Specificity in Transmembrane Helix-Helix Interactions Mediated by Aromatic Residues

Aromatic residues have been previously shown to mediate the self-assembly of different soluble proteins through π-π interactions (McGaughey, G. B., Gagne, M., and Rappe, A. K. (1998) J. Biol. Chem. 273, 15458–15463). However, their role in transmembrane (TM) assembly is not yet clear. In this study, we performed statistical analysis of the frequency of occurrence of aromatic pairs in a bacterial TM data base that provided an initial indication that the appearance of a specific aromatic pattern, Aromatic-XX-Aromatic, is not coincidental, similar to the well characterized QXXS motif. The QXXS motif was previously shown to be both critical and sufficient for stabilizing TM self-assembly. Using the ToxR system, we monitored the dimerization propensities of TM domains that contain mutations of interacting residues to aromatic amino acids and demonstrated that aromatic residues can adequately stabilize self-association. Importantly, we have provided an example of a natural TM domain, the cholera toxin secretion protein EpsM, whose TM self-assembly is mediated by an aromatic motif (WXXW). This is, in fact, the first evidence that aromatic residues are involved in the dimerization of a wild type TM domain. The association mediated by aromatic residues was found to be sensitive to the TM sequence, suggesting that aromatic residue motifs can provide a general means for specificity in TM assembly. Molecular dynamics provided a structural explanation for this backbone sequence sensitivity. Aromatic residues have been previously shown to mediate the self-assembly of different soluble proteins through π-π interactions (McGaughey, G. B., Gagne, M., and Rappe, A. K. (1998) J. Biol. Chem. 273, 15458–15463). However, their role in transmembrane (TM) assembly is not yet clear. In this study, we performed statistical analysis of the frequency of occurrence of aromatic pairs in a bacterial TM data base that provided an initial indication that the appearance of a specific aromatic pattern, Aromatic-XX-Aromatic, is not coincidental, similar to the well characterized QXXS motif. The QXXS motif was previously shown to be both critical and sufficient for stabilizing TM self-assembly. Using the ToxR system, we monitored the dimerization propensities of TM domains that contain mutations of interacting residues to aromatic amino acids and demonstrated that aromatic residues can adequately stabilize self-association. Importantly, we have provided an example of a natural TM domain, the cholera toxin secretion protein EpsM, whose TM self-assembly is mediated by an aromatic motif (WXXW). This is, in fact, the first evidence that aromatic residues are involved in the dimerization of a wild type TM domain. The association mediated by aromatic residues was found to be sensitive to the TM sequence, suggesting that aromatic residue motifs can provide a general means for specificity in TM assembly. Molecular dynamics provided a structural explanation for this backbone sequence sensitivity. Receptor self-assembly is a central process in a variety of signal transduction cascades. This assembly is mainly mediated by the extracellular or the intracellular domains. However, considerable data have been accumulated concerning the causal involvement of the transmembrane (TM) 2The abbreviation used is: TM, transmembrane. domains in this process as well (1von Heijne G. Q. Rev. Biophys. 1999; 32: 285-307Crossref PubMed Scopus (86) Google Scholar, 2Gouldson P.R. Higgs C. Smith R.E. Dean M.K. Gkoutos G.V. Reynolds C.A. Neuropsychopharmacology. 2000; 23: S60-S77Crossref PubMed Scopus (126) Google Scholar, 3Li R. Bennett J.S. Degrado W.F. Biochem. Soc. Trans. 2004; 32: 412-415Crossref PubMed Scopus (25) Google Scholar, 4Lemmon M.A. Flanagan J.M. Hunt J.F. Adair B.D. Bormann B.J. Dempsey C.E. Engelman D.M. J. Biol. Chem. 1992; 267: 7683-7689Abstract Full Text PDF PubMed Google Scholar, 5Arkin I.T. Biochim. Biophys. Acta. 2002; 1565: 347-363Crossref PubMed Scopus (49) Google Scholar). In contrast to the soluble regions of membrane proteins, our knowledge of the factors that control protein-protein interactions and recognition of the membrane-embedded domains is still limited. To date, the non-covalent association of native TM domains was reported to be mediated by (i) a heptad motif of leucines through their side chain residues packing interaction (6Gurezka R. Laage R. Brosig B. Langosch D. J. Biol. Chem. 1999; 274: 9265-9270Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar); (ii) a GXXXG motif, which was first found in the glycophorin A TM domain (4Lemmon M.A. Flanagan J.M. Hunt J.F. Adair B.D. Bormann B.J. Dempsey C.E. Engelman D.M. J. Biol. Chem. 1992; 267: 7683-7689Abstract Full Text PDF PubMed Google Scholar, 8Lemmon M.A. Treutlein H.R. Adams P.D. Brunger A.T. Engelman D.M. Nat. Struct. Biol. 1994; 1: 157-163Crossref PubMed Scopus (298) Google Scholar, 9Russ W.P. Engelman D.M. J. Mol. Biol. 2000; 296: 911-919Crossref PubMed Scopus (785) Google Scholar); or (iii) polar residues through the formation of hydrogen bonds (10Zhou F.X. Merianos H.J. Brunger A.T. