analyses suggested that atoSC is also involved in several other cellular activities, although we have recently reported a palindromic repeat within the atoDAEB promoter as the single, cis-regulatory binding site of the AtoC response regulator. In this work, we used a computational approach to explore the presence of yet unidentified AtoC binding sites within other parts of the E. coli genome. RESULTS: Through the implementation of a computational de novo motif detection workflow, a set of candidate motifs was generated, representing putative AtoC binding targets within the E. coli genome. In order to assess the biological relevance of the motifs and to select for experimental validation of those sequences related robustly with distinct cellular functions, we implemented a novel approach that applies Gene Ontology Term Analysis to the motif hits and selected those that were qualified through this procedure. The computational results were validated using Chromatin Immunoprecipitation assays to assess the in vivo binding of AtoC to the predicted sites. This process verified twenty-two additional AtoC binding sites, located not only within intergenic regions, but also within gene-encoding sequences. CONCLUSIONS: This study, by tracing a number of putative AtoC binding sites, has indicated an AtoC-related cross-regulatory function. This highlights the significance of computational genome-wide approaches in elucidating complex patterns of bacterial cell regulation.

Phosphorylation activity of the response regulator of the two-component signal transduction system AtoS-AtoC in E. coli.

Antizyme, long known to be a non-competitive inhibitor of ornithine decarboxylase, is encoded by the atoC gene in Escherichia coli. The present study reveals another role for AtoC, that of a response regulator of the AtoS-AtoC two component system regulating the expression of the atoDAEB operon upon acetoacetate induction. This operon encodes enzymes involved in short-chain fatty acid catabolism in E. coli. Evidence is presented to show that AtoS is a sensor kinase that together with AtoC constitutes a two-component signal transduction system. AtoS is a membrane protein which can autophosphorylate and then transfer that phosphoryl group to AtoC. This process can also be reproduced in vitro. AtoC contains in its amino acid sequence a conserved aspartic acid (D55), which is the putative phosphorylation site, as well as an unexpected "H box" consensus sequence (SHETRTPV), common to histidine kinases, with the histidine contained therein (H73) being a second potential target for phosphorylation. Substitution of either D55 or H73 in His10-AtoC diminished but did not abrogate AtoC phosphorylation suggesting that either both residues can be phosphorylated independently or that the phosphate group can be transferred between them. However, the D55 mutation in comparison to H73 had a more pronounced effect in vivo, on the activation of atoDAEB promoter after acetoacetate induction, although it was the presence of both mutations that rendered AtoC totally unresponsive to induction. These data provide evidence that the gene products of atoS and atoC constitute a two-component signal transduction system, with some unusual properties, involved in the regulation of the atoDAEB operon.