Price to be paid for two-metal catalysis: magnesium ions that accelerate chemistry unavoidably limit product release from a protein kinase.

Incorporation of divalent metal ions into an active site is a fundamental catalytic tool used by diverse enzymes. Divalent cations are used by protein kinases to both stabilize ATP binding and accelerate chemistry. Kinetic analysis establishes that Cyclin-dependent kinase 2 (CDK2) requires simultaneous binding of two Mg(2+) ions for catalysis of phosphoryl transfer. This tool, however, comes with a price: the rate-acceleration effects are opposed by an unavoidable rate-limiting consequence of the use of two Mg(2+) ions by CDK2. The essential metal ions stabilize ADP product binding and limit the overall rate of the reaction. We demonstrate that product release is rate limiting for activated CDK2 and evaluate the effects of the two catalytically essential Mg(2+) ions on the stability of the ADP product within the active site. We present two new crystal structures of CDK2 bound to ADP showing how the phosphate groups can be coordinated by either one or two Mg(2+) ions, with the occupancy of one site in a weaker equilibrium. Molecular dynamics simulations indicate that ADP phosphate mobility is more restricted when ADP is coordinated by two Mg(2+) ions compared to one. The structural similarity between the rigid ADP·2Mg product and the cooperatively assembled transition state provides a mechanistic rational for the rate-limiting ADP release that is observed. We demonstrate that although the simultaneous binding of two Mg(2+) ions is essential for efficient phosphoryl transfer, the presence of both Mg(2+) ions in the active site also cooperatively increases ADP affinity and opposes its release. Evolution of protein kinases must have involved careful tuning of the affinity for the second Mg(2+) ion in order to balance the needs to stabilize the chemical transition state and allow timely product release. The link between Mg(2+) site affinity and activity presents a chemical handle that may be used by regulatory factors as well as explain some mutational effects.

Briefly bound to activate: transient binding of a second catalytic magnesium activates the structure and dynamics of CDK2 kinase for catalysis.

We have determined high-resolution crystal structures of a CDK2/Cyclin A transition state complex bound to ADP, substrate peptide, and MgF(3)(-). Compared to previous structures of active CDK2, the catalytic subunit of the kinase adopts a more closed conformation around the active site and now allows observation of a second Mg(2+) ion in the active site. Coupled with a strong [Mg(2+)] effect on in vitro kinase activity, the structures suggest that the transient binding of the second Mg(2+) ion is necessary to achieve maximum rate enhancement of the chemical reaction, and Mg(2+) concentration could represent an important regulator of CDK2 activity in vivo. Molecular dynamics simulations illustrate how the simultaneous binding of substrate peptide, ATP, and two Mg(2+) ions is able to induce a more rigid and closed organization of the active site that functions to orient the phosphates, stabilize the buildup of negative charge, and shield the subsequently activated γ-phosphate from solvent.

A Fast Implementation of a Scan Statistic for Identifying Chromosomal Patterns of Genome Wide Association Studies.

In order to take into account the complex genomic distribution of SNP variations when identifying chromosomal regions with significant SNP effects, a single nucleotide polymorphism (SNP) association scan statistic was developed. To address the computational needs of genome wide association (GWA) studies, a fast Java application, which combines single-locus SNP tests and a scan statistic for identifying chromosomal regions with significant clusters of significant SNP effects, was developed and implemented. To illustrate this application, SNP associations were analyzed in a pharmacogenomic study of the blood pressure lowering effect of thiazide-diuretics (N=195) using the Affymetrix Human Mapping 100K Set. 55,335 tagSNPs (pair-wise linkage disequilibrium R(2)<0.5) were selected to reduce the frequency correlation between SNPs. A typical workstation can complete the whole genome scan including 10,000 permutation tests within 3 hours. The most significant regions locate on chromosome 3, 6, 13 and 16, two of which contain candidate genes that may be involved in the underlying drug response mechanism. The computational performance of ChromoScan-GWA and its scalability were tested with up to 1,000,000 SNPs and up to 4,000 subjects. Using 10,000 permutations, the computation time grew linearly in these datasets. This scan statistic application provides a robust statistical and computational foundation for identifying genomic regions associated with disease and provides a method to compare GWA results even across different platforms.

KGraph: a system for visualizing and evaluating complex genetic associations.

UNLABELLED: The KGraph is a data visualization system that has been developed to display the complex relationships between the univariate and bivariate associations among an outcome of interest, a set of covariates, and a set of genetic factors, such as single nucleotide polymorphisms (SNPs). It allows for easy viewing and interpretation of genetic associations, correlations among covariates and SNPs, and information about the replication and cross-validation of the associations. The KGraph allows the user to more easily investigate multicollinearity and confounding through visualization of the multidimensional correlation structure underlying genetic associations. It emphasizes gene-environment and gene-gene interaction, both important components of any genetic system that are often overlooked in association frameworks. AVAILABILITY: http://www.epidkardia.sph.umich.edu/software/kgrapher

ChromoScan: a scan statistic application for identifying chromosomal regions in genomic studies.

UNLABELLED: ChromoScan is an implementation of a genome-based scan statistic that detects genomic regions, which are statistically significant for targeted measurements, such as genetic associations with disease, gene expression profiles, DNA copy number variations, as well as other genome-based measurements. A Java graphic user interface (GUI) is provided to allow users to select appropriate data transformations and thresholds for defining the significant events. AVAILABILITY: ChromoScan is freely available from http://www.epidkardia.sph.umich.edu/software/chromoscan/