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2250-2255Crossref PubMed Scopus (318) Google Scholar, 11Choma C. Gratkowski H. Lear J.D. DeGrado W.F. Nat. Struct. Biol. 2000; 7: 161-166Crossref PubMed Scopus (348) Google Scholar, 12Dawson J.P. Weinger J.S. Engelman D.M. J. Mol. Biol. 2002; 316: 799-805Crossref PubMed Scopus (217) Google Scholar, 13Gratkowski H. Lear J.D. DeGrado W.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 880-885Crossref PubMed Scopus (289) Google Scholar, 14Sal-Man N. Gerber D. Shai Y. J. Biol. Chem. 2005; 280: 27449-27457Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). However, the involvement of additional motifs or key factors that may mediate protein-protein interactions within the membrane merit further investigation. Examination of the assembly of soluble proteins reveals that aromatic residues serve as key structural elements that mediate the molecular recognition and the self-assembly of amyloid polypeptides as well as bacterial toxins and several proteins such as acetylcholinesterase (15Belbeoc'h S. Falasca C. Leroy J. Ayon A. Massoulie J. Bon S. Eur. J. Biochem. 2004; 271: 1476-1487Crossref PubMed Scopus (32) Google Scholar, 16Ramachandran R. Tweten R.K. Johnson A.E. Nat. Struct. Mol. Biol. 2004; 11: 697-705Crossref PubMed Scopus (152) Google Scholar, 17Azriel R. Gazit E. J. Biol. Chem. 2001; 276: 34156-34161Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 18Soto C. Sigurdsson E.M. Morelli L. Kumar R.A. Castano E.M. Frangione B. Nat. Med. 1998; 4: 822-826Crossref PubMed Scopus (800) Google Scholar, 19Pallitto M.M. Ghanta J. Heinzelman P. Kiessling L.L. Murphy R.M. Biochemistry. 1999; 38: 3570-3578Crossref PubMed Scopus (198) Google Scholar, 20Findeis M.A. Musso G.M. Arico-Muendel C.C. Benjamin H.W. Hundal A.M. Lee J.J. Chin J. Kelley M. Wakefield J. Hayward N.J. Molineaux S.M. Biochemistry. 1999; 38: 6791-6800Crossref PubMed Scopus (229) Google Scholar). The interactions are formed between the planar aromatic rings and are referred to as π-π interactions (21Bernstein E.R. Sun S. J. Phys. Chem. 1996; 100: 13348-13366Crossref Scopus (76) Google Scholar, 22McGaughey G.B. Gagne M. Rappe A.K. J. Biol. Chem. 1998; 273: 15458-15463Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar, 23Gazit E. FASEB J. 2002; 16: 77-83Crossref PubMed Scopus (943) Google Scholar). Even a single mutation of aromatic amino acid in the sequence of the short amyloid peptide abolishes the ability of the peptide to form amyloid fibrils (17Azriel R. Gazit E. J. Biol. Chem. 2001; 276: 34156-34161Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). Sequence analysis of membrane immunoglobulin and the T-cell receptor reveals highly conserved aromatic amino acids within the TM domains of these proteins, which may indicate the involvement of these residues in TM-TM interactions (24Campbell K.S. Backstrom B.T. Tiefenthaler G. Palmer E. Semin. Immunol. 1994; 6: 393-410Crossref PubMed Scopus (85) Google Scholar). An additional piece of evidence that supports this assumption is the recent finding of Langosch and co-workers (25Ridder A. Skupjen P. Unterreitmeier S. Langosch D. J. Mol. Biol. 2005; 354: 894-902Crossref PubMed Scopus (62) Google Scholar) that demonstrated, using a randomized TM library, tryptophan is enriched in self-interacting TM domains containing a heptad repeat backbone sequence. In this study, we have investigated the role of aromatic residues in TM self-assembly. We performed a statistical analysis that revealed that an Aromatic-XX-Aromatic pattern (which was chosen based on the QXXS motif) is over-represented in a bacterial TM data base. Using the ToxR system, we measured the ability of aromatic residues to stabilize oligomerization when inserted into the well defined interaction interface of the N-terminal TM domain of the Escherichia receptor This TM domain, which is of the bacterial was previously shown to form a through polar residues by hydrogen bonds N. Gerber D. Shai Y. J. Biol. Chem. 2005; 280: 27449-27457Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). indicate that aromatic residues can self-assembly when the of the polar QXXS motif. Importantly, we have demonstrated that the TM sequence, which been found in the statistical analysis to contain a native aromatic sequence was in of dimerization by mutations in these specific This protein was chosen was previously to form J. A.E. K. M. J. Mol. Biol. 2004; PubMed Scopus Google data that are not for membrane analysis of the of occurrence of the and performed on a bacterial TM domain data base. The of the TM was the data base to The data base bacterial TM domains between and amino The of the different amino acid in the data base The frequency of was by the of specific pattern in randomized data of the data base was by the of the TM domain of a into of which was and into the of the ToxR residues of the wild type TM domain or the TM domain inserted between the ToxR and the E. protein within the The TM domain of was inserted into a a sequence of which is a of TM domains. mutations of in the the and mutations of in or both residues and The QXXS motif backbone and motif backbone contain the or the