Structural Insights Into Specific Recognition Of Methylated H3K4 By Tandem Tudor Domains Of PHIP

Background:PHIP（pleckstrin homology domain-interacting protein）is a multi-domain protein,containing eight WD40 repeats（1-8 WD40）,a CryptoTudor domain（CTD）and two bromodomains（BRD1-BRD2）,and plays vital roles in insulin signal pathway.PHIP is overexpressed in many cancers,such as triple-negative breast,melanoma,and lung cancer and takes part in the proliferation,invasion and metastasis of these cancer cells,which makes it become an attractive therapeutic target for these cancer types.Recently,it is reported that PHIP interacts with methylated H3K4 sites and then plays a role in epigenetic regulation and cell cycle progression,however,the molecular mechanism of these interactions is still unknown.Aim:To elucidate the molecular mechanism of PHIP specific recognition of methylated histone H3K4,and to provide a structural basis for subsequent studies on PHIP inhibitors screening.Methods:1.The expression of protein was induced with 0.25 mM IPTG for 24 h at 15℃.The proteins were purified by nickel affinity chromatography,and then purified by ion exchange chromatography and gel filtration chromatography to obtain the protein with a purity above 90%.2.ITC experiment was used to analyze the binding affinity between different truncated forms of PHIP and different methylated histone H3K4 in vitro.At the same time,we analyzed the influence of other histone modifications around the H3K4 site on the binding ability of PHIP and methylated H3K4（H3K4me）.3.The buffer for FP experiments was optimized in terms of additive,buffer solution,pH value,salt concentration to obtain stable and reliable FP buffer conditions,and then we detected the binding ability of PHIP with different peptides with fluorescein-labeled at C termini.4.The puritied protein was mixed with different lengths and different methylated levels of H3K4 peptide at different molar ratios to crystallize using the sitting drop vapor diffusion method at 18℃.Diffraction data were collected at SSRF beamline BL17U1 and structure was solved.5.The binding sites of PHIP and H3K4me were predicted by comparing the PHIP apo structure with the published complex structure of the tandem Tudor domains and methylated histone,and the predictied binding sites were verified by point mutations.Results:1.26 of the 46 recombinant plasmids could be expressed and purified to obtain more than 90%purity of protein.2.ITC experiments showed that the binding affinity between PHIP CryptoTudor and methylated H3K4 peptides is in a micro molar range,while CryptoTudor+BRD significantly enhances the binding affinity compared with CryptoTudor.PHIP preferentially binds to the lower methylated H3K4.Further analysis of the effect of other histone post-translational modifications around the H3K4 site on the binding ability of PHIP-H3K4me revealed that the combination of H3K4 methylation and H3K9 or H3K14 acetylation weakens the binding affinity.Nevertheless,the interaction is completely destroyed by dual modifications of methylated H3K4 and methylated H3R2 or phosphorylated H3T3.3.Our FP experiment indicated that the applicable FP assay buffer for PHIP contains 20 mM Bis-tris pH 6.0,150 mM NaCl,1 mM DTT and 0.01%Triton X-100.FP results suggested that PHIP specifically binds to methylated histone H3K4 not to other methylated histone peptides.4.The PHIP₅ apo crystal was obtained in a buffer containing 1.8 M triammonium citrate,pH 7.0,with crystal resolution at 2.65 A.Structural analysis revealed that the CryptoTudor of PHIP is a new form of tandem Tudor domains,namely TTDs.5.Based on the comparison of the PHIP TTDs apo structure with the published complex structure of other TTDs and methylated histone,it was predicted that PHIP binds to H3K4me through its TD2.The predicted residue sites were mutated to alanine and ITC analysis verified our prediction,revealing that PHIP forms hydrophobic pocket to bind to methylated K4 by W1107,W1081 and 11076,while D1111 may further stabilize the interaction through hydrogen bonds with K4me.Conclusion:PHIP tandem Tudor domains specifically recognize methylated histone H3K4 and prefer binding to lower methylated H3K4.BRD enhances the binding affinity between PHIP and H3K4me,while the dual modifications near H3K4 site weaken or destroy the interaction.Based on homologous structure comparison,it is predicted that PHIP binds to H3K4me through TD2,and W1107 is the key residue in the interaction between PHIP and H3K4me,which is confirmed by point mutation.