Exploration On Structure And Assembly Of Septum Forming Protein (SepF) And Its Interaction With FtsZ

In Gram-positive bacteria,Septum Forming protein(SepF)forms assemblies participating in membrane tethering of FtsZ ring(Z ring).Meanwhile,the curvature of SepF assembly is considered to regulate the thickness of the bacterial nascent septa.Since many researches have suggested the necessity of the assembling of SepF for biological functions,several issues needed to be explored.First of all,all crystal structures of SepF in the database are dimeric,however,the structure as well as the contact interfaces in an assembly state is lacking.Secondly,it is unknown how the curvature changes of SepF assembly affect its function.Thirdly,the binding sites of assembled SepF to FtsZ need further investigation.Herein,by combining solid-state NMR(ss NMR)with other techniques(biochemical experiments,cryo-electron microscopy,solution-state NMR,molecular simulation,etc),the assembly structure,curvature change,and interaction with FtsZ of SepF in an assembled state were studied.The specific results are as follows:1)Appropriate sample preparations of Bacillus subtilis SepF(Bs-SepF)for ss NMR studies were obtained.Based on the chemical shift assignments of SepF in the assembly state,the structure models of monomeric and dimeric SepF were calculated.The results revealed that the angle of α-helix changed subtly during SepF assembly.In addition,we found an unexpected influence of the N-terminal first 12 amino acids on assembly of SepF mediated by the salt of Na Cl.2)Mutations in the conserved amino acid of G137,which is located in β-sheet 5 of SepF,affect the assembly of SepF,thus resulting in diverse morphologies of SepF including ring,fiber and oligomers.By performing ss NMR experiments,the chemical shift perturbations of SepF from a ring structure to the linear fiber were characterized,and the main changes were located at α-helix 1,α-helix 2,β-sheet 1,β-sheet 4 and β-sheet 5 of SepF.Subsequently,the structure of SepF fibril was obtained by combining ss NMR with CryoEM,confirming that the α-α interface was decisive in the morphology transformation.On the basis of these data,we propose the following mechanism for the curvature change of the SepF assembly.The angle of α-helix interface was changed via conformational changes caused by the mutation of G137 in β-sheet interface.Furthermore,this effect was amplified with the assembly of SepF,thus resulting in the formation of fibrils.3)Interaction of SepF assembly with the C-terminal domain of FtsZ(FtsZ-CTD)leads to further aggregation of the ring.ss NMR experiments revealed that the changes were mainly observed in α-helix 1,α-helix 2,β-sheet 3 and β-sheet 4 of SepF.Solution-state NMR experiments on dimeric SepF proved a prerequisite role of α-helix(α-α)interface formation in SepF for FtsZ binding.Finally,a new interaction mechanism between FtsZ and SepF assembly was proposed.FtsZ binds to a pocket at the α-helix interfaces of SepF assembly.The pocket is formed by α-helix 2,β-sheet 3,and β-sheet 4 of one monomer andα-helix 1 of the neighboring one.Both sides of SepF can be contacted because of the symmetry in the polymer structure.In summary,the assembly structure,curvature change mechanism of SepF and interaction between SepF and FtsZ in assembled state were investigated by combining multiple techniques.These results revealed that the monomer conformation of SepF did not change much during the SepF self-assembly.The mechanism for curvature changes of SepF assembly and its interaction with FtsZ were proposed.All of the above results provide a deeper understanding of SepF anchoring FtsZ to the membrane in its assembled state and the regulation of septa thickness by the curvature formation.