Tryptophan Supports Interaction of Transmembrane Helices

doi:10.1016/j.jmb.2005.09.084

J. Mol. Biol. (2005) 354, 894–902

Tryptophan Supports Interaction of Transmembrane Helices Anja Ridder, Paulina Skupjen, Stephanie Unterreitmeier and Dieter Langosch* Technische Universita¨t Mu¨nchen, Lehrstuhl fu¨r Chemie der Biopolymere Weihenstephaner Berg 3, D-85354 Freising-Weihenstephan Germany

Interactions of transmembrane helices play an important role in folding and oligomerisation of integral membrane proteins. The interfacial residues of these helices frequently correspond to heptad repeat motifs. In order to uncover novel mechanisms underlying these interactions, we randomised a heptad repeat pattern with a complete set of amino acids. Those sequences that were capable of high-affinity self-interaction upon integration into bacterial inner membranes were selected by means of the POSSYCCAT system. A comparison between selected and non-selected sequences reveals that high-affinity sequences were strongly enriched in tryptophan residues that accumulated at specific positions of the heptad motif. Mutation of Trp in selected clones significantly reduced selfinteraction of the transmembrane segments without affecting their efficiency of membrane integration. Conversely, grafting Trp onto artificial transmembrane segments strongly enhanced their interaction. We conclude that tryptophan supports interaction of transmembrane segments. q 2005 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: tryptophan; transmembrane segment; interaction; ToxR; POSSYCCAT

Introduction Many membrane proteins have several a-helical transmembrane segments (TMSs) that associate with each other to form the finally folded, functional protein. In addition, many integral membrane proteins fulfill their function as part of oligomeric complexes that frequently assemble by sequence-specific TMS–TMS interactions. Thus, interactions between TMSs are of prime importance for membrane protein assembly and function. Selfinteraction of a-helical TMSs has been found to depend on multiple van der Waals’ interactions between their well-packed surfaces.1–3 Also, hydrogen bonding4–7 or charge–charge interactions8 can Present address: A. Ridder, Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands. Abbreviations used: CM, chloramphenicol; b-gal, b-galactosidase; CAT, chloramphenicol acetyltransferase; MalE, maltose binding protein; MU, Miller units; TMS, transmembrane segment; POSSYCCAT, positive selection system based on chromosomally integrated CAT. E-mail address of the corresponding author: [email protected]

drive TMS oligomerisation. For maximal packing, pairs of interacting helices favour either a positive or a negative crossing angle.9,10 The interfaces of helix–helix pairs with positive crossing angles, i.e. left-handed pairs, are characterised by a heptad repeat pattern of amino acids ([a..de.g]n), which is known from soluble leucine zipper interaction domains10 (Figure 1). A de novo designed hydrophobic heptad repeat motif, denoted AZ2, where Leu residues occupy the interfacial a, d, e and g positions, does indeed self-assemble and was therefore proposed to represent an artificial membranespanning leucine zipper.11 A very efficient way of investigating the sequence dependence of TMS–TMS interactions is to randomise canonical interfacial amino acid motifs and select self-interacting TMSs from the corresponding combinatorial libraries. For selection, genetic tools based on the membrane-spanning ToxR transcription activator such as POSSYCCAT12 or TOXCAT13 are in use. Previously, the heptad repeat pattern characteristic of left-handed helix–helix pairs was randomised with limited sets of amino acids. Selection yielded TMSs most of which self-interacted with medium affinity and accumulated aliphatic sidechains relative to low-affinity unselected

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

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Tryptophan Mediates TMS–TMS Interactions

Figure 1. Design of the library. (a) The interfacial a, d, e and g positions, indicated by X, of the heptad repeat pattern were randomised with all naturally occuring amino acid types. Invariant b, c and f positions, represented by dots, are occupied by Ala. Lower case letters denote flanking sequences of the ToxR protein. The position numbers of the randomised positions are shown above the sequence, and the corresponding heptad repeat positions are shown below it. (b) Helical wheel representation of a leucine zipper illustrating the order of the different heptad positions.

