A hydrophilic microenvironment in the substrate-translocating groove of the YidC membrane insertase is essential for enzyme function

Abstract

The YidC family of proteins are membrane insertases that catalyze the translocation of the periplasmic domain of membrane proteins via a hydrophilic groove located within the inner leaflet of the membrane. All homologs have a strictly conserved, positively charged residue in the center of this groove. In Bacillus subtilis, the positively charged residue has been proposed to be essential for interacting with negatively charged residues of the substrate, supporting a hypothesis that YidC catalyzes insertion via an early-step electrostatic attraction mechanism. Here, we provide data suggesting that the positively charged residue is important not for its charge but for increasing the hydrophilicity of the groove. We found that the positively charged residue is dispensable for Escherichia coli YidC function when an adjacent residue at position 517 was hydrophilic or aromatic, but was essential when the adjacent residue was apolar. Additionally, solvent accessibility studies support the idea that the conserved positively charged residue functions to keep the top and middle of the groove sufficiently hydrated. Moreover, we demonstrate that both the E. coli and Streptococcus mutans YidC homologs are functional when the strictly conserved arginine is replaced with a negatively charged residue, provided proper stabilization from neighboring residues. These combined results show that the positively charged residue functions to maintain a hydrophilic microenvironment in the groove necessary for the insertase activity, rather than to form electrostatic interactions with the substrates. The YidC family of proteins are membrane insertases that catalyze the translocation of the periplasmic domain of membrane proteins via a hydrophilic groove located within the inner leaflet of the membrane. All homologs have a strictly conserved, positively charged residue in the center of this groove. In Bacillus subtilis, the positively charged residue has been proposed to be essential for interacting with negatively charged residues of the substrate, supporting a hypothesis that YidC catalyzes insertion via an early-step electrostatic attraction mechanism. Here, we provide data suggesting that the positively charged residue is important not for its charge but for increasing the hydrophilicity of the groove. We found that the positively charged residue is dispensable for Escherichia coli YidC function when an adjacent residue at position 517 was hydrophilic or aromatic, but was essential when the adjacent residue was apolar. Additionally, solvent accessibility studies support the idea that the conserved positively charged residue functions to keep the top and middle of the groove sufficiently hydrated. Moreover, we demonstrate that both the E. coli and Streptococcus mutans YidC homologs are functional when the strictly conserved arginine is replaced with a negatively charged residue, provided proper stabilization from neighboring residues. These combined results show that the positively charged residue functions to maintain a hydrophilic microenvironment in the groove necessary for the insertase activity, rather than to form electrostatic interactions with the substrates. The YidC/Oxa1/Alb3 proteins are found in bacteria, mitochondria, and chloroplast where they play a pivotal role in membrane protein biogenesis (1Dalbey R.E. Kuhn A. How YidC inserts and folds proteins across a membrane.Nat. Struct. Mol. Biol. 2014; 21: 435-436Google Scholar, 2Hennon S.W. Soman R. Zhu L. Dalbey R.E. YidC/Alb3/Oxa1 family of insertases.J. Biol. Chem. 2015; 290: 14866-14874Google Scholar). More recently, homologs have also been found in the eukaryotic endoplasmic reticulum (ER) membrane (3Chen Y. Dalbey R.E. Oxa1 superfamily: New members found in the ER.Trends Biochem. Sci. 2018; 43: 151-153Google Scholar, 4Dalbey R.E. Kuhn A. Membrane insertases are present in all three domains of life.Structure. 