motifs within a of and The the motif the sequence of The of of the by The of the TM domains the amino acids and of the TM domain or and of the TM of the wild type of TM acids are to their in the protein amino acids in the of the dimerization motif are in in the TM domain are of the TM domains the amino acids and of polar residues motif, TM domains inserted into a amino acid sequence, a sequence of residues that is of of TM acids are to their in the protein The amino acids in the of the dimerization motif are in in the TM domain are The of the TM domains the amino acids and of polar residues motif, The TM domains inserted into a amino acid sequence, a sequence of residues that is of of TM domains in a In of of TM within the ToxR can be used to protein-protein interactions within the E. A TM domain the was between the ToxR and the protein in the The was into E. which contain the control of a of the TM domains in this in association of the ToxR which and is to the D. Brosig B. H. H.J. J. Mol. Biol. 1996; PubMed Scopus Google Scholar). of the of was by the of the and by to the protein The of a control which a was of the and referred to as The in the of for in and the for of by a Molecular D. Brosig B. H. H.J. J. Mol. Biol. 1996; PubMed Scopus Google Scholar, H. C. G. K. H.J. Biol. Chem. 1994; PubMed Scopus Google Scholar). the of the performed for of of a different a for on and to the The used was The was a and as previously B. Langosch D. Sci. 1998; 7: PubMed Scopus Google Scholar). the different The and used to a The of the was measured different by a Molecular for the was for the TM sequence. and using the of the The as The for the was of the TM in was and the of the was within a to the The to and in that by for further analysis we of the for the formation of the The for TM the to a to in This was by of The to their structural The of the interaction and the hydrogen bonds for the The using the and The the the was as the of TM sequence for further In this study, we have investigated the involvement of aromatic residues in TM self-assembly. To this we used the well characterized interaction interface of the formed by the N-terminal TM domain of the E. receptor we have of or both polar residues (which are to the interaction by aromatic residues the frequency of occurrence of aromatic residue pairs to these motifs may be involved in the association of TM domains. The pattern Aromatic-XX-Aromatic, was chosen based on the QXXS A between or of a specific pattern in TM domains and interactions was previously found A. M. Engelman D.M. J. Mol. Biol. 2000; 296: PubMed Scopus Google Scholar). The analysis was in a of bacterial TM domains. indicate that the aromatic pairs to be over-represented their The pattern was found within a data base of of TM occurrence was and the pattern occurrence was The of occurrence of the and is the of and is the the and motifs these that the and may as The of occurrence of the motifs or within the of the may indicate that pairs not serve as association is in TM domains and and residues within TM domains are not the TM sequence and to be the interface S. J. Biol. 1996; PubMed Scopus Google Scholar). we the frequency of occurrence of the aromatic in the and of the TM domains. The of occurrence of the sequence was and for the and The of the and and for the and are the data the TM sequence and that the of the and motifs is not to the of and residues to to the of Aromatic the ability of aromatic residues to mediate TM-TM we the ToxR assembly system, which can within the membrane of E. D. Brosig B. H. H.J. J. Mol. Biol. 1996; PubMed Scopus Google Scholar). The wild type the interaction interface of the and to and by and for their of in which both and by of the dimerization of the of residue of the motif for for of and for of are the different of residue of the motif to the dimerization of was by and by N. Gerber D. Shai Y. Biochemistry. 2004; PubMed Scopus Google The of the residues to the membrane can serve as an additional that the dimerization ability of the and be and dimerization propensities that the association mediated by aromatic residues not to their To the that the between the dimerization of the different of the proteins, or a of the to into the we performed and and The of the proteins similar to the that to the protein using the of the proteins into the membrane of E. was by the ability of the to a E. B. Langosch D. Sci. 1998; 7: PubMed Scopus Google Scholar). are to on containing as the that the protein in the the be to and similar of membrane A a TM domain as a was to in the and was to the To this pattern of dimerization is specific for or a general aromatic we additional that contain aromatic residue or both of the polar dimerization motif The an pattern similar to the of the proteins and their not we that aromatic residues can and stabilize association within the membrane when to the through of the TM domain used to the involvement of aromatic residues in TM of TM acids are to their in the protein amino acids in the of the dimerization motif are in in the TM domain are of the TM domains the amino acids and of polar residues motif, TM domains inserted into a amino