A Heptad Motif of Leucine Residues Found in Membrane Proteins Can Drive Self-assembly of Artificial Transmembrane Segments

Specific interactions between α-helical transmembrane segments are important for folding and/or oligomerization of membrane proteins. Previously, we have shown that most transmembrane helix-helix interfaces of a set of crystallized membrane proteins are structurally equivalent to soluble leucine zipper interaction domains. To establish a simplified model of these membrane-spanning leucine zippers, we studied the homophilic interactions of artificial transmembrane segments using different experimental approaches. Importantly, an oligoleucine, but not an oligoalanine, se- quence efficiently self-assembled in membranes as well as in detergent solution. Self-assembly was maintained when a leucine zipper type of heptad motif consisting of leucine residues was grafted onto an alanine host sequence. Analysis of point mutants or of a random sequence confirmed that the heptad motif of leucines mediates self-recognition of our artificial transmembrane segments. Further, a data base search identified degenerate versions of this leucine motif within transmembrane segments of a variety of functionally different proteins. For several of these natural transmembrane segments, self-interaction was experimentally verified. These results support various lines of previously reported evidence where these transmembrane segments were implicated in the oligomeric assembly of the corresponding proteins. Specific interactions between α-helical transmembrane segments are important for folding and/or oligomerization of membrane proteins. Previously, we have shown that most transmembrane helix-helix interfaces of a set of crystallized membrane proteins are structurally equivalent to soluble leucine zipper interaction domains. To establish a simplified model of these membrane-spanning leucine zippers, we studied the homophilic interactions of artificial transmembrane segments using different experimental approaches. Importantly, an oligoleucine, but not an oligoalanine, se- quence efficiently self-assembled in membranes as well as in detergent solution. Self-assembly was maintained when a leucine zipper type of heptad motif consisting of leucine residues was grafted onto an alanine host sequence. Analysis of point mutants or of a random sequence confirmed that the heptad motif of leucines mediates self-recognition of our artificial transmembrane segments. Further, a data base search identified degenerate versions of this leucine motif within transmembrane segments of a variety of functionally different proteins. For several of these natural transmembrane segments, self-interaction was experimentally verified. These results support various lines of previously reported evidence where these transmembrane segments were implicated in the oligomeric assembly of the corresponding proteins. transmembrane segment 3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonic acid erythropoietin receptor maltose-binding protein Miller units In any type of cell, a multitude of integral membrane proteins is simultaneously synthesized and integrated into various membranes followed by association to homo- or heterooligomeric complexes. To ensure specific assembly, their subunits must present complementary recognition domains to each other. These domains may be located on the ectodomains and/or the transmembrane segments (TMSs).1 Interactions between TMSs are currently intensely studied, since they usually form autonomous α-helices and have been found to direct subunit assembly or support correct folding of many membrane proteins (1Lemmon M.A. Engelman D.M. Q. Rev. Biophys. 1994; 27: 157-218Crossref PubMed Scopus (177) Google Scholar, 2Dieckmann G.R. DeGrado W.F. Curr. Opin. Struct. Biol. 1997; 7: 486-494Crossref PubMed Scopus (50) Google Scholar). Biochemical and functional analyses, molecular modeling, and structural studies indicated that the self-assembly of transmembrane helices is driven by a close packing of their characteristically shaped surfaces. These packing interactions may result in pairs of α-helices with a right-handed twist as exemplified by glycophorin A (3Lemmon M.A. Flanagan J.M. Treutlein H.R. Zhang J. Engelman D.M. Biochemistry. 