2015; 23: 1559-1560Google Scholar, 5McDowell M.A. Heimes M. Sinning I. Structural and molecular mechanisms for membrane protein biogenesis by the Oxa1 superfamily.Nat. Struct. Mol. Biol. 2021; 28: 234-239Google Scholar). This includes the Get1, EMC3, and TMCO1 proteins that function within large membrane complexes that play a key role in ER membrane protein biogenesis (5McDowell M.A. Heimes M. Sinning I. Structural and molecular mechanisms for membrane protein biogenesis by the Oxa1 superfamily.Nat. Struct. Mol. Biol. 2021; 28: 234-239Google Scholar). In bacteria, YidC can function on its own (6Samuelson J.C. Chen M. Jiang F. Moller I. Wiedmann M. Kuhn A. Phillips G.J. Dalbey R.E. YidC mediates membrane protein insertion in bacteria.Nature. 2000; 406: 637-641Google Scholar, 7Chen M. Samuelson J.C. Jiang F. Muller M. Kuhn A. Dalbey R.E. Direct interaction of YidC with the Sec-independent Pf3 coat protein its membrane protein Biol. Chem. Scholar, M. is a of the YidC for membrane protein Biol. Scholar, J.C. Jiang F. L. Chen M. of YidC for the insertion of protein in E. of that show in membrane and Biol. Chem. Scholar, L. Escherichia coli YidC is a membrane insertase for Sec-independent 23: Scholar, E. is by the to YidC in the E. coli inner or in with the to proteins the membrane L. Jiang F. Chen M. A. Dalbey R.E. YidC is strictly for membrane insertion of a and of the and of the Scholar, L. Kuhn A. Dalbey R.E. Membrane biogenesis of of for insertion of and Mol. Biol. Scholar, L. Kuhn A. Dalbey R. YidC and are for translocation of the periplasmic and of a Mol. Biol. Scholar). YidC the of proteins from the I. L. M. A. R. YidC and form a protein translocation and in the of of membrane proteins L. Dalbey R.E. YidC a molecular for protein via the protein Biol. Chem. Scholar, G.J. L. of and the in Escherichia coli Biol. Chem. Scholar). In the of YidC from Bacillus M. A. Y. Y. Y. Y. F. of Sec-independent membrane protein insertion by 2014; and Escherichia coli A. Y. R. of Escherichia coli a membrane protein and 2014; at These that the YidC protein a domain in the of the E. coli with an hydrophilic located within the inner leaflet of the membrane that is from the and but not from the In YidC a conserved in the of and that is in of YidC Pf3 coat M. Kuhn A. Muller the and insertion of a protein by an 2021; of and the that to YidC A. L. I. F. The interaction of the YidC insertase with the and the 2018; Scholar). a with the of the domain of E. coli is by conserved in of the of E. coli and in of These with the in E. coli to the of E. coli function to the from the domain the hydrophilic groove. The E. coli YidC residues that have been the of insertion found to on the of and that a (1Dalbey R.E. Kuhn A. How YidC inserts and folds proteins across a membrane.Nat. Struct. Mol. Biol. 2014; 21: 435-436Google Scholar, Kuhn A. of the Pf3 coat protein YidC Biol. Chem. the hydrophilic of the was the hydrophilic groove to translocation across the membrane Kuhn A. Dalbey R.E. the of a protein in Mol. Biol. Scholar). The a strictly conserved positively charged residue that is essential in Bacillus M. A. Y. Y. Y. Y. F. of Sec-independent membrane protein insertion by 2014; Scholar). This residue has been proposed to in an electrostatic to the negatively charged of the protein across the membrane M. A. Y. Y. Y. Y. F. of Sec-independent membrane protein insertion by 2014; Scholar). the conserved charge is not essential for the insertase of the E. coli YidC or an Y. Soman R. Kuhn A. Dalbey R.E. The role of the strictly conserved positively charged residue the and chloroplast YidC Biol. Chem. 2014; Scholar). molecular of E. coli YidC in a the protein to be than the and in the of the protein Y. Zhu L. Dalbey R.E. YidC insertase of Escherichia accessibility and membrane Scholar). The of the membrane and the of the hydrophilic groove the for is a in of proteins a Biol. 2021; Scholar). The strictly conserved arginine in the hydrophilic groove of the E. coli YidC is in to and 517 at the top of the groove Y. Zhu L. Dalbey R.E. YidC insertase of Escherichia accessibility and membrane and is via These residues are of an at the the in the groove and the of YidC in the leaflet of the membrane. the role of in the YidC insertase activity, we the function of this conserved positively charged residue in the hydrophilic groove of the E. coli YidC and Streptococcus mutans We found that the charge is important for the E. coli YidC when a at position 517 is to an with an is not essential when the is to a hydrophilic or results also found with mutans accessibility show that the arginine is to keep the of the groove solvent when is replaced with an both E. coli YidC and mutans can function