sequences.12 In contrast, randomisation of the righthanded glycophorin A TMS–TMS interface with limited sets of residues mainly yielded high-affinity sequences that contained a GxxxG motif.13 This motif constitutes the most critical part of the interface of the dimeric glycophorin A TMS.14–16 Excluding Gly from this randomised pattern resulted in enrichment of Ser, Thr and Pro residues.17 Here, we extend these previous approaches by randomising the heptad repeat pattern with a complete set of amino acids. Interestingly, we find that Trp is now enriched within high-affinity sequences in a position-specific manner. Mutational analysis shows that Trp is indeed responsible for TMS–TMS assembly.

Results Library design and selection of self-interacting TMSs The goal of this study was to uncover novel mechanisms underlying TMS–TMS interactions. To this end, we randomised the a, d, e and g positions of a heptad repeat motif with every naturally occurring amino acid (Figure 1). Thereby, we avoided the intrinsic limitations of previous randomisation strategies that employed only limited sets of residues.13,17,12 Ala was chosen as the invariant residue at b, c and f positions since an oligo-alanine sequence does not self-interact to a significant degree.11 A library of 1.2!107 independent clones was generated via PCR with a partly degenerate primer and re-ligation of the PCR product into the pToxRIV

vector.12 The distribution of codons at the randomised positions within the library closely matched the theoretical distribution as shown by sequencing the randomised parts from 26 randomly chosen plasmids (data not shown). Self-interacting TMSs were selected from the library using the POSSYCCAT system. This system makes use of a chimeric protein consisting of the cytoplasmic ToxR transcription activator domain that is connected by any given TMS to the periplasmic maltose binding protein (MalE) domain. Depending on the capacity of a particular TMS to self-interact, the chimeric protein selfassembles in the inner membrane of expressing Escherichia coli cells18,16 and thus activates the cholera toxin (ctx) promoter via its ToxR moiety. In the Chr3 strain, the ctx promoter drives expression of the chloramphenicol acetyltransferase (cat) gene. The resulting degree of chloramphenicol (Cm) resistance of the cell is thus a measure for TMS–TMS affinity. By growing the bacteria in the presence of Cm, one can therefore select for selfinteracting sequences in vivo.12 The library was transformed into the Chr3 selection strain and plated on medium that contained the inducer arabinose and Cm. As expected, increasing the Cm concentration in the plates resulted in a decreasing number of surviving clones. At the highest Cm concentration used (180 mg/ml) only around 300 clones survived, corresponding to w0.0025% of the original library. Under these conditions, Chr3 cells expressing the parental membrane-spanning leucine zipper AZ2 are also able to survive, albeit only at low probability. We expected that a significant fraction of the random sequences would not integrate into the membrane due to the presence of too many polar amino acids. To eliminate these sequences, 78 selected plasmids were individually transformed into the MalE deletion strain PD28. Transformed PD28 cells can utilise maltose only when the chimeric ToxR protein is integrated into the inner membrane with its MalE domain exposed to the periplasm. A total of 55 clones were able to complement the MalE deficiency of PD28 (see Materials and Methods) and only these were included in further analyses. A quantitative measure for self-interaction of the selected clones was obtained by expressing them in the E. coli FHK12 strain that has a lacZ reporter gene fused to the ctx promoter. The resulting b-galactosidase activity thus reflects the mutual affinity of the respective TMS. For all selected clones, the measured b-galactosidase activity was comparable to AZ2 or higher (Table 1). Tryptophan is enriched in high-affinity sequences The TMSs of the 54 selected and membraneintegrated ToxR proteins were sequenced and compared to those of 22 unselected clones that

