The Core of the Tetrameric Mycobacterial Porin MspA Is an Extremely Stable β-Sheet Domain

Abstract

MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the cell wall and is the prototype of a new family of tetrameric porins with a single central pore of 10 nm in length. Infrared and circular dichroism spectroscopy revealed that MspA consists mainly of antiparallel β-strands organized in a coherent domain. Heating to 92 and 112 °C was required to dissociate the MspA tetramer and to unfold the β-sheet domain in the monomer, respectively. The stability of the MspA tetramer exceeded the remarkable stability of the porins of Gram-negative bacteria for every condition tested and was not reduced in the presence of 2% SDS and at any pH from 2 to 14. These results indicated that the interactions between the MspA subunits are different from those in the porins of Gram-negative bacteria and are discussed in the light of a channel-forming β-barrel as a core structure of MspA. Surprisingly, the channel activity of MspA in 2% SDS and 7.6m urea at 50 °C was reduced 13- and 30-fold, respectively, although the MspA tetramer and the β-sheet domain were stable under those conditions. Channel closure by conformational changes of extracellular loops under those conditions is discussed to explain these observations. This study presents the first experimental evidence that outer membrane proteins not only from Gram-negative bacteria but also from mycobacteria are β-sheet proteins and demonstrates that MspA constitutes the most stable transmembrane channel protein known so far. Thus, MspA may be of special interest for biotechnological applications. MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the cell wall and is the prototype of a new family of tetrameric porins with a single central pore of 10 nm in length. Infrared and circular dichroism spectroscopy revealed that MspA consists mainly of antiparallel β-strands organized in a coherent domain. Heating to 92 and 112 °C was required to dissociate the MspA tetramer and to unfold the β-sheet domain in the monomer, respectively. The stability of the MspA tetramer exceeded the remarkable stability of the porins of Gram-negative bacteria for every condition tested and was not reduced in the presence of 2% SDS and at any pH from 2 to 14. These results indicated that the interactions between the MspA subunits are different from those in the porins of Gram-negative bacteria and are discussed in the light of a channel-forming β-barrel as a core structure of MspA. Surprisingly, the channel activity of MspA in 2% SDS and 7.6m urea at 50 °C was reduced 13- and 30-fold, respectively, although the MspA tetramer and the β-sheet domain were stable under those conditions. Channel closure by conformational changes of extracellular loops under those conditions is discussed to explain these observations. This study presents the first experimental evidence that outer membrane proteins not only from Gram-negative bacteria but also from mycobacteria are β-sheet proteins and demonstrates that MspA constitutes the most stable transmembrane channel protein known so far. Thus, MspA may be of special interest for biotechnological applications. n-octylpolyoxyethylene recombinant MspA dissociation temperature 2-(N-morpholino)ethanesulfonic acid attenuated total reflection Fourier transform infrared The impermeability of their cell wall renders mycobacteria intrinsically resistant against many compounds that are toxic to other bacteria. This is of particular medical importance in the case ofMycobacterium tuberculosis, because it strongly limits the number of drugs available for treatment of tuberculosis (1Kremer L.S. Besra G.S. Exp. Opin. Investig. Drugs. 2002; 11: 1033-1049Google Scholar). The central part of the mycobacterial cell wall is an asymmetrical bilayer (2Nikaido H. Kim S.H. Rosenberg E.Y. Mol. Microbiol. 1993; 8: 1025-1030Google Scholar), consisting of an inner leaflet of very long-chain fatty acids, the mycolic acids, and of many other unusual lipids, which form the outer leaflet (3Barry III, C.E. Lee R.E. Mdluli K. Sampson A.E. Schroeder B.G. Slayden R.A. Yuan Y. Prog. Lipid Res. 1998; 37: 143-179Google Scholar). This outer membrane is of exceptionally low fluidity (4Liu J. Barry III, C.E. Besra G.S. Nikaido H. J. Biol. Chem. 1996; 271: 29545-29551Google Scholar) and is estimated to be about 10 nm thick (5Paul T.R. Beveridge T.J. Zentralbl. Bakteriol. 