1992; 31: 12719-12725Crossref PubMed Scopus (467) Google Scholar, 4MacKenzie K.R. Prestegard J.H. Engelman D.M. Science. 1997; 276: 131-133Crossref PubMed Scopus (873) Google Scholar) and probably by synaptobrevin II (5Laage R. Langosch D. Eur. J. Biochem. 1997; 249: 540-546Crossref PubMed Scopus (73) Google Scholar). Other TMS interactions involve a leucine zipper type of side-chain packing as known from certain soluble proteins. Within soluble leucine zippers, the interacting residues form repeated heptad (abcdefg) motifs. Residues at a- and d-positions constitute the hydrophobic core of the interfaces; side-chains at the e- and g-positions are frequently charged, form salt bridges to each other, and make hydrophobic contacts to the core (6Lupas A. Trends Biochem. Sci. 1996; 21: 375-382Abstract Full Text PDF PubMed Scopus (1008) Google Scholar). Heptad motifs were also suggested to form the TMS interfaces of phospholamban (7Arkin I.T. Adams P.D. MacKenzie K.R. Lemmon M.A. Brünger A.T. Engelman D.M. EMBO J. 1994; 13: 4757-4764Crossref PubMed Scopus (174) Google Scholar, 8Simmerman H.K.B. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) and the M2 proton channel (9Pinto L.H. Dieckmann G.R. Gandhi C.S. Papworth C.G. Braman J. Shaugnessy M.A. Lear J.D. Lamb R.A. DeGrado W.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11301-11306Crossref PubMed Scopus (319) Google Scholar). Based on a quantitative evaluation of high resolution structures, we recently confirmed previous observations (10Rees D.C. Komiya H. Yeates T.O. Allen J.P. Feher G. Annu. Rev. Biochem. 1989; 58: 607-633Crossref PubMed Scopus (174) Google Scholar, 11Cohen C. Parry D.A.D. Proteins. 1990; 7: 1-15Crossref PubMed Scopus (666) Google Scholar) in demonstrating that TMSs primarily interact via a leucine zipper type of packing within bacteriorhodopsin, the photosynthetic reaction center, and cytochrome c oxidase. There, the heptads are repeated on average 2–3 times, and the motif gaxxdexgaxxdexga covers the central parts of the membrane-spanning interfaces. Salt bridges are absent due to the hydrophobic nature of most membrane-embedded residues (12Langosch D. Heringa J. Proteins. 1998; 31: 150-160Crossref PubMed Scopus (122) Google Scholar). To establish a simplified model of membrane-spanning leucine zipper domains, we designed artificial TMSs on the basis of leucine and alanine residues. We show that an oligoleucine sequence or agaxxdexgaxxdexga motif of leucine residues elicits specific self-assembly in membranes and in detergent solution. Interestingly, variants of this motif are found within the TMSs of a diverse set of natural membrane proteins, where they appear to be important for oligomeric assembly. Construction of plasmids pToxRΔTM and pSNiRΔTM was described previously (5Laage R. Langosch D. Eur. J. Biochem. 1997; 249: 540-546Crossref PubMed Scopus (73) Google Scholar, 13Brosig B. Langosch D. Protein Sci. 1998; 7: 1052-1056Crossref PubMed Scopus (196) Google Scholar). All other pToxR constructs were made by ligating synthetic oligonucleotide cassettes encoding the desired sequences into the plasmid pHKToxR(TMIl4)MalE (14Kolmar H. Hennecke F. Götze K. Janzer B. Vogt B. Mayer F. Fritz H.-J. EMBO J. 1995; 14: 3895-3904Crossref PubMed Scopus (64) Google Scholar) previously cut with NheI and BamHI. For the nuclease A fusions, the oligonucleotide cassettes were ligated into plasmids pSNiR (5Laage R. Langosch D. Eur. J. Biochem. 1997; 249: 540-546Crossref PubMed Scopus (73) Google Scholar) or pSNiR2 previously cut withNheI and BamHI. Details on the pSNiR and pSNiR2 plasmids will be described elsewhere. All constructs were verified by dideoxy sequencing. Transcription activation was determined upon expression of the pToxR constructs in the indicator strain FHK12 as described (15Langosch D.L. Brosig B. Kolmar H. Fritz H.