acid sequence, a sequence of residues that is of of TM acids are to their in the protein The amino acids in the of the dimerization motif are in in the TM domain are The of the TM domains the amino acids and of polar residues motif, The TM domains inserted into a amino acid sequence, a sequence of residues that is of of TM domains in a of the TM of the the involvement of aromatic motif in TM-TM interactions of an TM domain, we the cholera toxin secretion protein EpsM, which been found to contain a sequence in the statistical analysis This protein a single TM domain and was previously to form J. A.E. K. M. J. Mol. Biol. 2004; PubMed Scopus Google Scholar) is for membrane The aromatic residues of the TM domain are conserved in proteins of the bacterial secretion not This further that these residues have a role in the of the A sequence of residues of the TM domain was inserted within the and dimerization was of the residue by a on the dimerization and on the dimerization for of both residues by on the dimerization that the aromatic residues of the TM domain are critical for self-association. the of a single aromatic residue is sufficient to assembly. This is the first evidence that aromatic residues the dimerization of TM domains. of the residues by a different aromatic a on the association that the aromatic of the residues is the critical for of the an aromatic residue is sufficient for TM-TM we the association of that contain the pattern within the of the sequence, which as a for for of the or within a of leucines and are shown in previously shown to have a dimerization and inserted within the membrane N. Gerber D. Shai Y. J. Biol. Chem. 2005; 280: 27449-27457Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The motif backbone dimerization the TM domain, the demonstrated dimerization In the QXXS motif, which is to stabilize dimerization of TM sequence, a dimerization similar to the TM domain when inserted within the backbone the QXXS motif within the sequence was to into the membrane N. Gerber D. Shai Y. J. Biol. Chem. 2005; 280: 27449-27457Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and is not the indicate the QXXS motif as a general dimerization motif, the aromatic can stabilize association in a similar to the TM domain and into the membrane not used to the of the aromatic residue motif to the backbone sequence The TM domains inserted into a sequence, which is of for TM a sequence of The amino acids in the of the dimerization motif are and in of inserted TM motif motif motif in a A for the of the Aromatic provide a structural explanation for the of the aromatic residue pattern we a of of for different TM contain the QXXS or the motifs within the sequence or within the The the a between the packing of the in and the the residues association when the motif was within the backbone sequence. However, in the of the sequence, this pattern a packing ability similar to the sequence. We an in the between the of the motif within the backbone sequence, the TM between the of the residues The which the between the of of of an in The motif packing interaction in the TM domain, by a A was for the motif in the as by the frequency of the structural for the different TM well the dimerization which indicate that the association of the is sensitive to backbone the wild type QXXS motif is residues are the interface C. R.A. J. Mol. Biol. PubMed Scopus Google Scholar). To the dimerization of a TM domain that an aromatic residue is by the of the aromatic residue to the we the association of of TM domains. a single and wild type polar residue of the of the dimerization motif and An additional of TM domains pairs of and residues these interacting are shown in we similar dimerization for the motif and the TM dimerization motif suggesting that the of and to TM domain is N. Gerber D. Shai Y. Biochemistry. 2004; PubMed Scopus Google Scholar). The TM domains containing aromatic on the a association ability for TM domains that to the interface and TM domains that contain in the and that the association mediated aromatic residues is A of to within the TM domain of an motif within the of the membrane This dimerization to the the that the aromatic residues stabilize TM dimerization by interaction the polar or the The dimerization for the motif to the TM domain to the of the polar used to the of aromatic residues on TM of TM acids are to their in the protein amino acids in the of the motif are in in the TM domain are of the TM domains the amino acids and of polar residues motif, TM domains inserted into a amino acid sequence, a sequence of residues that is of of TM in in in in acids are to their in the protein The amino acids in the of the motif are in in the TM domain are The of the TM domains the amino acids and of polar residues motif, The TM domains inserted into a amino acid sequence, a sequence of residues that is of of TM domains in a The first in interactions is to the interaction interface and the specific residues that are involved in TM-TM In this we the well characterized dimerization interface of the to the involvement of aromatic residues in the dimerization of TM data revealed that mutation of or both