1993; 279: 450-457Google Scholar, 6Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Scopus (1563) Google Scholar). Pore-forming proteins mediate the uptake of hydrophilic compounds across this outer membrane. The existence of such porins in mycobacteria was discovered 10 years ago (7Trias J. Jarlier V. Benz R. Science. 1992; 258: 1479-1481Google Scholar), but only two mycobacterial porins were identified so far. OmpATb of M. tuberculosis has low channel activity in vitro (8Senaratne R.H. Mobasheri H. Papavinasasundaram K.G. Jenner P. Lea E.J. Draper P. J. Bacteriol. 1998; 180: 3541-3547Google Scholar) and is important for the adaptation of M. tuberculosis to low pH and for survival in macrophages and mice (9Raynaud C. Papavinasasundaram K.G. Speight R.A. Springer B. Sander P. Bottger E.C. Colston M.J. Draper P. Mol. Microbiol. 2002; 46: 191-201Google Scholar). MspA was identified as a channel-forming protein in chloroform/methanol extracts of Mycobacterium smegmatis(10Niederweis M. Ehrt S. Heinz C. Klöcker U. Karosi S. Swiderek K.M. Riley L.W. Benz R. Mol. Microbiol. 1999; 33: 933-945Google Scholar). Recently, it was shown that MspA is the major porin of M. smegmatis. The permeability of the cell wall of M. smegmatis for glucose is reduced 4-fold in anmspA deletion mutant (11Stahl C. Kubetzko S. Kaps I. Seeber S. Engelhardt H. Niederweis M. Mol. Microbiol. 2001; 40: 451-464Google Scholar). Electron microscopy and cross-linking experiments revealed that MspA is a tetrameric protein forming a central pore of 10 nm length (12Engelhardt H. Heinz C. Niederweis M. J. Biol. Chem. 2002; 277: 37567-37572Google Scholar). This is drastically different from the trimeric porins of Gram-negative bacteria, which have three pores per molecule and are 4 nm long (13Koebnik R. Locher K.P. van Gelder P. Mol. Microbiol. 2000; 37: 239-253Google Scholar). These properties classify MspA as the prototype of a new family of pore proteins, since three other porins MspB, MspC, and MspD were identified in M. smegmatis, which differ only by a few amino acids from MspA (11Stahl C. Kubetzko S. Kaps I. Seeber S. Engelhardt H. Niederweis M. Mol. Microbiol. 2001; 40: 451-464Google Scholar). Crystal structures of outer membrane proteins from Gram-negative bacteria revealed a β-barrel as a common architectural principle (13Koebnik R. Locher K.P. van Gelder P. Mol. Microbiol. 2000; 37: 239-253Google Scholar, 14Koronakis V. Sharff A. Koronakis E. Luisi B. Hughes C. Nature. 2000; 405: 914-919Google Scholar, 15Vandeputte-Rutten L. Kramer R.A. Kroon J. Dekker N. Egmond M.R. Gros P. EMBO J. 2001; 20: 5033-5039Google Scholar). However, nothing is known about the secondary structure of mycobacterial outer membrane proteins in general and of mycobacterial porins in particular. It was noted earlier that MspA, which was purified by preparative not dissociate in the presence of such as urea in M. Ehrt S. Heinz C. Klöcker U. Karosi S. Swiderek K.M. Riley L.W. Benz R. Mol. Microbiol. 1999; 33: 933-945Google Scholar). of MspA by of smegmatis in the presence of to °C indicated that MspA is a very stable channel-forming protein C. Niederweis M. Biochem. 2000; Scholar). a stable pore protein be of as a in H. Nature. 2001; Scholar) and as a for of and M. Heinz C. K. S.H. 2002; Scholar), a of purified MspA is to the stability with this the secondary structure of MspA by circular dichroism and infrared the drastically different with porins of Gram-negative bacteria, antiparallel were identified as the secondary structure of MspA to those of other that the β-sheet structure of purified MspA is stable against tetrameric MspA and very and conditions. This of a mycobacterial porin revealed properties of a new of channel-forming were from at the The SDS were from and respectively. was from was from U. S. MspA was from M. smegmatis a and purified as C. Niederweis M. Biochem. 