-J. J. Mol. Biol. 1996; 263: 525-530Crossref PubMed Scopus (220) Google Scholar). 0.4 mm isopropyl 1-thio-β-d-galactopyranoside was added to the cultures to enhance the dynamic range of the produced β-galactosidase signals (in Miller units (MU), means ± S.D.) elicited by the different constructs in several independent experiments. This effect is thought to result from isopropyl 1-thio-β-d-galactopyranoside-induced expression of an F′-plasmid-encoded truncated β-galactosidase, which competes with full-length enzyme in the formation of functional tetramers. The previously (15Langosch D.L. Brosig B. Kolmar H. Fritz H.-J. J. Mol. Biol. 1996; 263: 525-530Crossref PubMed Scopus (220) Google Scholar) described construct pToxR/GPA13 elicited 1240 ± 298 MU under these conditions. pSNiR and pSNiR2 fusion proteins were expressed in BL21(DE3)pLysS cells (Novagen), solubilized in 25 mm HEPES, pH 7.9, 0.5 m NaCl, 2% CHAPS, 1 mm EDTA and quantitated as described (5Laage R. Langosch D. Eur. J. Biochem. 1997; 249: 540-546Crossref PubMed Scopus (73) Google Scholar). Volumes of 300 μl at concentrations of 4 or 20 μm fusion protein were separated on a Superdex 200HR 10/30 column (Amersham Pharmacia Biotech FPLC system) using a flow rate of 0.5 ml/min and 25 mmHEPES, pH 7.9, 0.5 m NaCl, 1% CHAPS, 1 mm EDTA as running buffer. Fractions of 0.5 ml were collected and analyzed for fusion protein with a dot blot procedure (16Becker C.-M. Hoch W. Betz H. J. Neurochem. 1989; 53: 124-131Crossref PubMed Scopus (47) Google Scholar) using the 9E10 monoclonal antibody directed against the c-myc marker epitope for detection. The elution profiles were constructed from the antigen content, and the apparent molecular weights were calculated with reference to standards given in the legend to Fig. 3. The Swiss-Prot data base (release 35.0) was searched with the LLXXLLXLLXXLLXLL motif using the Findpatterns option of the HUSAR sequence analysis package made available by the German Cancer Research Center (Heidelberg). Up to three mismatches were allowed. To selectively retrieve TMSs, any amino acid except the charged residues lysine, arginine, glutamate, aspartate, or the helix-breaker proline was allowed for those positions not occupied by leucine. Western blotting was done as described with an antiserum recognizing the maltose-binding protein (MalE) moiety of the constructs, and the bands were quantitated densitometrically (13Brosig B. Langosch D. Protein Sci. 1998; 7: 1052-1056Crossref PubMed Scopus (196) Google Scholar, 15Langosch D.L. Brosig B. Kolmar H. Fritz H.-J. J. Mol. Biol. 1996; 263: 525-530Crossref PubMed Scopus (220) Google Scholar). The ability of our constructs to complement the MalE deficiency of PD28 cells was tested by measuring the cell densities of transformed bacteria in minimal medium containing maltose at 640 nm after different growth periods (13Brosig B. Langosch D. Protein Sci. 1998; 7: 1052-1056Crossref PubMed Scopus (196) Google Scholar). NaOH extraction was done as described (17Chen H. Kendall D.A. J. Biol. Chem. 1995; 270: 14115-14122Crossref PubMed Scopus (68) Google Scholar) by vortexing whole bacteria with cold 0.1m NaOH followed by centrifugation to separate soluble from membrane-bound proteins. Leucine is the most prevalent amino acid within the interface of leucine zippers (18Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3469) Google Scholar), which is probably related to its ability to adopt multiple conformations (19Schrauber H. Eisenhaber F. Argos P. J. Mol. Biol. 1993; 230: 592-612Crossref PubMed Scopus (204) Google Scholar). We therefore reasoned that the flexible leucine side chain may be particularly well suited to form a well packed membrane-spanning leucine zipper. The methyl side chain of alanine, in contrast, is expected to be too small for efficient interaction with other alanine residues. This prediction was tested by comparing the self-association of oligoleucine and oligoalanine sequences, which are known to form stable α-helices (20Zhang Y.