896

Tryptophan Mediates TMS–TMS Interactions

Sequence

Relative activity

C41 C7 C9 C27 C67 C21 C10 C26 C3 C8 C2 C76 C47 C29 C23 C31 C103 C19 C52 C15 C56 C5 C85 C70 C87 C48 C106 C95 C50 C28 C25 C42 C34 C63 C104 C100 C75 C65 C24 C80 C6 C57 C38 C97 C94 C33 C43 C44 C60 C64 C30 C14 C73 C49 C55

LI..LF.TD..ML.FV EW..WS.FL..HL.LF NL..CV.FG..LL.WA WA..HL.LL..LL.WG CQ..ML.WS..LI.WY MV..FS.FY..GL.TM WA..IV.WL..VF.LI SQ..LL.LS..LG.LL VL..ST.WW..VF.WS VT..VV.LV..IG.YL EA..MC.SL..IV.WA VF..LG.WT..LV.LI YS..VV.VV..FS.WL MV..VC.WF..SS.WL LV..FL.GV..GL.NV CW..VV.IS..FV.WL AV..LW.SV..NV.LL IL..CV.TC..VL.WL IV..IL.FM..LP.WW LF..SV.VG..LG.LV EW..VL.VM..LT.WL MW..LV.WA..MV.WI VM..TW.LG..MG.LT LL..GC.IV..IL.WP WA..LI.WV..FF.WI VL..MT.WV..GV.HL NG..WL.VM..TS.WI YM..VG.WL..IL.AV FC..AA.MA..VL.WV WI..IL.YN..VL.WL YL..LL.WC..LF.HV TL..VG.WL..VF.FI LV..LI.II..VL.WL HM..GL.CL..LF.WS LI..AL.CS..VL.WM LA..VF.GV..LG.WL WM..VV.CL..LV.WY MV..AT.VS..II.WA LV..IV.WN..IL.WV VL..HC.LS..IS.FV FI..FA.WG..ML.WW LM..VI.NM..WP.LL IL..LL.WG..FY.WL LL..IF.WL..ML.WW WV..FL.FL..SV.WL EY..FV.IM..VW.WV FL..LF.WG..LC.WS VA..LL.WL..TT.WV IA..CV.TS..LV.WV LV..FF.GI..TS.LM FM..AI.WF..IA.WS WC..AS.WL..SA.WY WC..FV.IL..VL.WF WV..VI.WL..FW.NV WL..GV.MF..LL.WV

0.6 0.9 1.1 1.1 1.1 1.2 1.4 1.4 1.4 1.4 1.6 1.7 1.8 1.9 1.9 2.0 2.0 2.0 2.1 2.2 2.2 2.2 2.3 2.3 2.3 2.3 2.3 2.3 2.3 2.3 2.3 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.5 2.5 2.6 2.6 2.7 2.7 2.8 2.9 2.9 2.9 2.9 3.1 3.2 3.2 3.6 3.6 3.6

Invariant Ala residues are represented by dots and Trp residues at positions 1, 8 and 15 are shown in bold. All clones were capable of membrane integration. The ToxR activities of these sequences are expressed relative to AZ2 (Z1).

were also membrane-integrated. These unselected reference sequences were obtained by a selection strategy based on MalE complementation as detailed in Materials and Methods. By dividing the frequency of occurrence of each residue type in selected sequences by its frequency in unselected ones we obtained the normalised frequencies of occurrence given in Figure 2(a). Remarkably, Trp

