The Affinity of GXXXG Motifs in Transmembrane Helix-Helix Interactions Is Modulated by Long-range Communication

Sequence motifs are responsible for ensuring the proper assembly of transmembrane (TM) helices in the lipid bilayer. To understand the mechanism by which the affinity of a common TM-TM interactive motif is controlled at the sequence level, we compared two well characterized GXXXG motif-containing homodimers, those formed by human erythrocyte protein glycophorin A (GpA, high-affinity dimer) and those formed by bacteriophage M13 major coat protein (MCP, low affinity dimer). In both constructs, the GXXXG motif is necessary for TM-TM association. Although the remaining interfacial residues (underlined) in GpA (LIXXGVXXGVXXT) differ from those in MCP (VVXXGAXXGIXXF), molecular modeling performed here indicated that GpA and MCP dimers possess the same overall fold. Thus, we could introduce GpA interfacial residues, alone and in combination, into the MCP sequence to help decrypt the determinants of dimer affinity. Using both in vivo TOXCAT assays and SDS-PAGE gel migration rates of synthetic peptides derived from TM regions of the proteins, we found that the most distal interfacial sites, 12 residues apart (and ∼18 Å in structural space), work in concert to control TM-TM affinity synergistically. Sequence motifs are responsible for ensuring the proper assembly of transmembrane (TM) helices in the lipid bilayer. To understand the mechanism by which the affinity of a common TM-TM interactive motif is controlled at the sequence level, we compared two well characterized GXXXG motif-containing homodimers, those formed by human erythrocyte protein glycophorin A (GpA, high-affinity dimer) and those formed by bacteriophage M13 major coat protein (MCP, low affinity dimer). In both constructs, the GXXXG motif is necessary for TM-TM association. Although the remaining interfacial residues (underlined) in GpA (LIXXGVXXGVXXT) differ from those in MCP (VVXXGAXXGIXXF), molecular modeling performed here indicated that GpA and MCP dimers possess the same overall fold. Thus, we could introduce GpA interfacial residues, alone and in combination, into the MCP sequence to help decrypt the determinants of dimer affinity. Using both in vivo TOXCAT assays and SDS-PAGE gel migration rates of synthetic peptides derived from TM regions of the proteins, we found that the most distal interfacial sites, 12 residues apart (and ∼18 Å in structural space), work in concert to control TM-TM affinity synergistically. After their biosynthesis and subsequent integration into a membrane, many transmembrane (TM) 1The abbreviations used are: TM, transmembrane; GpA, glycophorin A; MCP, M13 major coat protein; TOXCAT assay, transmembrane helix association coupled with the expression of chloramphenicol acetyltransferase; MCP-TM, M13 major coat protein transmembrane segment; GpA-TM, glycophorin A transmembrane segment; RMS, root-mean-square; Fmoc, N-(9-fluorenyl)methoxycarbonyl; CAT, chloramphenicol acetyltransferase; RMSD, root-mean-square deviation; wt, wild type. helices associate with other pre-formed helices to form functional membrane protein domains (1Popot J.L. Engelman D.M. Annu. Rev. Biochem. 2000; 69: 881-922Crossref PubMed Scopus (541) Google Scholar). Specificity for a given helix-helix interaction is achieved through the appropriate presentation of complementary side chains, which serve as recognition elements between associating helices. The most highly studied, and apparently widespread, mode of association is mediated by the so-called GXXXG motif, which is known to act as a universal scaffold for the assembly of both TM helices (2Deber C.M. Khan A.R. Li Z. Joensson C. Glibowicka M. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11648-11652Crossref PubMed Scopus (89) Google Scholar, 3Lemmon M.A. Flanagan J.M. Treutlein H.R. Zhang J. Engelman D.M. Biochemistry. 