2000; Scholar). MspA from the exchange at and was from porin in the MspC, which at C. E. Niederweis M. and Scholar). MspA was in were for by and for channel-forming activity by bilayer MspA was in from the and the form of was purified be were acid Biochem. Scholar) and as of MspA were at a protein of in a and a with an length of The was were under conditions and from the The as were the These were to by the the of the is the number of is the of the protein in and is the cell in available the at of MspA in was for 2 against to the The protein to the of (12Engelhardt H. Heinz C. Niederweis M. J. Biol. Chem. 2002; 277: 37567-37572Google Scholar). were a which was in a a were with a from to the exchange was by the with for The of was by every and by of the and were of at a of 2 MspA was in of at a of about The in a with and which the of from °C to °C of were every 10 °C in the between and The secondary structure was secondary and Fourier for H. Scholar), and were in the for and of of MspA in were for at from to by to 4 were in 10 °C a which a of The temperature was as °C by the and °C in and of of MspA in were to of 2% SDS urea and as was by against of for at temperature to in of MspA in urea the for the stability of MspA, that not to a of MspA. of MspA were and as The stability of purified MspA at different pH was of of MspA in which were in the to a of and for at The pH were as M. Scholar) by and to pH and to pH acid to pH and to pH and to pH and and to pH and respectively. The stability of MspA in extracts at different pH was of 2 of of M. in pH C. Niederweis M. Biochem. 2000; Scholar). The were with of the as for MspA and for at The proteins in these were by and the channel activity was by bilayer as stability of the MspA of of purified MspA were for at different which are indicated in of MspA of was a MspA as a The was and the It be noted that the MspA was with the were in which were with C. Niederweis M. Biochem. 2000; Scholar). were with pH and for 10 at temperature a H. Biochem. Scholar) not of the stability the were and protein were from the the of the the at The and the of the were to the of the of the were The of of MspA was as the temperature at which the of tetrameric MspA was reduced by as with that at The channel activity of was in bilayer experiments as C. Niederweis M. Biochem. 2000; Scholar). bilayer the bilayer was from and the and 10 The temperature was at °C a MspA extracts were at different pH and with the C. Niederweis M. Biochem. 2000; Scholar) for the pH stability The MspA in the was estimated to MspA pores in were in single channel the of urea the pore forming activity of MspA, of of MspA were at different for in and in the presence of 2% SDS and the were with to a of MspA. of MspA from was to the the The of MspA in the bilayer was were a an and of was to of the MspA 10 were in the presence of 7.6m urea for at in 10 °C from to The were to and this temperature was the of MspA was at and was from to The of of MspA in the presence of urea was as the temperature the of MspA was of that at MspA was with and in was with the in which not any The was and were at °C for a MspA was under the but the were were by to a of 2 which such as and K. were with and a The was a and MspA was the M. Ehrt S. Heinz C. Klöcker U. Karosi S. Swiderek K.M. Riley L.W. Benz R. Mol. Microbiol. 1999; 33: 933-945Google Scholar), a secondary and the The major porin of M. smegmatis, MspA, was by for in a with to lipids, and purified by exchange and to compounds and proteins as earlier C. Niederweis M. Biochem. 2000; Scholar). This is the only that MspA in the tetrameric MspA pore has a structure drastically different from that of other porins (12Engelhardt H. Heinz C. Niederweis M. J. Biol. Chem. 2002; 277: 37567-37572Google Scholar) and is the first outer membrane protein from mycobacteria that is to the secondary structure of purified MspA was dichroism a a at at which the and a single at nm These are of β-sheet structures N. 8: Scholar) and were to those for β-barrel proteins such as the porins of Gram-negative bacteria M. Scholar). spectroscopy is known to a of the β-sheet of proteins R. van M. Biochem. 1993; Scholar). of MspA were for the of the which is for the secondary structure of proteins H. Biochem. 258: Scholar), and with that of porin from The has a β-sheet of as from structure K. K. Engelhardt H. 2000; 8: Scholar). of a β-sheet of in with the of MspA a which is for proteins with Fourier for and of to MspA exchange by revealed a secondary structure of and structure of and The of in MspA with that of porin is with a at The at in the is of antiparallel H. Biochem. 