in membrane protein insertion when the arginine is replaced with a negatively charged residue, provided that a is also the groove results support the hypothesis that the positively charged residue is to keep the groove hydrophilic and rather than to the charge of the to be A. Y. R. of Escherichia coli a membrane protein and 2014; and studies Y. Zhu L. Dalbey R.E. YidC insertase of Escherichia accessibility and membrane that the residues at and 517 in the E. coli YidC are located arginine at the top of the hydrophilic groove the of residues. or both and to The of the YidC with the and a of the of the at the (6Samuelson J.C. Chen M. Jiang F. Moller I. Wiedmann M. Kuhn A. Phillips G.J. Dalbey R.E. YidC mediates membrane protein insertion in bacteria.Nature. 2000; 406: 637-641Google Scholar). The YidC or the YidC by on a or at We found that the of and or the YidC when was to or was important for the but not or of combined with not the YidC and the of and we the of the by the membrane insertion of we the protein to with its by of the periplasmic domain of J.C. Jiang F. L. Chen M. of YidC for the insertion of protein in E. of that show in membrane and Biol. Chem. Scholar). inserts across the is by and to The and YidC in the was results with the substrate, has the residues to the of Pf3 coat and an arginine the of to translocation of the domain Y. Soman R. Kuhn A. Dalbey R.E. The role of the strictly conserved positively charged residue the and chloroplast YidC Biol. Chem. 2014; inserts across the the of and to a that was with the or insertion was with the is that of insertion with the was This has also been Y. Soman R. Kuhn A. Dalbey R.E. The role of the strictly conserved positively charged residue the and chloroplast YidC Biol. Chem. 2014; to the insertion mechanisms by the substrates. all the the protein was by The of the charge was by with positively charged or negatively charged residues in the of in the is the YidC The combined results show that we to the arginine in E. coli YidC essential by the 517 of residue 517 is for YidC we 517 with an aromatic, or residue and YidC is functional when the charge is studies that the or not the YidC when combined with an for or was for the and and In when the residue at position 517 a hydrophilic or and we YidC and the residue, was was a or a hydrophilic residue not a of YidC activity, we membrane insertion and and the of protein by Membrane insertion of and was when the 517 was to an and arginine was to an or residue the results we found that the strictly conserved arginine in mutans to in E. is essential the protein was when the residue in the hydrophilic groove was with a or negatively charged residue Y. Soman R. Kuhn A. Dalbey R.E. The role of the strictly conserved positively charged residue the and chloroplast YidC Biol. Chem. 2014; Scholar). of residues to of the E. coli YidC to residues to of mutans the conserved arginine at position the at residue 517 can the is essential or not in the E. coli we on to of the residue to the residue in E. in the mutans the of to in E. when the residue at the top of the groove of was with with hydrophilic or the YidC the positively charged residue in the groove not with an at or the of the mutans and for the and was for the and results a that to both E. coli YidC and mutans the conserved positively charged residue is for when is an residue at position 517 in the E. coli YidC or at position in the mutans but not when is a hydrophilic residue at The results show a the of the conserved charge and the the top of the hydrophilic groove in both E. coli YidC and mutans We that the positively charged residue is to maintain a hydrophilic in the groove. this we the solvent accessibility of groove residues and in the at a Y. Zhu L. Dalbey R.E. YidC insertase of Escherichia accessibility and membrane Scholar). this of the groove residues to residues. the is in a be the is the is in a the not with the is the YidC is by we the in a with to a in the molecular of not with a in the position of YidC on the when is to the of to is with when is the is solvent The with the YidC and to the where and are when own solvent accessibility is the is to an residue and is with a that the solvent of is when the is to a is when is to the solvent accessibility is for when and are with an and on is in the groove. the and the solvent of and residues are but to a than