-P. Lewis R.N.A.H. Hodges R.S. McElhaney R.N. Biochemistry. 1992; 31: 11579-11588Crossref PubMed Scopus (157) Google Scholar, 21Marqusee S. Robbins V.H. Baldwin R.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5286-5290Crossref PubMed Scopus (735) Google Scholar). One of the experimental approaches we is on an of the This protein is by a TMS of within the membrane of where is thought to in a The form to the expression of a in a strain H. Hennecke F. Götze K. Janzer B. Vogt B. Mayer F. Fritz H.-J. EMBO J. 1995; 14: 3895-3904Crossref PubMed Scopus (64) Google Scholar). expression is therefore of self-assembly in the We previously this as a to TMS interactions using the structurally well glycophorin A TMS for reference (13Brosig B. Langosch D. Protein Sci. 1998; 7: 1052-1056Crossref PubMed Scopus (196) Google Scholar, 15Langosch D.L. Brosig B. Kolmar H. Fritz H.-J. J. Mol. Biol. 1996; 263: 525-530Crossref PubMed Scopus (220) Google Scholar). we found that a sequence of leucine residues elicited activation ± ± In contrast, a sequence of alanine residues elicited a ± A and This that the oligoleucine sequence in the the oligoalanine sequence the be as host for a leucine zipper Based on motif the central parts of most transmembrane helix-helix interfaces within crystallized membrane proteins (12Langosch D. Heringa J. Proteins. 1998; 31: 150-160Crossref PubMed Scopus (122) Google Scholar), a simplified of a membrane-spanning leucine zipper interaction was In this and positions are occupied by leucine and by The construct with this sequence to a ± as the protein To that the leucine residues within constitute the helix-helix we of to alanine and the for of the made the not when a or and positions were simultaneously the by ± or ± the leucine residues are for the interaction most make the Further, to be of for helix-helix a into the of the sequence the interaction ± with the known of α-helices by J. 1992; PubMed Scopus Google Scholar) and their by proline G. J. Mol. Biol. 1991; PubMed Scopus Google Scholar) residues. We also the leucines of by three different random sequences consisting of the most residues found within TMSs B. Argos P. J. Mol. Biol. 1994; PubMed Scopus Google Scholar) and side-chain and Scholar). with these random sequences also self-assembled the of the leucine side chain for helix-helix packing ± Fig. A and and data not The in of the mutants with are the concentrations of our constructs by Western blot analysis indicated that most of were expressed at was for the construct we the cells with NaOH to separate membrane proteins from soluble proteins (17Chen H. Kendall D.A. J. Biol. Chem. 1995; 270: 14115-14122Crossref PubMed Scopus (68) Google Scholar), constructs with the membranes as expected except which be a of the protein to in a soluble which is probably due to the of the oligoalanine sequence. This is thought not to with the To correct of the proteins into the we tested their ability to functionally complement the MalE deficiency of PD28 to a MalE this strain is to in minimal medium with maltose as the H. P. Eur. J. Biochem. PubMed Scopus Google Scholar). In cells membrane proteins with the moiety the and the MalE to the Fig. the MalE maltose and cell growth (13Brosig B. Langosch D. Protein Sci. 1998; 7: 1052-1056Crossref PubMed Scopus (196) Google Scholar, H. Hennecke F. Götze K. Janzer B. Vogt B. Mayer F. Fritz H.