258: Scholar) and very to of porins from Gram-negative bacteria. structure the MspA and different a about and in with the and spectroscopy indicated as the secondary structure of MspA. earlier experiments indicated a very stability of MspA M. Ehrt S. Heinz C. Klöcker U. Karosi S. Swiderek K.M. Riley L.W. Benz R. Mol. Microbiol. 1999; 33: 933-945Google Scholar, C. Niederweis M. Biochem. 2000; Scholar), to the of the MspA tetramer against dissociation with the stability of secondary structure and channel first was to the stability of MspA in the which was for MspA was for at different and that MspA with an of were at that is not to MspA. Thus, the temperature required for of MspA to is at °C that of dissociation of the E. porin which at about °C and is to be an stable protein Scholar, A. R. 1998; 37: Scholar). of the revealed that about of the MspA were at 92 °C It be noted that not the MspA in extracts smegmatis to °C for C. Niederweis M. Biochem. 2000; Scholar), that a was the The of SDS to a of 2% to the purified MspA tetramer not stability in to of was reduced to °C in the presence of SDS stability of MspA in the presence of were as at which the of MspA to the of the of tetrameric MspA was reduced to and tetrameric MspA was respectively. These were by the of 2 The temperature at which the β-sheet and the of the were to was as and is to the temperature from to experimental not be at and of MspA were the β-sheet of MspA was estimated to to that of other proteins The of MspA SDS was to M. not in a new were as at which the of MspA to the of the of tetrameric MspA was reduced to and tetrameric MspA was respectively. These were by the of 2 The temperature at which the β-sheet and the of the were to was as and is to the temperature from to experimental not be at and of MspA were the β-sheet of MspA was estimated to to that of other proteins The of MspA SDS was to M. not the secondary structure of the MspA tetramer was at to that the MspA tetramer dissociation in the presence of MspA in was for in a special at in of 10 °C and in of °C were at temperature the of the in the at and was to The of the to °C and drastically to the of MspA The of the was for of the secondary structure by of as The changes of the β-sheet of MspA with are in B. a β-sheet of in with the shown The to a of at °C but at of the Thus, the of the and the β-sheet indicated of MspA at The of 2% SDS to MspA not to in the as with those in A. revealed changes of the of the at °C and changes at °C The β-sheet of MspA in the presence of 2% SDS to at 50 to and to at These results that SDS a but the stability of the secondary structure of MspA. Lipid bilayer experiments were to the activity of MspA as a channel protein with the of MspA, which a low of in the of 10 the of MspA at a of of of in the bilayer 4 of of MspA were for at and in the channel activity of MspA was at to the temperature to °C reduced the channel activity about but a activity of was 4 This is in with the existence of MspA and a reduced β-sheet of about at °C by and respectively. Surprisingly, the of 2% SDS reduced the channel activity of MspA at of the channel activity was at 50 °C and although the MspA tetramer was stable at this temperature and the β-sheet was not reduced experiments were to the stability of MspA also to other which with protein A. M. 1999; Scholar). revealed that the MspA tetramer was stable in a 7.6m urea at °C of the MspA were at Thus, the temperature of MspA not of 2% SDS urea were However, the MspA was at which was °C the MspA and dissociation of the MspA tetramer was at °C in to the MspA the of that urea reduced the stability of MspA MspA at a of 10 a at nm at nm of MspA was of the and was to the of MspA in the presence of urea in The of MspA the temperature in 10 °C to °C for the of MspA was to nm that the of MspA a from a to a M.R. Scholar). revealed a of at °C in with the These results a major of MspA with dissociation and under these conditions. The channel activity of MspA was not by urea at °C in bilayer However, the temperature to 50 °C reduced the channel activity of MspA 4 although the tetramer was stable and the not any major changes of the of the at this temperature Thus, SDS and strongly reduced the channel structure the of MspA major changes by and spectroscopy the stability of MspA under and of of purified MspA were at pH from to for at Surprisingly, that MspA were stable at any pH very low pH of the MspA were and of of MspA was at pH 2 and the purified MspA tetramer was stable against MspA in extracts of M. smegmatis, the pH stability of MspA and was MspA is the most protein in extracts since the is for porins C. Niederweis M. Biochem. 