1992; 31: 12719-12725Crossref PubMed Scopus (469) Google Scholar, 4Mendrola J.M. Berger M.B. King M.C. Lemmon M.A. J. Biol. Chem. 2002; 277: 4704-4712Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 5Op De Beeck A. Montserret R. Duvet S. Cocquerel L. Cacan R. Barberot B. Le Maire M. Penin F. Dubuisson J. J. Biol. Chem. 2000; 275: 31428-31437Abstract Full Text Full Text PDF PubMed Google Scholar, 6Russ W.P. Engelman D.M. J. Mol. Biol. 2000; 296: 911-919Crossref PubMed Scopus (790) Google Scholar, 7Senes A. Gerstein M. Engelman D.M. J. Mol. Biol. 2000; 296: 921-936Crossref PubMed Scopus (515) Google Scholar, 8Arselin G. Giraud M.F. Dautant A. Vaillier J. Brethes D. Coulary-Salin B. Schaeffer J. Velours J. Eur. J. Biochem. 2003; 270: 1875-1884Crossref PubMed Scopus (112) Google Scholar, 9McClain M.S. Iwamoto H. Cao P. Vinion-Dubiel A.D. Li Y. Szabo G. Shao Z. Cover T.L. J. Biol. Chem. 2003; 278: 12101-12108Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) and soluble α-helices (10Kleiger G. Grothe R. Mallick P. Eisenberg D. Biochemistry. 2002; 41: 5990-5997Crossref PubMed Scopus (175) Google Scholar). The GXXXG motif, where two glycine residues are separated by any three amino acids on a helical framework, gives rise to a flat surface region on one face of the helix. This arrangement of Gly residues permits the close approach of interacting helices, whereupon extensive packing interactions take place between pairs of surrounding residues. It has been proposed that a portion of the interactive strength of GXXXG-mediated associations may originate from inter-helix hydrogen bonds between Cα hydrogens and carbonyl oxygen atoms on the adjacent helix (11Senes A. Ubarretxena-Belandia I. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9056-9061Crossref PubMed Scopus (441) Google Scholar). The glycophorin A transmembrane (GpA-TM) segment is a well characterized transmembrane helix dimer that associates with high affinity, principally by using a central GXXXG motif (3Lemmon M.A. Flanagan J.M. Treutlein H.R. Zhang J. Engelman D.M. Biochemistry. 1992; 31: 12719-12725Crossref PubMed Scopus (469) Google Scholar, 12MacKenzie K.R. Prestegard J.H. Engelman D.M. Science. 1997; 276: 131-133Crossref PubMed Scopus (877) Google Scholar). The details of side chain-side chain packing for GpA are known in considerable detail, having been gleaned originally from extensive mutagenesis experiments (3Lemmon M.A. Flanagan J.M. Treutlein H.R. Zhang J. Engelman D.M. Biochemistry. 1992; 31: 12719-12725Crossref PubMed Scopus (469) Google Scholar), computer modeling (13Treutlein H.R. Lemmon M.A. Engelman D.M. Brunger A.T. Biochemistry. 1992; 31: 12726-12732Crossref PubMed Scopus (157) Google Scholar, 14Adams P.D. Engelman D.M. Brunger A.T. Proteins. 1996; 26: 257-261Crossref PubMed Scopus (158) Google Scholar) and, subsequently, from a high-resolution structural analysis using nuclear magnetic resonance (NMR) for the GpA dimer in both detergent micelles (12MacKenzie K.R. Prestegard J.H. Engelman D.M. Science. 1997; 276: 131-133Crossref PubMed Scopus (877) Google Scholar) and lipid bilayers (15Smith S.O. Song D. Shekar S. Groesbeek M. Ziliox M. Aimoto S. Biochemistry. 2001; 40: 6553-6558Crossref PubMed Scopus (171) Google Scholar). Despite the high occurrence of the GXXXG motif in transmembrane helices (7Senes A. Gerstein M. Engelman D.M. J. Mol. Biol. 2000; 296: 921-936Crossref PubMed Scopus (515) Google Scholar), GpA-TM is the only GXXXG peptide dimer with a structure determined to high resolution (11Senes A. Ubarretxena-Belandia I. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9056-9061Crossref PubMed Scopus (441) Google Scholar). Thus, it is not clear whether other membrane proteins containing this motif adopt a similar dimeric fold to GpA or have alternate structure. Moreover, it is not known how residues surrounding GXXXG motifs “tailor” the affinity of their helix-helix interactions for the required structural and functional purposes. For instance, the transmembrane helix of the major coat protein of the M13 bacteriophage (MCP-TM) contains a GXXXG motif (2Deber C.M. Khan A.R. Li Z. Joensson C. Glibowicka M. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11648-11652Crossref PubMed Scopus (89) Google Scholar) and has been shown to self-associate using both in vitro (2Deber C.M. Khan A.R. Li Z. Joensson C. Glibowicka M. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11648-11652Crossref PubMed Scopus (89) Google Scholar, 16Makino S. Woolford Jr, J.L. Tanford C. Webster R.E. J. Biol. Chem. 1975; 250: 4327-4332Abstract Full Text PDF PubMed Google Scholar, 17Cavalieri S.J. Goldthwait D.A. Neet K.E. J. Mol. Biol. 1976; 102: 713-722Crossref PubMed Scopus (27) Google Scholar, 18Henry G.D. Sykes B.D. J. Mol. Biol. 1990; 212: 11-14Crossref PubMed Scopus (19) Google Scholar, 19Wang C. Deber C.M. J. Biol. Chem. 2000; 275: 16155-16159Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 20Melnyk R.A. Partridge A.W. Deber C.M. J. Mol. Biol. 2002; 315: 63-72Crossref PubMed Scopus (61) Google Scholar) and in vivo (21Haigh N.G. Webster R.E. J. Mol. Biol. 1998; 279: 19-29Crossref PubMed Scopus (30) Google Scholar) techniques. However, unlike the highly stable GpA-TM dimer, the affinity of MCP-TM helix-helix self-association is comparatively weak (22Dawson J.P. Melnyk R.A. Deber C.M. Engelman D.M. J. Mol. Biol. 2003; 331: 255-262Crossref PubMed Scopus (72) Google Scholar). Indeed, such low affinity seems to be a requirement for phage viability (18Henry G.D. Sykes B.D. J. Mol. Biol. 1990; 212: 11-14Crossref PubMed Scopus (19) Google Scholar), thus raising the intriguing notion that the MCP homodimer may not be optimized for stability, but is instead designed to be in flux between the monomeric and dimeric states during the life cycle of the phage (20Melnyk R.A. Partridge A.W. Deber C.M. J. Mol. Biol. 2002; 315: 63-72Crossref PubMed Scopus (61) Google Scholar). The GpA and MCP systems thus afford the opportunity to directly investigate the detailed mechanism by which the stability of a GXXXG-mediated interaction is either optimized (in the case of GpA) or not (in the case of MCP). In the present work, we initially establish, by using a method developed to model TM helix oligomers (23Kim S. Chamberlain A.K. Bowie J.U. J. Mol. Biol. 2003; 329: 831-840Crossref PubMed Scopus (67) Google Scholar), that the primary sequence of MCP is compatible with the dimers of the type formed by GpA. Then, through systematic replacement of the interfacial residues of MCP with those of GpA, individually or in combination, we assess the relative contributions of the interfacial sites in stabilizing TM helix-helix interactions, using both in vitro (TM peptides) and in vivo (TOXCAT) techniques. These combined approaches provide a model in which TM helix-helix interactive motifs are modulated through the propagation of long range cooperative interactions along a 12-residue segment of the oligomeric interface. Computation of MCP Oligomerization—The structures of MCP-TM helix (amino acids 25-42, AWAMVVVIVGATIGIKLF) and mutants were built as uniform α-helices having a backbone torsion angle of ϕ = -65° and ψ = -40° (24Smith L.J. Bolin K.A. Schwalbe H. MacArthur M.W. Thornton J.M. Dobson C.M. J. Mol. Biol. 1996; 255: 494-506Crossref PubMed Scopus (370) Google Scholar) using the Insight II Biopolymer package (Accelrys). We used the backbone-dependent rotamer library program, SCWRL (25Bower M.J. Cohen F.E. Dunbrack Jr, R.L. J. Mol. Biol. 1997; 267: 1268-1282Crossref PubMed Scopus (488) Google Scholar), to chose the side chain rotamers. Structure prediction of MCP dimer was carried out as described (23Kim S. Chamberlain A.K. Bowie J.U. J. Mol. Biol. 2003; 329: 831-840Crossref PubMed Scopus (67) Google Scholar). Briefly, the dimer optimization started with 200 pairs of helices placed in random orientations with respect to each other. Then, the packing interaction of each helix pair was optimized by using the MC minimization method. The internal backbone and side-chain