the in the of the groove. was when the accessibility was the of the groove at and when the and of the results is in with the top the and the the residues that are and residues that are not solvent The top that the groove is a of and residues can be at the of the groove by the that the top of the groove is not in we also studies to solvent accessibility of a the hydrophilic groove. YidC in with and to at in The for in the of or are in from the and the in a a was at position and to be solvent by the was with a hydrophilic residue position the at was when the residue was at position 517 was also to the top of the hydrophilic groove and with at that in all position 517 was to a of the that when the charge was or 517 solvent than when was present that the charge at to the groove hydrated. Additionally, we a of of E. coli YidC and the at in a the of at within the the of within of of groove residues the was Y. Zhu L. Dalbey R.E. YidC insertase of Escherichia accessibility and membrane and to the of the residues of proteins Y. Zhu L. Dalbey R.E. YidC insertase of Escherichia accessibility and membrane Scholar). that the accessibility residues and in the middle and of the groove for the to the The combined results support hypothesis that the charge at in E. coli is to maintain a hydrophilic microenvironment in the groove when is with an We that to be solvent in studies to in to the or of the the arginine in the groove to the groove sufficiently a charge also function in this studies that the charge YidC Y. Soman R. Kuhn A. Dalbey R.E. The role of the strictly conserved positively charged residue the and chloroplast YidC Biol. Chem. 2014; Scholar). is to a functional E. coli YidC with a charged residue the arginine at we for of the we YidC in the This the YidC to the YidC The is located in the hydrophilic groove and with the 517 residue the YidC membrane insertion of and of and and in a in we the of and YidC to a Pf3 coat protein a was in the at position in the and YidC The and was to residue The YidC proteins with to the Pf3 coat protein was at residue with the in the the Pf3 coat protein was to the the YidC proteins in increasing The of Pf3 coat protein to the YidC was by with a of of for the and for at be for the the was in the of the Pf3 coat YidC and the of Escherichia coli YidC protein with a charge in its groove is of the Escherichia coli YidC in a and are not at in interaction the of and the residue at 517 is the This interaction groove. and of the electrostatic of E. coli YidC at with the protein and in and and in The to the electrostatic of a to the membrane the center of the protein are the of All or the are at the The electrostatic for the is the We to a that the of the mutans in to in E. coli Kuhn A. of the Pf3 coat protein YidC Biol. Chem. This mutans a of the residue to the E. coli YidC when we the in the mutans we found that this mutans residue with in E. coli was also functional at but at Moreover, a with a of the residue, was also found to be with at but functional at The of that the negatively charged residue in the groove the protein at and the membrane insertion results of and that is with the the insertion of with mutans was was with the this is to the insertion mechanisms by the substrates. the molecular that the of YidC we a at the to the 517 of the in the the groove The electrostatic from the that the of the groove of the is negatively charged for the YidC the groove is positively charged We from results that YidC is functional with a negatively charged residue in the groove when is a that the electrostatic attraction the arginine in the groove and the negatively charged residues in the is not for The hydrophilic groove of all YidC family members a strictly conserved positively charged residue that has been proposed to with the hydrophilic of the M. A. Y. Y. Y. Y. F. of Sec-independent membrane protein insertion by 2014; Scholar, A. Y. R. of Escherichia coli a membrane protein and 2014; Scholar). for this electrostatic attraction from the that both the positively charged residue in the and the residues in the of the are for insertion M. A. Y. Y. Y. Y. F. of Sec-independent membrane protein insertion by 2014; Scholar). In a positively charged residue in the groove is for the insertase of the mutans Y. Soman R. Kuhn A. Dalbey R.E. The role of the strictly conserved positively charged residue the and