2000; Scholar). was of MspA at any pH from to for at °C although the was the pores at an estimated protein of the was at pH and 14. It be noted that the of the bilayer of MspA at pH was with at other pH in the single channel of of the pores was in with earlier results M. Ehrt S. Heinz C. Klöcker U. Karosi S. Swiderek K.M. Riley L.W. Benz R. Mol. Microbiol. 1999; 33: 933-945Google Scholar). These results that MspA in extracts was resistant against at any pH from to 14. of at pH and a to single channel a in the channel activity of these not that the structure and the of the MspA pore were not by of and was also of MspA smegmatis with a of and M. Ehrt S. Heinz C. Klöcker U. Karosi S. Swiderek K.M. Riley L.W. Benz R. Mol. Microbiol. 1999; 33: 933-945Google Scholar) and to be an of MspA (12Engelhardt H. Heinz C. Niederweis M. J. Biol. Chem. 2002; 277: 37567-37572Google Scholar). these results that MspA in extracts was stable purified MspA. the stability of purified MspA was also that in extracts it is that a was of MspA. were from MspA by the C. Niederweis M. Biochem. 2000; Scholar), which explain the reduced stability of purified MspA against purified MspA is stable against pH changes the E. which trimeric structure at pH 4 and M. Scholar, Scholar). of purified MspA were with for at °C not the from to and the to not to of tetrameric MspA with in a 4 is to and is under these conditions and It be noted that tetrameric which in E. and channel forming activity as earlier M. Ehrt S. Heinz C. Klöcker U. Karosi S. Swiderek K.M. Riley L.W. Benz R. Mol. Microbiol. 1999; 33: 933-945Google Scholar), was resistant to under these conditions The of tetrameric be to a of with the experiments with MspA. this may that tetramer in E. is not with MspA smegmatis. The channel structure of the major porin MspA smegmatis is drastically different from that of other known central pore in to the trimeric porins of Gram-negative bacteria, which pore per (12Engelhardt H. Heinz C. Niederweis M. J. Biol. Chem. 2002; 277: 37567-37572Google Scholar). 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Scholar), in to MspA and other general which not The stability experiments and the of MspA to the trimeric This to that the of MspA is part of channel-forming β-barrel in the MspA tetramer to the structures of in and L. M.R. C. S. H. Science. 1996; Scholar, K. Nature. Scholar). This is the observations. proteins are known for their remarkable the of of E. from are stable to °C and °C M. M. G.S. P. A. L. 2001; 40: Scholar, H. S. S. A. J. Biol. Scholar). It is to in this that the stability of the β-barrel against is not to of those proteins in the outer since also β-barrel proteins such as the protein very of and S. A. Chem. 2002; Scholar, S. J. R. 1999; Scholar). the β-sheet the MspA is stable 112 the interactions between subunits 92 may be for in MspA was stable at any pH from to that not to a major in interactions This as the major between the MspA subunits and a coherent β-barrel which is by Chem. 1996; Scholar). This is also in the pH stability of other β-barrel proteins such as 2002; Scholar). The of the channel wall of MspA as in the a β-barrel structure a structure of (12Engelhardt H. Heinz C. Niederweis M. J. Biol. Chem. 2002; 277: 37567-37572Google Scholar). The of nm forming the of the is in with the of long in the inner membrane are of a of the β-barrel was in outer membrane proteins (13Koebnik R. Locher K.P. van Gelder P. Mol. Microbiol. 2000; 37: 239-253Google Scholar). This was by the that the from the inner membrane and the to the outer membrane Microbiol. Rev. 1993; Scholar). This principle also to the which have an outer membrane in to the membrane (2Nikaido H. Kim S.H. Rosenberg E.Y. Mol. 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Biochem. 2000; the extracts only of proteins other MspA, it that the stability of MspA and were by in the of C. Niederweis M. Biochem. 2000; Scholar). This is to porins of Gram-negative bacteria, which are by A. A. R. A. U. Scholar). for for the and for the