positions were kept fixed during the minimization, but the relative positions of the helices were given all six degrees of freedom. The energy function used was a softened van der Waals potential without any electrostatic component. The simulations were stopped after a maximum of 100,000 MC steps or if 15,000 steps occurred without moving to a lower energy. All six orientation parameters were changed during a step; a step of the same magnitude and direction was repeated if the previous step resulted in lower energy. The step size in each parameter was randomly selected. The temperature was initially set at 500 K and decreased linearly to K the In this we a of optimized After the MC the dimer structures were to structures with the dimer Then, we the remaining structures by Cα root-mean-square using M.J. 1996; PubMed Scopus Google Scholar). the model from the was as structure. This method the that the structure is to in a energy well the other structures D. D. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar) and was in TM helix structures (23Kim S. Chamberlain A.K. Bowie J.U. J. Mol. Biol. 2003; 329: 831-840Crossref PubMed Scopus (67) Google Scholar). to MCP residues three were with on a peptide the of was during using cycle The pair was used with a amino A was used to were with a of peptides were with were in a in and peptide was in and of peptide were a The major from a was and was used to the molecular of the and the was used to peptide was to the of peptides as were using a peptide were in containing with or were at peptide between and were using a with a of were performed from with a of a of and a of were to gel using were for to were on a were in a were from to = for and of were using the were at and in lipid from in was into containing peptide was by using a and were using 500 of and for were formed using were clear TOXCAT TOXCAT of of a transmembrane and the were by the MCP by J. using the mutagenesis using pairs The was in the of the were into were used to expression of the were on and analysis was carried out using were developed using The of in the was by on were by 200 of at of in 500 of with of and and one of at for The was used in the Briefly, were at with chloramphenicol and A. After the was by the into The was and using the All were performed shown are on of MCP and GpA whether the MCP-TM to form a structure similar to the known GpA-TM dimer (12MacKenzie K.R. Prestegard J.H. Engelman D.M. Science. 1997; 276: 131-133Crossref PubMed Scopus (877) Google Scholar, S.O. Song D. Shekar S. Groesbeek M. Ziliox M. Aimoto S. Biochemistry. 2001; 40: 6553-6558Crossref PubMed Scopus (171) Google Scholar) or whether structures be we shown to be at modeling TM helix the of is known (23Kim S. Chamberlain A.K. Bowie J.U. J. Mol. Biol. 2003; 329: 831-840Crossref PubMed Scopus (67) Google Scholar). Using this program, a model for the packing of MCP-TM was In this the residues from the GXXXG motif are in the dimer thus close helix-helix of the structure of the MCP-TM dimer a to that of the GpA-TM dimer, in side chain of the interfacial residues a helix-helix angle of The Cα between this model of the MCP-TM and the GpA-TM from is only and for GpA-TM from the is Å between the structure from and is Thus, the side on MCP-TM be to be compatible with the side in the GpA-TM dimer structure and of MCP and GpA MCP-TM and GpA-TM dimers adopt similar the in their relative helix-helix affinity be to in interactions the residues in the of MCP and GpA with the interfacial residues a through It is that both in to the GXXXG motif at the and a of amino acids It has been proposed that residues, which have in the of in TM proteins (7Senes A. Gerstein M. Engelman D.M. J. Mol. Biol. 2000; 296: 921-936Crossref PubMed Scopus (515) Google Scholar). Despite in all interfacial residues differ between MCP and GpA in the to TOXCAT in investigate how in the interfacial residues of the GpA and MCP dimers to helix-helix we how in MCP to the residues in GpA the affinity of in bilayers using the TOXCAT W.P. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the in the regions of all and affinity in the TOXCAT In the which is to be to that of We that the GXXXG motif in GpA the of GpA, in MCP, the GXXXG motif is the a the and of the TM are not and the relative positions of the GXXXG motifs are not To that the in not from the positions of the GXXXG motifs relative to the we MCP in which the of MCP was to the GpA residues in the association is with that of the for may the of the dimer in the as described These the notion that the GpA is optimized for TM-TM affinity relative to that of the MCP interface. The for the MCP mutants shown in were thus to the affinity of the as this is the maximum affinity for the MCP of the to the MCP dimer by each of the and if in affinity as by the TOXCAT only and on These were for in MCP model the helices are on the the in the of the MCP this not be to to dimer affinity. Moreover, at in in it was shown that both in GpA and (in the a in MCP, or on dimer stability for GpA and MCP, (3Lemmon M.A. Flanagan J.M. Treutlein H.R. Zhang J. Engelman D.M. Biochemistry. 1992; 31: 12719-12725Crossref PubMed Scopus (469) Google Scholar). The of affinity for MCP mutants and was given the the have in the GpA Thus, if interfacial residues only individually to the stability of interacting helices, the thus that of three mutants in any of the MCP the and mutants were combined to form the MCP a in stability was in a of the affinity of this the of the two mutants that and work in to the dimer in a The in affinity as one from the MCP which is of the thus to a but for the and positions in to dimer To investigate the mechanism the by the was in which the was to the was For this the size of the side chain was but the was The for the was not for the a of the in stabilizing TM dimer affinity. of characterized the of of the mutants in the of synthetic TM afford the not in the TOXCAT constructs, to on the TM MCP-TM peptides were designed to the TM of MCP along with three residues, which peptide and (20Melnyk R.A. Partridge A.W. Deber C.M. J. Mol. Biol. 2002; 315: 63-72Crossref PubMed Scopus (61) Google Scholar). We that MCP-TM peptides designed in this were in between the monomeric and dimeric states (20Melnyk R.A. Partridge A.W. Deber C.M. J. Mol. Biol. 2002; 315: 63-72Crossref PubMed Scopus (61) Google Scholar). The of all of the present TM peptide were in both and in and helical The occurrence of a in all MCP peptides to to assess whether or to the the stable of peptides into and shown in was and in both MCP and in both detergent and SDS-PAGE has to be a to assess the association of TM for many (3Lemmon M.A. Flanagan J.M. Treutlein H.R. Zhang J. Engelman D.M. Biochemistry. 1992; 31: 12719-12725Crossref PubMed Scopus (469) Google Scholar, P.D. K.R. Lemmon M.A. Brunger A.T. Engelman D.M. J. PubMed Scopus Google Scholar, J.M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, I. P. Lemmon M.A. G. Sci. 1996; PubMed Scopus Google Scholar, I. A. G. J. Mol. Biol. 1997; PubMed Scopus Google Scholar, Engelman D.M. J. Mol. Biol. PubMed Scopus (158) Google Scholar, C.M. R.A. PubMed Scopus (61) Google Scholar, S. Y. Bowie J.U. J. Mol. Biol. PubMed Scopus Google Scholar, R. J. B. D. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus (112) Google Scholar, C. H. Biol. 2000; PubMed Scopus Google Scholar, G. Biochemistry. 2000; PubMed Scopus Google Scholar, Bowie J.U. Biochemistry. 2000; PubMed Scopus Google Scholar, Brunger A.T. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: PubMed Scopus Google Scholar). the migration of the MCP peptide on We found that the migration of the TM peptides well with the affinity by the TOXCAT the a between monomeric and dimeric is in of the dimer as a function of both the of and the relative of the monomeric In the low affinity peptide dimer the high-affinity rates of other mutants are with determined by TOXCAT The GXXXG motif a scaffold responsible for TM helix-helix Indeed, on GpA found that the central GXXXG portion was the most of the interaction motif, as by to (3Lemmon M.A. Flanagan J.M. Treutlein H.R. Zhang J. Engelman D.M. Biochemistry. 