Studies On The Self-assembly Mechanisms Of Kit Ligand And Neuraminidase Transmembrane Domains

Kit ligand(KITL)and neuraminidase(NA)are both single transmembrane(TM)proteins.The former mediates many life activities such as cell proliferation,survival and differentiation through the Kit/KITL pair;the latter plays a variety of roles in the different stages of influenza virus infection.The prerequisite for the realization of these functions is to form the correct self-assemblies.Previous studies have shown that KITL forms a non-covalent dimer via its TM helix,and NA uses the tetramer conformation of its TM domain to match the oligomeric state of the distal head and further promotes the maturation of the entire NA molecule.However,the potential self-assembly mechanisms of KITL and NA TM domain are not clear.In this paper,the self-assembly mechanisms of KITL and NA TM domains were studied by using both coarse-grained(CG)and all-atom(AA)molecular dynamics(MD)simulation methods.For KITL,the wild-type KITL TM helices formed a stable dimer,which was mediated by a conserved SxxxGxxxG motif;the site-directed mutations of the interface residues showed that the dimer capacities of S11I,G15I and G191 were significantly decreased relative to the wild-type,which further validated the importance of the SxxxGxxxG motif for KITL TM dimer.Comparing the spatial probability density distributions of N-terminal and C-terminal between the wild-type and Y22I,we found that the C-terminal in the wild-type KITL had the smallest spatial degree of freedom;the radial distribution of phosphoric acid relative to the 22nd residue indicated that phosphoric acid appeared most frequently at 1 nm away from the tyrosine residue in the wild-type,whereas no phosphoric acid was accumulated near the 22nd residue in the mutant Y22I.So we concluded that Tyr22 promoted the formation of KITL TM dimer by anchoring phosphoric acid to it.Pro7 induced the decline of the dimer ability of the wild-type KITL TM helix through changing the local helix structure.For NA,NA TM helices begin with the formation of the dimer,which finally assemble into a homologous tetramer by the stepwise addition of a monomer;the two diagonal distances are equal with an average value of 12.7± 0.2 A after the tetramer forms,and the crossing angles for adjacent helices in aN5(avian neuraminidase subtype 5)TM helix bundle are calculated at approximately-30°,these two results indicate that NA TM tetramer is a symmetric right-handed conformation;the results of the mutants S14LN21LH25LS28L and H25Q reveal the dynamic factors of NA TM tetramer,i.e.the polar interfaces composed of Ser14,Asn21,His25 and Ser28 are essential for NA TM four-helix bundle;in addition,the side chains of the His25 residues form a confinement ring that towards the polar center so as to promote the stabilization of NA TM four-helix bundle.