Normalised frequency of occurance

Clone

(a) 5 4 3 2 1 0 A C D E F

G H

I

K

L

M N P Q R S T

V W Y

Amino acid (b) 35 30 25

number

Table 1. Selected transmembrane sequences and their relative ToxR activities

20 15 10 5 0

A C D E F G H I K L M N P Q R S T V W Y 12

amino acid

8 56

16 1315 912

n

itio

pos

Figure 2. Analysis of selected sequences. (a) Overall enrichment of amino acids in TMS sequences that survived positive selection on Cm-containing media (180 mg/ml) relative to unselected clones. The values were obtained by dividing the frequency at which a particular amino acid was found in selected clones (nZ55) by the respective frequency in unselected clones (nZ22). A normalised frequency of occurance of 1 thus means that the respective amino acid is neither enriched nor depleted by the selection procedure. The numbers used here are given in Supplementary Table 2A. (b) Positional specificity of amino acids in selected clones as obtained by comparing their absolute numbers found at each randomised position. The numbers used here are given in Supplementary Table 2B. All sequences evaluated here were able to complement MalE deficiency of PD28 cells, indicating efficient membrane integration.

residues are four times more abundant in selected sequences, suggesting that Trp favours interaction of TMSs. Of the 55 TMSs shown in Table 1, 46 (or 85%) contain one or more Trp residues. Further, selfinteracting and membrane-integrated TMSs appear to be depleted for Lys, Pro, Gln and Arg residues. As these polar amino acids are quite rare in the membrane-integrated sequences (Supplementary Table 2A), the statistical significance of this latter finding is not clear, however. Next, we evaluated the frequency of occurrence of amino acids at each randomised heptad position of self-interacting TMSs. Most amino acids are rather equally distributed over the entire length of the TMS. The apparent abundance of Leu and Val is apparently due to overrepresentation of the respective codons (due to the library design) in the random sequences and is therefore not seen in the normalised representation shown in Figure 2(a).

Tryptophan Mediates TMS–TMS Interactions

897

Interestingly, however, Trp residues are rather frequent at the first position and dominate at the eighth and 15th position. These sites correspond to position g of the heptad repeat pattern. This positional specificity of Trp is very high, since 78% of the selected clones have a Trp at one or more of these positions (ten at position 1, 20 at position 8, 36 at position 15) (Table 1). Of the remaining sequences, three (C15, C23, C85) contain a GxxxG motif within the randomised part. Two additional ones (C8, C26) contain a GxxxG motif when the invariant Gly residue that follows the randomised part is also considered (see Figure 1); sequence C26 has a SxxxG motif in addition. Sequence C64 has an SxxxG motif and sequence C80 has an SxxxSxxxG motif. These are motifs that have previously been selected from randomised TMS libraries that were, however, based on the right-handed dimerisation motif of glycophorin A.13 Tryptophan mediates interaction of selected and artificial TMSs To confirm the role of Trp in self-interaction, two TMSs containing Trp at g positions were subjected to site-directed mutagenesis (Figure 3(a)). Selfinteraction of the mutants was compared to the respective parental TMS, to AZ2 and to a ToxR protein were the TMS had been deleted (ToxRDTM). When the single Trp at position 15 of the strongly self-interacting clone C65 was replaced by Ala or Leu, ToxR activity dropped to the level of AZ2, corroborating that Trp is indeed responsible for the efficient self-interaction of C65 (Figure 3(b)). Similar effects were seen upon mutation of Trp15 to Val, Cys, Ser or Gly (data not shown). Clone C75 contains two Trp residues at positions 1 and 15. Trp at position 15 seems to be primarily responsible for self-interaction of this clone, since mutation of this residue to Ala decreases affinity more strongly than mutation of Trp at position 1 (Figure 3(b)). These results are in agreement with the positional specificity found in all selected clones where position 1 displays only a modest number of Trp, while most clones have a Trp at position 15 (Figure 2(b) and Table 1). It is known that Trp residues that flank TMSs are able to interact with the lipid headgroup region of the membrane and thereby anchor membrane proteins within the lipid bilayer.19–22 In principle, the membrane anchoring capacity of Trp could contribute to ToxR activity by increasing the concentration of ToxR proteins in the bacterial membrane. To exclude this possibility, we first ascertained that the concentrations of ToxR proteins in the cells were similar for the parental AZ2 TMS, the Trp-containing sequences (C65 and C75) and their mutants by Western blot analysis of bacterial lysates (Figure 3(b)). Second, we compared the efficiencies of membrane integration of these sequences by the PD28 growth assay. Since all mutants were able to utilise maltose at least as well as the parental clones, this experiment