-J. EMBO J. 1995; 14: 3895-3904Crossref PubMed Scopus (64) Google Scholar). expression of constructs the MalE deficiency of PD28 cells to In contrast, a construct where the TMS is to support cell growth as expected from its In equivalent of proteins analyzed for self-assembly appear to be integrated into the and the β-galactosidase be To self-assembly of our artificial TMSs by an independent their oligomeric were in detergent The and sequence segments were to the of a fusion moiety nuclease a soluble The fusion proteins were in solubilized with CHAPS, and to at concentrations of 4 or 20 at 20 and fusion proteins as with apparent molecular of at 4 the were in of the between of the proteins not the assembly to is currently not the most that may at apparent molecular weights due to detergent molecular In to the and constructs to at and at These are with and may be by different of detergent on the and the of the hydrophobic segments. independent experimental approaches that the oligoleucine sequence and the model leucine zipper motif in a in membranes as well as in detergent solution. the self-assembly of the we TMSs with leucine in proteins. The Swiss-Prot data base was searched for hydrophobic sequence segments with the motif LLXXLLXLLXXLLXLL for to three This search sequences, TMSs within membrane proteins, and TMSs from membrane proteins when proteins from different were the sequences and TMSs of proteins were not the TMS sequences corresponding to the proteins are shown in of a was with the The TMS the was from the erythropoietin receptor followed by the TMSs of the and of Other TMSs corresponding to receptor and receptor to of self-assembly 4 A and A Western blot for expression 4 motifs of leucine residues in natural sequence are as protein from erythropoietin from receptor recognition receptor protein from from those parts of the TMSs that the The sequence positions of the residues are and leucine residues within the heptad given the sequences are in as determined with the ± ± not The Swiss-Prot proteins sequences the search The proteins with the search ± ± ± ± ± ± Swiss-Prot sequence are as protein from erythropoietin from receptor recognition receptor protein from from those parts of the TMSs that the The sequence positions of the residues are and leucine residues within the heptad given the sequences are in as determined with the ± not The Swiss-Prot proteins sequences the search The proteins with the search in a The data that these TMSs are important for oligomerization of the corresponding proteins. A of previously reported experimental evidence and our indicated this to be the for several of these proteins or related as We that an artificial TMS of leucine residues efficiently in membranes and in detergent solution. A heptad motif of leucine residues to which therefore is thought to be driven by the type of side-chain packing known from leucine zipper interaction domains. The of our results are establish a simplified model of membrane-spanning leucine that interaction domains may a in recognition of certain natural membrane proteins. We that the and TMSs form α-helical upon Self-assembly is thought to involve that which each other via a type of side-chain packing of leucine zippers (6Lupas A. Trends Biochem. Sci. 1996; 21: 375-382Abstract Full Text PDF PubMed Scopus (1008) Google Scholar). The flexible leucine side chain (19Schrauber H. Eisenhaber F. Argos P. J. Mol. Biol. 1993; 230: 592-612Crossref PubMed Scopus (204) Google Scholar) may be particularly well suited for this type of packing with this heptad motifs have previously been in the of transmembrane J.D. DeGrado W.F. Science. PubMed Scopus (467) Google Scholar) or of a membrane protein P. G. Struct. Biol. 1994; PubMed Scopus Google Scholar). the other leucine helices have frequently been as experimental to TMS interactions with For of these studies (20Zhang Y.-P. Lewis R.N.A.H. Hodges R.S. McElhaney R.N. Biochemistry. 1992; 31: 11579-11588Crossref PubMed Scopus (157) Google Scholar, M. Biophys. J. 1992; Full Text PDF PubMed Scopus Google Scholar, J.M. Biochemistry. 1998; PubMed Scopus Google Scholar, Lewis R. McElhaney R.N. Hodges R.S. A. Biochemistry. 1998; PubMed Scopus Google Scholar), of the leucine helices were with residues interaction may in a Lewis R. McElhaney R.N. Hodges R.S. A. Biochemistry. 1998; PubMed Scopus Google Scholar). In other (17Chen H. Kendall D.A. J. Biol. 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