Disrupting the Constitutive, Homodimeric Protein-Protein Interface in CK2β Using a Biophysical Fragment-Based Approach.

Identifying small molecules that induce the disruption of constitutive protein-protein interfaces is a challenging objective. Here, a targeted biophysical screening cascade was employed to specifically identify small molecules that could disrupt the constitutive, homodimeric protein-protein interface within CK2β. This approach could potentially be applied to achieve subunit disassembly of other homo-oligomeric proteins as a means of modulating protein function.This research was supported by the Agency for Science, Technology and Research (A*STAR) Singapore (Ph.D. sponsorship, W.G.S.) and the Wellcome Trust Strategic Award (090340/Z/09/Z)

Mass Spectrometry Reveals Protein Kinase CK2 High-Order Oligomerization via the Circular and Linear Assembly.

CK2 is an intrinsically active protein kinase that is crucial for cellular viability. However, conventional kinase regulatory mechanisms do not apply to CK2, and its mode of regulation remains elusive. Interestingly, CK2 is known to undergo reversible ionic-strength-dependent oligomerization. Furthermore, a regulatory mechanism based on autoinhibitory oligomerization has been postulated on the basis of the observation of circular trimeric oligomers and linear CK2 assemblies in various crystal structures. Here, we employ native mass spectrometry to monitor the assembly of oligomeric CK2 species in an ionic-strength-dependent manner. A subsequent combination of ion mobility spectrometry-mass spectrometry and hydrogen-deuterium exchange mass spectrometry techniques was used to analyze the conformation of CK2 oligomers. Our findings support ionic-strength-dependent CK2 oligomerization, demonstrate the transient nature of the α/β interaction, and show that CK2 oligomerization proceeds via both the circular and linear assembly.This research was supported by the Wellcome Trust Strategic Award (090340/Z/09/Z), the Agency for Science Technology and Research (A*STAR) Singapore (Ph.D. sponsorship, W.G.S.), and the Croucher Foundation and the Cambridge Overseas Trust (Croucher Cambridge International Scholarship, D.S.-H.C).This is the author accepted manuscript. The final version is available from the American Chemical Society via http://dx.doi.org/10.1021/acschembio.6b0006

Characterization of the histone methyltransferase PRDM9 using biochemical, biophysical and chemical biology techniques

PRDM proteins have emerged as important regulators of disease and developmental processes. To gain insight into the mechanistic actions of the PRDM family, we have performed comprehensive characterization of a prototype member protein, the histone methyltransferase PRDM9, using biochemical, biophysical and chemical biology techniques. In the present paper we report the first known molecular characterization of a PRDM9-methylated recombinant histone octamer and the identification of new histone substrates for the enzyme. A single C321P mutant of the PR/SET domain was demonstrated to significantly weaken PRDM9 activity. Additionally, we have optimized a robust biochemical assay amenable to high-throughput screening to facilitate the generation of small-molecule chemical probes for this protein family. The present study has provided valuable insight into the enzymology of an intrinsically active PRDM protein

Disrupting the Constitutive, Homodimeric Protein-Protein Interface in CK2β Using a Biophysical Fragment-Based Approach.

Identifying small molecules that induce the disruption of constitutive protein-protein interfaces is a challenging objective. Here, a targeted biophysical screening cascade was employed to specifically identify small molecules that could disrupt the constitutive, homodimeric protein-protein interface within CK2β. This approach could potentially be applied to achieve subunit disassembly of other homo-oligomeric proteins as a means of modulating protein function.This research was supported by the Agency for Science, Technology and Research (A*STAR) Singapore (Ph.D. sponsorship, W.G.S.) and the Wellcome Trust Strategic Award (090340/Z/09/Z)

Mass Spectrometry Reveals Protein Kinase CK2 High-Order Oligomerization <i>via</i> the Circular and Linear Assembly

CK2 is an intrinsically active protein kinase that is crucial for cellular viability. However, conventional kinase regulatory mechanisms do not apply to CK2, and its mode of regulation remains elusive. Interestingly, CK2 is known to undergo reversible ionic-strength-dependent oligomerization. Furthermore, a regulatory mechanism based on autoinhibitory oligomerization has been postulated on the basis of the observation of circular trimeric oligomers and linear CK2 assemblies in various crystal structures. Here, we employ native mass spectrometry to monitor the assembly of oligomeric CK2 species in an ionic-strength-dependent manner. A subsequent combination of ion mobility spectrometry–mass spectrometry and hydrogen–deuterium exchange mass spectrometry techniques was used to analyze the conformation of CK2 oligomers. Our findings support ionic-strength-dependent CK2 oligomerization, demonstrate the transient nature of the α/β interaction, and show that CK2 oligomerization proceeds <i>via</i> both the circular and linear assembly

2-Aminothiazole derivatives as selective allosteric modulators of the protein kinase CK2. Part 1: Identification of an allosteric binding site

International audienc