Figure 3. Mutational analysis of selected clones. (a) Amino acid sequences of two selected clones and point mutants derived thereof. The mutated Trp residues are shown in bold and their position numbers are indicated above the sequences. (b) Self-interaction of selected sequences and their mutants as determined by ToxR activity assays. Data represent average values of bgalactosidase activity (in MU) normalised to AZ2 (Z100%). nZ12–40 data points, meansGSD. Western blots of FHK12 cells, that were probed with anti-MalE antibody, are shown below. (c) PD28 growth assay of C65, C75 and their mutants as compared to AZ2 and DTM. PD28 containing the indicated clones were grown in minimal medium containing maltose as the only carbon source. The A650 after 48 h of growth (the averages of a duplicate experiment) indicates similar levels of membrane-integration of AZ2, C65 and C75 and that mutating Trp does not diminish integration. ToxRDTM does not integrate into the membrane and was therefore used as negative control.

demonstrates that removing the Trp residues does not decrease the ability of the sequences to integrate into the membrane (Figure 3(c)). Third, replacement of the Trp in C65 by Tyr, a residue that is also able to

898 strongly interact with the membrane–water interface,19 resulted in a decrease in ToxR activity that was just as strong as when it was replaced with Ala or Leu (Figure 3(b)). Taken together, these results indicate that the effect of Trp is not due to improved membrane integration. To examine whether Trp can enhance interaction of a given TMS, sequences were designed to contain multiple Trp residues at the different heptad positions of AZ2 (Figure 4(a)). When Trp residues were placed at the a or e positions, self-interaction was not affected (Figure 4(b)). At position d, they actually decreased ToxR activity; this is apparently due to a strongly reduced expression level that is shown by Western blot analysis and has unknown reasons. However, when placed at g positions, Trp increased ToxR activity about threefold. This shows again that the effect of Trp residues is highly position-specific and is consistent with the observed enrichment of this amino acid at g positions in the clones selected from the library. Finally, we investigated whether the effect of Trp depends on sequence context by placing it at g positions in a TMS solely composed of Leu residues, L16.11 In this background, Trp also increased ToxR activity, albeit only about twofold, i.e. to a lesser extent than in AZ2 (Figure 4(b)). This result suggests

Figure 4. Effect of Trp on self-interaction of artificial TMSs. (a) Amino acid sequences of Trp mutants of the membrane-spanning leucine zipper AZ2 and of L16. (b) Self-interaction of Trp mutants as determined by ToxR activity assays are compared to those of ToxRDTM, AZ2 and C65. Data represent average values of b-galactosidase activity (in MU) normalised to AZ2 (Z100%) (meansGSD, nZ12–40 data points). Western blots of FHK12 cells expressing these clones are shown for control of expression levels.

Tryptophan Mediates TMS–TMS Interactions

that the effect of Trp residues on self-interaction of TMSs may not only depend on position, but also on the nature of the surrounding residues.