polar residues of this dimerization motif to aromatic residues the for an Importantly, we demonstrated that the residues within the wild type TM sequence are for dimerization as mutations of both residues to abolishes self-association. mutations of to on the on dimerization and suggesting that the of a single residue is sufficient for stabilizing is that in the of residue the residue can to of the residues to a different aromatic on the self-assembly of the TM domain, our assumption that the aromatic of the residue is the critical that for self-association. In this we the first evidence that aromatic residues are involved in the dimerization of a TM domain. The similar dimerization pattern for aromatic amino acids that this interaction is mediated through π-π However, and are polar residues and stabilize association formation of hydrogen We that the of the TM-TM association on (i) the residue the of the interaction the aromatic (ii) the backbone sequence in which the aromatic is and (iii) the of the aromatic residue to the The of the the of the QXXS on the dimerization motif by aromatic amino acid and in a of formation the wild type TM domain and This that the interactions of the aromatic residue the formed by the The of the the N. Gerber D. Shai Y. Biochemistry. 2004; PubMed Scopus Google additional for this that the of was similar to that of the However, this is by the of to the which is in which the QXXS motif, was by an aromatic residue and formation the TM domain and are the different of residue of the QXXS motif to as previously shown was by and by the N. Gerber D. Shai Y. Biochemistry. 2004; PubMed Scopus Google The dimerization of and the that the association mediated by aromatic residues is to the aromatic residue pairs and as well as single aromatic and dimerization the TM domain and the ability of aromatic residues to TM self-assembly in the of additional polar The similar assembly pattern of aromatic residues that this interaction is through π-π which is a of the aromatic The of the TM on the the can TM-TM interactions in a similar to the QXXS motif, we association ability within backbone and In contrast to the QXXS motif, which similar dimerization within the sequence and within the backbone N. Gerber D. Shai Y. J. Biol. Chem. 2005; 280: 27449-27457Abstract Full Text Full Text PDF PubMed Scopus (51) Google the motif association within the sequence, association within the sequence, and in association within the backbone that dimerization by aromatic residues is and may specificity in TM-TM A recent that is enriched within in a is in our (25Ridder A. Skupjen P. Unterreitmeier S. Langosch D. J. Mol. Biol. 2005; 354: 894-902Crossref PubMed Scopus (62) Google Scholar). A for the Sequence of the structural a explanation for the association of the as to the QXXS motif, which was shown to be to backbone We a in the packing of the motif within the backbone the motif within the sequence This was in a between the of the TM shown in the molecular In this of the between the can of the which in can further the association by interacting the interface The of the Aromatic to the the this study, we aromatic residues into or both interacting of the QXXS motif. The indicate that the dimerization of the TM domain was by the of the aromatic residue to the TM domains that aromatic residues to the interface association ability TM domains an amino acid in the membrane A TM domain that an aromatic motif in the membrane the that the dimerization mediated by aromatic residues is mainly performed by interactions of the aromatic residues the polar or the to our we that the of the interface supports the mediated by the aromatic the association this is that within the membrane This the finding that randomized the different of the heptad repeat motif frequency the interface (25Ridder A. Skupjen P. Unterreitmeier S. Langosch D. J. Mol. Biol. 2005; 354: 894-902Crossref PubMed Scopus (62) Google Scholar). of Aromatic analysis of the frequency of occurrence of the aromatic a general role for this motif in the assembly of bacterial TM domains. A the TM proteins that contain this aromatic revealed the pattern in membrane proteins different for which to and protein which in the control of and which is involved in the of the extracellular through the that the motif is not by or The is that of the proteins that contain this motif to the motif is involved in the assembly of these proteins, this the of a membrane protein assembly. In we have demonstrated the involvement of aromatic amino acids in the association of TM domains. we have provided an example of a wild type TM sequence that native aromatic (WXXW). The interaction mediated by aromatic motifs was found to be by the sequence may serve as a means for specificity in TM-TM The of association of the was the polar motif The found the motif to be over-represented within a of bacterial TM domains and several TM domains of bacterial proteins that contain this motif. TM proteins are for our our of the oligomerization process within the membrane and may to the of membrane protein This in in the to the of membrane proteins for We D. Langosch for the A and as well as the and E.