1992; 31: 12719-12725Crossref PubMed Scopus (469) Google Scholar), and to the assembly of monomeric B. D. Sci. 1998; PubMed Scopus Google Scholar). The of this motif was in a were used to randomly high-affinity TM on the motif of GpA W.P. Engelman D.M. J. Mol. Biol. 2000; 296: 911-919Crossref PubMed Scopus (790) Google Scholar). to this motif, it was found that of the high-affinity the GXXXG The structure of GpA how the of the two Gly residues in the the close approach of helices, extensive and side chain-side chain interactions to (12MacKenzie K.R. Prestegard J.H. Engelman D.M. Science. 1997; 276: 131-133Crossref PubMed Scopus (877) Google Scholar). experiments have shown that of any interfacial along the GpA-TM to is Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: PubMed Scopus Google Scholar). This is most with and where the dimer is by and It has been that the GXXXG motif may of interactive energy from the Cα hydrogen of the Gly residues hydrogen bonds with the backbone of the adjacent which have strength in the lipid (11Senes A. Ubarretxena-Belandia I. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9056-9061Crossref PubMed Scopus (441) Google Scholar). we a analysis of two TM and both of which are in but with (22Dawson J.P. Melnyk R.A. Deber C.M. Engelman D.M. J. Mol. Biol. 2003; 331: 255-262Crossref PubMed Scopus (72) Google Scholar), and to understand how the interfacial residues control the affinity of association. their common GXXXG motif, is sequence between MCP and GpA at the remaining interfacial and However, on previous mutagenesis of GpA (2Deber C.M. Khan A.R. Li Z. Joensson C. Glibowicka M. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11648-11652Crossref PubMed Scopus (89) Google Scholar) that positions in extensive we that the at the and positions have only a on affinity. the at sites in the high-affinity by W.P. Engelman D.M. J. Mol. Biol. 2000; 296: 911-919Crossref PubMed Scopus (790) Google Scholar) to principally on the a and positions for the present We found that it was necessary to both a and positions of MCP to the GpA residues to the stability that residues, which are separated by 12 residues in the sequence and ∼18 as in the GpA-TM act to the interface. in we that the between two residues be by the in structure by which along the helical to the residues in the into close of hydrogen either between two as by S.O. M. Song D. Groesbeek M. G. Ziliox M. Aimoto S. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), between the and a backbone carbonyl The low affinity for the to both size and as the for the stability by the at the The of the a for this is by the that the with at the a in dimer affinity relative to In the a as a molecular that whether interaction (in this instance, take place at the and by the affinity of GXXXG-mediated Thus, three sequence present high TM-TM a central GXXXG a at the a and either a or at the The sequence seems to a scaffold used by TM helices to high-affinity and assembly between TM helices are controlled by a set of sequence the GXXXG motif is one of the most motifs in TM (7Senes A. Gerstein M. Engelman D.M. J. Mol. Biol. 2000; 296: 921-936Crossref PubMed Scopus (515) Google Scholar). Using the approach a dimer be to a dimer in a we were not only to the from each to dimer stability, but to understand how side with each other long to control interaction affinity. were by the of the model structure of the MCP-TM dimer to the GpA-TM The 12-residue of the a and positions along the TM helix that interactions a in the of TM domains in membrane Thus, the GXXXG Gly residues are determinants of interfacial it is the surrounding interfacial residues that control and the affinity and of TM helix-helix

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