Discussion Our results reveal that Trp can strongly support self-assembly of TMSs. This conclusion is based on a striking enrichment of Trp in sequences that were selected for high-affinity self-interaction in bacterial membranes. The importance of Trp for TMS–TMS interaction is confirmed by the observed drastic reductions of affinity upon mutating it to other residues. Previous attempts to select high-affinity TMSs from random libraries used restricted sets of codons that excluded this residue 13,12,17 and thus did not detect the importance of Trp. Trp and Tyr residues were previously shown to have a high affinity for the membrane–water interface and are therefore enriched at the termini of natural TMSs.23,19,20 These residues do not affect the efficiency of membrane insertion, but rather stabilise a TMS in the membrane through interactions with lipid headgroups.21,24,22 In principle, the level of self-interaction of a membranespanning ToxR protein does not only depend on the mutual affinity of its TMS but, according to the law of mass action, also on its concentration in the membrane. Therefore, one possible explanation of the effect of Trp could be that it increases the amount of membrane-inserted ToxR proteins. However, there are several observations that argue against this possibility. First, mutating Trp in those selected sequences that were investigated in detail (C65, C75) diminished ToxR activity without affecting the efficiency of membrane insertion. Second, in C65, the mutation of Trp to Tyr, a residue that is also able to strongly interact with the membrane–water interface,19 reduced ToxR activity as efficiently as other exchanges. Third, five out of 55 selected sequences only had a Trp at the central g position, whereas location at a helix terminus would be required for an anchoring function. Moreover, Trp is almost exclusively found at g positions of the selected heptad patterns and increases self-interaction of the AZ2 sequence only when occupying g positions. This points at a function that is more specific than mere membrane anchoring. It also demonstrates that the ability of Trp to drive TMS–TMS interactions is highly dependent on its precise location within the leucine zipper interface. Apart from those sequences containing Trp, we also isolated a number of TMSs whose selfinteraction appears to be mediated by other amino acid patterns. Among these, seven TMSs contained GxxxG, SxxxG, GxxxS and/or SxxxS motifs. The GxxxG motif is well known to drive self-interaction of the glycophorin A TMS14–16,18 and of other TMSs.25–28 Previous randomisation of the glycophorin A TMS–TMS interface followed by selection produced high-affinity isolates that contained

Tryptophan Mediates TMS–TMS Interactions

mostly GxxxG, but also SxxxG, GxxxS, SxxxS and other motifs.13 Several mechanisms have been proposed by which a GxxxG motif may drive TMS–TMS interaction. These are probably not mutually exclusive and include formation of a flat helix surface, reduced loss of side-chain entropy upon association13 as well as hydrogen bond formation between their Ca-hydrogen atoms and the backbone of the partner helix.29,7 The glycophorin A TMSs form a right-handed helix–helix pair 15 and it is thus likely that these (small)xxx(small) motifs30 are specific for the corresponding interfaces. Although our randomised heptad repeat pattern is designed to form lefthanded TMS–TMS assemblies, it is possible that the presence of a GxxxG motif produces right-handed pairs. On the other hand, a heptad repeat motif where the a and d positions are occupied by Gly (also resulting in a GxxxG motif) has recently also been shown to self-associate; it is not clear, however, whether this designed sequence still forms a left-handed pair.31 Surprisingly, we did not observe enrichment of Asn or Gln residues in high-affinity sequences. These amino acids had previously been shown to stabilise TMS–TMS interactions by virtue of hydrogen-bond formation between their carboxamide side-chains.6,4,32 The reason for non-enrichment of these amino acids is not clear. It may be due to the fact that Asn, Gln and other polar residues are quite rare in the membrane-integrated sequences (Supplementary Table). Moreover, Asn and Gln function in TMS–TMS interaction has been reported to be modulated by sequence context.33 To our knowledge, direct evidence for a function of Trp in TMS–TMS interaction has not been reported before. On the other hand, a prominent role of Trp in protein–protein interactions is well established. Previously, statistical analysis of known structures of interfaces between soluble proteins has revealed that Trp is one of the most prevalent amino acids at “hot-spots”, i.e. those sites that critically determine interaction strength.34 In addition, there is circumstantial evidence for a role of Trp in folding of multi-spanning membrane proteins. For example, Trp is the amino acid that displays the highest relative abundance in the interface of membrane-spanning leucine zippers.10 Statistical analyses of TMSs also suggest that Trp is generally buried within their interfaces35 and exhibits a correlated distribution six amino acid residues apart, i.e. at heptad repeat distance.36 Further, Trp has the highest propensity of all amino acids to participate in interhelical contacts in soluble and membrane-spanning proteins.37 The latter analyses focussed on TMS–TMS packing in multi-spanning membrane proteins and did not include homotypic TMS–TMS interactions. Interestingly, an analysis of polytopic membrane protein structures recently revealed that Trp residues located in the hydrocarbon region of the membrane are as frequently exposed to lipids as to protein. The authors argue that the large hydrophobic surface of

