All change: protein conformation and the ubiquitination reaction cascade

The structures of enzymes that collectively modify proteins by covalent addition of ubiquitin-like protein moieties have provided significant insights into the regulatory pathways they compose and have highlighted the importance of protein flexibility for the mechanism and regulation of the ubiquitination reaction

Halogen Bonds Form the Basis for Selective P-TEFb Inhibition by DRB

SummaryCdk9, the kinase of the positive transcription elongation factor b, is required for processive transcription elongation by RNA polymerase II. Cdk9 inhibition contributes to the anticancer activity of many Cdk inhibitors under clinical investigation and hence there is interest in selective Cdk9 inhibitors. DRB (5,6-dichlorobenzimidazone-1-β-D-ribofuranoside) is a commonly used reagent for Cdk9 inhibition in cell biology studies. The crystal structures of Cdk9 and Cdk2 in complex with DRB reported here describe the molecular basis for the DRB selectivity toward Cdk9. The DRB chlorine atoms form halogen bonds that are specific for the Cdk9 kinase hinge region. Kinetic and thermodynamic experiments validate the structural findings and implicate the C-terminal residues of Cdk9 in contributing to the affinity for DRB. These results open the possibility to exploit halogen atoms in inhibitor design to specifically target Cdk9

Identification and characterization of an irreversible inhibitor of CDK2

Irreversible inhibitors that modify cysteine or lysine residues within a protein kinase ATP binding site offer, through their distinctive mode of action, an alternative to ATP-competitive agents. 4-((6-(Cyclohexylmethoxy)- 9H-purin-2-yl)amino)benzenesulfonamide (NU6102) is a potent and selective ATP-competitive inhibitor of CDK2 in which the sulfonamide moiety is positioned close to a pair of lysine residues. Guided by the CDK2/NU6102 structure, we designed 6-(cyclohexylmethoxy)-N-(4-(vinylsulfonyl)phenyl)-9H-purin-2-amine (NU6300), which binds covalently to CDK2 as shown by a co-complex crystal structure. Acute incubation with NU6300 produced a durable inhibition of Rb phosphorylation in SKUT-1B cells, consistent with it acting as an irreversible CDK2 inhibitor. NU6300 is the first covalent CDK2 inhibitor to be described, and illustrates the potential of vinyl sulfones for the design of more potent and selective compounds

Identification of a novel ligand for the ATAD2 bromodomain with selectivity over BRD4 through a fragment growing approach

Structure-guided expansion of a fragment hit for the ATAD2 bromodomain enabled improvement in ATAD2 inhibition and selectivity over BRD4.</p

Structural and functional characterization of Rpn12 identifies residues required for Rpn10 proteasome incorporation

The ubiquitin–proteasome system targets selected proteins for degradation by the 26S proteasome. Rpn12 is an essential component of the 19S regulatory particle and plays a role in recruiting the extrinsic ubiquitin receptor Rpn10. In the present paper we report the crystal structure of Rpn12, a proteasomal PCI-domain-containing protein. The structure helps to define a core structural motif for the PCI domain and identifies potential sites through which Rpn12 might form protein–protein interactions. We demonstrate that mutating residues at one of these sites impairs Rpn12 binding to Rpn10 in vitro and reduces Rpn10 incorporation into proteasomes in vivo

The CDK9 C-helix Exhibits Conformational Plasticity That May Explain the Selectivity of CAN508

Correct regulation of transcription is essential for maintaining a healthy cellular state. During transcription RNA polymerase II (Pol II) proceeds in a regulated manner through several transitions to ensure appropriate control of synthesis and enable correct processing of the pre-RNA. Shortly after initiation Pol II is caused to pause by the binding of factors, DSIF and NELF. To enable transition of Pol II into the elongation phase CDK9/cyclin T phosphorylates the C-terminal domain (CTD) of Pol II, DSIF and NELF. This phosphorylation releases the paused state and provides an alternative set of post-transcriptional modifications on the CTD to generate a binding platform for elongation, histone modifying and termination factors. CDK9/cyclin T is itself regulated within multicomponent complexes. A small activated complex, containing Brd4, recruits CDK9/cyclin T to active sites of transcription, thereby promoting the elongation of transcription. The role of CDK9/cyclin T in the regulation of transcription has resulted in its validation as a drug target against several disease states including cancer, HIV and cardiac hypertrophy.In this thesis, I present the crystallographic structures of a series of 2-amino-4-heteroaryl-pyrimidine compounds and the roscovitine derivative, (S)-CR8, bound to CDK9/cyclin T and CDK2/cyclin A. In combination with thermal denaturation data and kinetic analysis, these structures have suggested chemical modifications that might be made to increase the CDK9 specificity of these compounds. I have also validated the use of a mutated form of cyclin T for use in the development of CDK9/cyclin T inhibitors.In addition, I present both structural and kinetic analysis of the Brd4-CDK9/cyclin T interaction. I show that C-terminal fragments of Brd4 enhance the in vitro kinase activity of CDK9/cyclin T against the Pol II CTD. Furthermore, I demonstrate that this enhancement may be inhibited by Plk1-mediated phosphorylation of Brd4. Finally, I show that Brd4 binds to a site that spans CDK9 and cyclin T and I propose detailed molecular models of the Brd4-cyclin T interaction.This thesis is not currently available via ORA