Recognition Of Kinesin KIF21A By KANK1/2 Protein And The Study Of SETD3-mediated Histidine Methylation Of ?-actin

In eukaryotic cells,cytoskeleton plays important roles in the maintenance of basic cell morphology,cell movement and differentiation,material transport,information transmission and other important life activities,and its concept is constantly developing.The major components of cytoskeleton include microtubules,microfilaments and intermediate fibers,among which microtubules and microfilaments are the most dynamic,and their polymerization and depolymerization processes are precisely regulated.Various types of post-translational modifications(PTMs)have been found to occur on the proteins that constitute microtubules and microfilaments.The PTMs might mediate the functions of microtubules and microfilaments in different ways,such as changing their internal characteristics or recruiting new effector proteins to regulate downstream pathways.Therefore,the molecular basis for the installment and removal of the specific PTM on a critical residue,can not only provide insight into the significance of PTM-mediated biological functions for cytoskeleton proteins,but also facilitate our understanding of the complexity of proteomics in cytoplasm.Our research in this paper focuses on the molecular mechanism of the role of human KANK1/2 in the regulation of microtubule growth in peri cellular matrix,and the molecular basis for the actin His73 N3-methylation by human SETD3.In the first part of this paper,we introduce the Kank1/2 family protein which can prevent microtubule catastrophe by recognizing the kinesin KIF21A.We examined the in vitro binding between KIF21A and KANK1/2 by GST pull down and isothermal calorimetric titration(ITC).By analyzing the crystal structure,we found that upon binding to KANK1/2,KIF21A adopts a helix-linker-helix conformation with the two helix formed at both ends.In the complex structures,KANK1/2 bind to the KIF21A peptide via an acidic patch and a hydrophobic pocket,and the extensive interactions between KANK1/2 and KIF21A demonstrate that KANK1/2 recognize the KIF21A fragment in a sequence-dependent manner.Mutagenesis and ITC binding assay further validated the binding interface between KANK1/2 and the KIF21A peptide.The key residues of KANK1/2 involved in KIF21A were identified,which are conserved among different species.Furthermore,by sequence alignment and detailed structure analysis,we found that despite the high sequence identity between the ankyrin domains of KANK proteins.key KIF21A binding residues of KANK1/2 are not conserved in the other two KANK members,KANK3 and KANK4,suggesting that KANK family members might evolve distinct cellular functions during the evolution.Further work is required to reveal the biological functions of KANK3/4.In the second part of this paper,we studied the molecular basis of actin His73 N3-methylation mediated by SETD3.Biochemical evidence indicates that the N3 methylation of his73 maintains the poly state of F-actin by regulating the release process of ?-phosphate group after ATP hydrolysis.It has been more than 50 years since the discovery of the His73 N3-methylation,and until recently,SETD3 was found to serve as the actin histidine methyltransferase.Control experiments also demonstrate that SETD3 is not a methyltransferase for histone H3K4 or H3K36.Therefore,SETD3 is the first non-lysine methyltransferase in SET domain family.By ITC binding assay,we found that a fragment of actin containing His73 binds to SETD3 with high affinity.In contrast,neither H3K4 nor H3K36 peptide display SETD3 binding affinity.In addition,the mass spectrometry data also show that SETD3 efficiently catalyzes the methylation of the His73-containg actin peptide.To unravel the substrate recognition and catalysis mechanism by SETD3,we solved two SETD3 structures in the presence of S-adenosylhomocysteine(SAH)by X-ray crystallography,with one bound to an unmodified actin fragment and the other bound to its methylated form.The two structures represent the snapshots of the pre-and the post-catalysis,respectively.In the pre-catalysis complex,we found that His73 is positioned into a specific pocket and its orientation is fixed by the hydrogen bonding interactions with pocket residues.After the catalysis,the imidazole ring of His73 rotated by?90 degree to avoid the potential steric clash and for the subsequent product release.SETD3 recognizes actin in a sequence-dependent manner.The kinetic parameters of SETD3 and its variants were determined towards SAM and the full-length actin,indicating that either SAM-binding mutants or actin binding mutants remarkably impaired the activity of SETD3 towards full-length protein.Overall our structure not only uncovers the recognition and catalytic mechanism of actin by SETD3,but also sheds light on the future design of potent SETD3 inhibitors for the treatment of associated human diseases in near future.