899 Trp might allow for extensive interactions with side-chains from the neighbouring helices on the lipid-facing surfaces of the helical bundle structures. Such interactions are indeed often found between parallel helices that do not have an extensive interface. This is exemplified by the structure of subunit one of cytochrome c oxidase from Thermus thermophilus (PDB: 1EHK) where two Trp residues are exposed to the hydrocarbon phase, yet also pack against residues from neighbouring helices, thus stabilising the folded structure (Trp110 interacts with Tyr23 and Leu27; Trp157 interacts with Ser197). Another example comes from the cytochrome bc1 complex (PDB:1KB9) from Saccharomyces cerevisiae where Trp69 interacts with residues (Met351, Ile354, Ile358, Leu335) from cytochrome b (see Adamian et al.38). These results are interesting in light of our current results, which localise most enriched Trp at g positions, i.e. also at the boundary between helix–helix and helix–lipid interfaces. The importance of Trp for protein–protein interaction has been attributed to its multi-functional sidechain. Its indole-ring can form aromatic p-interactions to other aromatic ring systems, to positively charged side-chains and/or to adjacent C–H or N–H groups,36 whereas its indole amide can function as hydrogen-bond donor.39 For example, direct stacking interactions between cross-strand Trp side-chains were shown to stabilise b-hairpin structures.40 Recently, a soluble “Trp-zipper” has been designed where Trp is located at the a and d positions of a heptad repeat pattern. This protein formed a stable pentamer whose stability depended on Trp–Trp interactions within the core of the helix–helix interface while the potential effect of Trp at the peripheral e and g positions was not investigated.41 That Trp mediates interactions of soluble helices at a and d positions contrasts our present finding that locates Trp at g positions of membrane-embedded heptad motifs. One may wonder whether this apparent discrepancy is related to the different polarities of the respective environments. On the other hand, the structures of our TMS assemblies are currently not known. Therefore, it is possible in principle that the TMS–TMS interfaces are built from both randomised and invariant residue positions. In other words, we can presently not exclude that the enriched Trp residues occupy a specific position in the interfaces that does not correspond to the g position. Presently, we can only speculate on the precise mechanism by which Trp mediates TMS–TMS interactions. Trp may mediate its effect by selfinteraction of its side-chain within the helix–helix interface. In this case, the helices would have to move away from each other such that the indole could reach from the g position into the interface. Alternatively, the Trp side-chain may interact with other side-chains and/or the backbone of the partner helix. It is noteworthy that Trp residues grafted onto the oligo-Leu sequence had a smaller effect than within AZ2 that contains invariant Ala residues. This suggests that the function of Trp may depend on sequence context and involve other

900 residues. Future studies of peptide models will clarify the precise mechanism by which Trp stabilises membrane-spanning zipper domains.