chloroplast YidC Biol. Chem. 2014; Scholar). the charge of this residue is not essential for the E. coli YidC or A. chloroplast Y. Soman R. Kuhn A. Dalbey R.E. The role of the strictly conserved positively charged residue the and chloroplast YidC Biol. Chem. 2014; Scholar). This that an electrostatic attraction to the conserved arginine residue is not a for insertion the groove to Here, we show that the of residue 517 a of E. coli YidC the strictly conserved arginine is essential or not an residue is present at position the arginine at is but when an or residue is present at the arginine is results for the mutans to E. coli with a hydrophilic or residue, we to the essential dispensable that this mutans has to of the E. coli YidC to to of mutans of E. coli YidC the and large periplasmic is necessary for membrane insertion and of the mutans in E. coli Y. Soman R. Kuhn A. Dalbey R.E. The role of the strictly conserved positively charged residue the and chloroplast YidC Biol. Chem. 2014; Scholar). at the position of in the of E. coli YidC that is located at the of the hydrophilic groove and is of an at the the in the groove and the leaflet of the accessibility in the top of the groove is when is with an residue and is when the charge at in the middle of the groove is This was by the accessibility in the groove by the of residues to and by studies with a in the groove and the residue is or solvent and a in the of the groove residues and was by the of in a YidC in a by We that the in are by in the groove to the of the the to the top of the groove of the with the at results that the of positively charged residue in the groove is to maintain the hydrophilic of the groove is for YidC function by for the of the of This is in with results that the hydrophilic of the Pf3 coat protein the hydrophilic groove and the of with the groove residues and Kuhn A. Dalbey R.E. the of a protein in Mol. Biol. Scholar). The of hydrophilic microenvironment in the groove was also by microenvironment for the insertase function of YidC Sci. A. 2015; by residues in with or both in a in insertase YidC is functional when the strictly conserved arginine is replaced with a negatively charged residue with proper We have found in E. coli YidC and in mutans that YidC to be functional with a negatively charged residue in the groove. In mutans with an for in with or are at at in all the are of the we the negatively charged residue in the YidC groove In the of the YidC show that a to 517 and The combined results show negatively charged residues can function in the suggesting that the electrostatic attraction is not for We from and studies that the hydrophilic of the membrane the residue are to the protein translocation mechanism. This the hydrophilic of the to the membrane at within a hydrophilic Y. Zhu L. Dalbey R.E. YidC insertase of Escherichia accessibility and membrane Scholar). In important on is that YidC the in its Y. Zhu L. Dalbey R.E. YidC insertase of Escherichia accessibility and membrane Scholar). this the of translocation the by a hydrophilic to is Membrane has been to in J.C. Structural the biogenesis of membrane Scholar, J.C. is for in Sci. A. 2018; F. A. Structural for the of the Sci. A. and the of proteins a Biol. 2021; We that both membrane and the of the hydrophilic membrane are in YidC family members to the membrane to and from was from was from was from was from and from a of and was from to was from own was from the YidC has its and has a of the at the the of the (6Samuelson J.C. Chen M. Jiang F. Moller I. Wiedmann M. Kuhn A. Phillips G.J. Dalbey R.E. YidC mediates membrane protein insertion in bacteria.Nature. 2000; 406: 637-641Google Scholar). The was to of the E. coli YidC and the mutans Y. A. Dalbey R.E. but of of Streptococcus mutans and Escherichia coli YidC Scholar, F. L. M. Chen M. YidC can the YidC and membrane insertion of both and chloroplast Biol. Chem. Scholar). of proteins is of the in to to of protein The protein is of residues to of the E. coli YidC to residues to of mutans Y. A. Dalbey R.E. but of of Streptococcus mutans and Escherichia coli YidC Scholar). The was to L. A. Dalbey R.E. of membrane proteins that of YidC and in Escherichia Biol. Chem. or R. Kuhn A. Dalbey R.E. and charge of the periplasmic the YidC and for the Biol. Chem. 2014; the of the and to YidC in the was from for the All of the of YidC homologs