Structural And Functional Research Of Bacterial Inner Membrane Protein Insertase YidC

Membrane proteins account for more than one-third of proteins in a cell,and they function as ATPase,ion channel,and permease,etc.Thus,insertion of membrane proteins into lipid bilayer is a crucial and challenged procedure.The YidC/Alb3/Oxa1 family members have been shown to promote the insertion of membrane proteins into the inner membrane in bacteria,chloroplasts,and mitochondria,respectively.Besides inserting certain proteins into lipid bilayer independently,YidC also facilitates the folding and assembly of membrane proteins cooperateing with SecYEG,which is the most important translocase used to export proteins across or insert proteins into the memebrane bilayer.The exact mechanism of YidC largely remains elusive so far,and many previous studies show that distribution and the number of charged residues in nascent cytoplasmic membrane protein play an important role on its translocating across the bilayer lipid via YidC.In this work,we determined the crystal structure of YidC from Thermotoga maritima(TmYidC),at 3.8 A resolution,which presents a hydrophilic groove formed by C-terminal five-transmembrane(TM)helices which tightly packed together in the periplasmic leaflet,and loosely packedin the cytoplasmic leaflet.The hydrophilic residues,includingGln322,Arg260,Ser371,Thr374 and Asn413,result in a hydrophilic environment in the groove.The P1 domain of TmYidC(TmYidCP1),which protrudes from the core five-TM helices,was solved at a higher resolution at 2.5 A independently.Our functional analysis using in vivo photo-crosslinking assay demonstrates that coat protein Pf3,a SecYEG-independent YidC substrate,exits into the lipid bilayler laterally via the TM3-TM5 interface,which is formed by the two a helical bundles.By engineering the disulfide bonds into intra molecular of YidC,we found that significant rearrangement of the two a-helical bundles in the periplasmic leaflet of the hydrophilic groove appears to be essentialfor YidC function.P2 loop connecting the TM3 and TM4,which covers the periplasmic side of the core hydrophilic groove,contains strictly conserved negative charged residues,resulting a negatively potential environment in the bottom of the hydrophilic groove.We identified that these conserved negatively-charged residues are criticalfor YidC function,supporting an idea that these negative charged residues might play certain roles during substrate integration into the membrane.Above all,it clearly suggests that the periplasmic leaflet of YidC transmembrane region,including the P2 loop,is a functionally active part in YidC,although it doesn't form the hydrophilic groove.