Materials and Methods Library construction A library of ToxR chimeric proteins with a randomised heptad repeat motif in place of its TMS (see Figure 1) was prepared as follows. A PCR was performed on pToxRIVAZ2 12 with the following 5 0 -biotinylated primers (ThermoHybaid): 5 0 -GG AAT CGA ACT AGT NNS NNS GCA GCT NNS NNS GCA NNS NNS GCA GCT NNS NNS GCA NNS NNS GGG ATC CTG ATC AAC CCA AG -3 0 (sense) and 5 0 -GTT GTA ACG TAC GGC ATC CCA GG-3 0 (antisense), where N is an equimolar mixture of G/A/T/C, S is an equimolar mixture of G and C and the SpeI and Pfl23II restriction sites are underlined. The resulting PCR fragments were digested with SpeI and Pfl23II and purified over streptavidin-agarose (Sigma).42 The original pToxRIVAZ2 vector was cut with NheI and Pfl23II and ligated at a 1:2 molar ratio to the purified PCR fragments. Since this destroys the NheI and SpeI sites, the ligation mixture was treated with NheI and SpeI to remove any religated plasmids. The ligation was then purified by precipitation using SeeDNA (Amersham) according to the manufacturers’ instruction, dissolved in water, transformed into Escherichia coli Top10 cells (Invitrogen) by electroporation and plated on LB plates containing 33 mg/ml kanamycin. This resulted in a total library size of 1.2!107. The colonies were pooled in 2! LB medium containing 15% (w/v) glycerol and frozen in liquid nitrogen. DNA from the library was isolated, transformed into E. coli Chr3 cells12 and plated on LB plates containing 33 mg/ml kanamycin. This resulted in 4!107 colonies. These were again pooled in 2! LB containing 15% (w/v) glycerol and frozen in liquid nitrogen.

Tryptophan Mediates TMS–TMS Interactions

Western blotting and MalE complementation assays Western blotting was done as described18 with an antiserum recognising the MalE moiety of the ToxR proteins. ToxR proteins were assayed for membrane integration upon transformation into E. coli PD28 cells43 and growth in M9 minimal medium containing 0.4% (w/v) maltose.16 Cell density (A650) was measured at 24 h and 48 h (in duplicate) and the growth kinetics thus obtained were compared with that of a construct lacking the TMS (DTM) and AZ2. Clones were considered to be membrane-integrated when the A650 after 48 h of growth was consistently at least three times higher than that of DTM in a parallel experiment. Control clones that were membrane-integrated but not selected for self-interaction were obtained as follows. Library DNA was transformed into the MalE deletion strain PD28 and clones were picked randomly from LB plates (containing kanamycin). Clones that showed growth on M9 plates containing 0.4% maltose were verified by the MalE complementation assay in liquid culture. Site-directed mutagenesis Mutants AZ2-Wa, AZ2-Wd, AZ2-We, AZ2-Wg and L16-Wg were constructed by cloning respective oligonucleotide cassettes into the NheI and BamHI sites of pToxIVAZ2. Mutants of C65, and C75 were generated by QuikChange mutagenesis (Stratagene) and confirmed by DNA sequencing.

Acknowledgements We thank Eric Lindner for helpful discussions and Barbara Rauscher for excellent technical help. This work was supported by a fellowship of the Netherlands Organization for Scientific Research (NWO) to A.R. and the Deutsche Forschungsgemeinschaft (grant La699/9-1 to D.L.).

Library selection Selection for clones containing self-interacting TMSs was performed by plating dilutions of the library glycerol stock in Chr3 cells on LB plates containing 2% (w/v) glucose, 1% (w/v) arabinose, kanamycin (33 mg/ml), ampicillin (50 mg/ml) and different concentrations of chloramphenicol, ranging from 0–180 mg/ml. Plasmids of surviving clones were isolated and sequenced. In contrast to our previous work,12 ToxR expression was not pre-induced in liquid culture prior to plating. Therefore, ToxR concentration in the inner membrane is lower at the time of plating than upon pre-induction; consequently, colonies surviving at 180 mg/ml Cm are expected to contain ToxR plasmids with TMSs that are on average of higher affinity. ToxR activity assays ToxR activity was determined as described18 upon transformation of plasmids into E. coli FHK12 cells, growing for 20–24 h in LB medium containing 0.005% (w/v) L-arabinose, 0.4 mM IPTG, 100 mg/ml ampicillin, 33 mg/ml kanamycin and is expressed as b-galactosidase activity in Miller units (MU).

Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j. jmb.2005.09.084

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Tryptophan Mediates TMS–TMS Interactions

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Edited by G. von Heijne (Received 25 July 2005; received in revised form 27 September 2005; accepted 27 September 2005) Available online 21 October 2005