Structural And Functional Research For CENP-IN Recognition Of CENP-A Nucleosomes On Kinetochore And Structural Insights Into The Staphylococcus Aureus Phosphofructokinase

Accurate and equal segregation of sister chromatids during mitosis is essential for the maintenance of genomic stability and species continuity.The correct segregation of the chromosome depends on the kinetochore assembly in the centromere.Centromere is a region of chromatin,of which structure and function are both highly specialized.Histone H3 is replaced by the histone variant centromere protein A(CENP-A)in centromere.Kinetochore is a large protein complex assembled in centromere,which is responsible for the connection of the chromosome and the spindle microtubules.The kinetochore proteins are classified into two layers:inner CCAN protein network and outer KMN protein network.The CCAN network can be divided into five subcomples:CENP-C,CENP-LN,CENP-HIKM,CENP-TWSX and CENP-OPQUR.CENP-N can identify CENP-A nucleosomes specifically and recuit other proteins of kinetochore.In the first part,we determined the structure of CENP-LN/CENP-A-NCP complex by cryo-electron microscopy with an average resolution of 5.8 A.CENP-N interacts with CENP-L through its C-terminus(?100 a.a.)to form a CENP-LN heterodimer,which is consistent with thire oligomerization state in solution.The interface between CENP-A NCP and the N-terminal domain of CENP-N(CENP-NN,?200 a.a)covers both the histones and the adjacent nucleosomal DNA.The R80,G81 and D83 residues on L1 loop of CENP-A play an important role in the specific recognition of CENP-A NCP by CENP-N.CENP-N interacts with the phosphate backbones along two successive minor grooves(with one major groove in between).The interacting phosphate backbones on nucleosomal DNA can be divided into four groups,+37/+36 nt,-32/-31 nt,+26/+25 nt and-22/-21 nt.These four DNA sites interact with four distinct regions of CENP-N to form a large DNA-protein interface,indicating an important role of CENP-N-DNA interaction in the formation and stabilization of CENP-A NCP/CENP-LN complex.The key amino acid residues on CENP-N for DNA binding,including K10A,R11A,R44A and H77A were identified by in vitro assays.Mutations of these key residues significantly reduced kinetochore localization of CENP-N,CENP-A and CENP-L in vivo,demonstrating that CENP-N is essential for stable centromere localization of CENP-A and CENP-L.In all organisms,glycolysis is an essential reaction to produce ATP with glucose as a substrate.In this process,hexokinase,phosphofructokinase and pyruvate kinase are considered to be regulatory enzymes,and the reactions catalysed by them are irreversible.Among them,phosphofructokinase(Pfk)is the key regulatory enzyme due to its lowest catalytic efficiency.Bacterial Pfks are tetramers that catalyzes the key control step of glycolysis by converting D-fructose 6-phosphate(F6P)and ATP to fructose 1,6-bisphosphate and ADP.It is a classical allosteric enzyme that exhibit activity allosterically regulated via conformational changes between the R-and T-states.In the second part,we report that the Pfk from Staphylococcus aureus NCTC 8325(SaPfk)exists as both an active tetramer and inactive dimer in solution.Multiple effectors,including pH,ADP,ATP,and adenylyl-imidodiphosphate(AMP-PNP),cause equilibrium shifts from the tetramer to dimer,whereas the substrate F6P stabilizes SaPfk tetrameric assembly.Crystal structures of SaPfk in complex with different ligands and biochemical analysis reveal that the flexibility of the Gly150-Leu151 motif in helix ?7 plays a role in tetramer-dimer conversion.Thus,we propose a molecular mechanism for allosteric regulation of bacterial Pfk via conversion between tetramer and dimer in addition to the well-characterized R-/T-state mechanism.