by All of the by E. coli was with or and with YidC or The in in the of and of the YidC was by in the of for at The with and in at for of membrane proteins was with for by with for the membrane insertion of a was at an of was to The was with and in The to with to membrane insertion by of to was to membrane insertion of the by at and in with and and the was on for was and on for by the of to the of was and the by and The translocation by of the at The of translocation across the membrane was Y. Soman R. Kuhn A. Dalbey R.E. The role of the strictly conserved positively charged residue the and chloroplast YidC Biol. Chem. 2014; Scholar). The was in the in E. coli the at in with and In the the and The for at and and of on and or The at The of YidC or was with and in and of at of with of on for with and in The by with of E. coli YidC or mutans YidC was from the mutans was a from of Y. Zhu L. Dalbey R.E. YidC insertase of Escherichia accessibility and membrane and in of was and to was to YidC for The with and the was to and the was with the and These at for and with and three with YidC the with and by at for at and with for on The with and with The and with for at was with The protein by E. coli YidC or mutans was and on with at The and The of was and for on and The at for was from and by The and of YidC was in E. coli and in for in was to and was at at protein was by for and at on and in The on for at an of to the The at for at to and the membrane the was to at for at Membrane in by with The was for to all and the was with the for at with and of the was to the YidC on the and for at YidC was from the with and in The by and the YidC and with to and The YidC protein was by a was from and The was in and at for The was at a to form by the of the was to a membrane with a of a is a YidC a of was to the and The was to to the and to the to in I. A. and functional of the protein translocation the Mol. Biol. The of in the with the in and is by of the at The are by at for The are by at to form a and in of for with an of the YidC protein in a and Y. Zhu L. Dalbey R.E. YidC insertase of Escherichia accessibility and membrane was a for the The was with the A. molecular Mol. to three of the YidC and to the J.C. R. E. E. L. molecular with Chem. the for proteins and with the and the for M. of the to the protein of Scholar, I. of the for on Chem. Scholar, protein on to Chem. Scholar). of with a function at was for was with The was to electrostatic and with a of was to with a of at the with a and a was and in the to when of all in the of the electrostatic was the with an of for in was with the molecular A. molecular Mol. of that of of and of with the protein in the that of of and of with the protein in the found YidC was with a that of of and of with the protein in the that of of and of with the protein in the that of of and of with the protein in the that of of and of with the protein in the found YidC was with a in a on a of an ER with an ER of the and in and Scholar). The with a of to at a of to data was a of and a of to The of all was and the by for all the YidC three with a with a and with in the of a of the and in and Scholar). The with for at at a of and The to the all and the was at The of the was and the of the The data a is a is the where the is to of its and is a of the of of the In the and are and a The the is the in the for and and is the in the for and of the and in and Scholar). was at position of a YidC at the of the membrane the and the and The residues of the YidC to a with the of the Pf3 was with the of the was to the protein and for at The was from the protein by with a and the YidC proteins in The to on a The was at to the and the was from to The of the was at The coat protein was in to the The in of the and a of The an of the of the Pf3 coat of the of Pf3 coat to we the is the at of YidC and is the of the at the of The by a and Pf3 The of the by a of the the of the the was the is the at All data for this are within this This supporting The that they have of with the of this Y. and R. E. Y. M. I. M. A. A. and Y. A. and R. E. M. A. A. and R. E. M. A. A. and R. E. This was by from to R. E. and from the to A. at the to M. A. L. to the of and A. L. also to support from the I. also to support from the is to the support of and The is the of the and not the of the of

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