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Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation
Article IMF
YJMBI-64435; No. of pages: 10; 4C: 3, 4, 5, 6, 7
Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation Minttu Virkki 1, 2 , Carolina Boekel 1 , Kristoffer Illergård 1, 2 , Christoph Peters 1, 2 , Nanjiang Shu 1, 2 , Konstantinos D. Tsirigos 1, 2 , Arne Elofsson 1, 2 , Gunnar von Heijne 1, 2 and IngMarie Nilsson 1 1 - Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden 2 - Science for Life Laboratory, Stockholm University, SE-17177 Solna, Sweden
Correspondence to Gunnar von Heijne and IngMarie Nilsson: G. von Heijne is to be contacted at: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden. [email protected]; [email protected]. http://dx.doi.org/10.1016/j.jmb.2014.04.020 Edited by J. Bowie
Abstract While early structural models of helix-bundle integral membrane proteins posited that the transmembrane α-helices [transmembrane helices (TMHs)] were orientated more or less perpendicular to the membrane plane, there is now ample evidence from high-resolution structures that many TMHs have significant tilt angles relative to the membrane. Here, we address the question whether the tilt is an intrinsic property of the TMH in question or if it is imparted on the TMH during folding of the protein. Using a glycosylation mapping technique, we show that four highly tilted helices found in multi-spanning membrane proteins all have much shorter membrane-embedded segments when inserted by themselves into the membrane than seen in the high-resolution structures. This suggests that tilting can be induced by tertiary packing interactions within the protein, subsequent to the initial membrane-insertion step. © 2014 Elsevier Ltd. All rights reserved.
Introduction Nearly all helix-bundle-type integral membrane proteins are inserted co-translationally into their target membrane via a translocon, that is, a protein-conducting channel that can mediate both translocation of polar protein segments across the membrane and lateral insertion of transmembrane helices (TMHs) into the membrane [1]. While the general principles of translocon-mediated insertion of TMHs are fairly well understood [2], much less is known about the ensuing steps of folding and oligomerization in the membrane. One illustration of this lack of knowledge is that current bioinformatics algorithms can predict the number and orientation of TMHs in a multi-spanning protein quite accurately but per0022-2836/© 2014 Elsevier Ltd. All rights reserved.
form much less well in predicting the precise ends of the TMHs and their degree of tilting in the membrane [3,4]. In a recent study, we showed that TMHs can undergo rather large repositionings perpendicular to the membrane during folding and oligomerization [5]. We now report a similar study on TMHs that are highly tilted in the final folded structure and show, in analogy to the results mentioned above, that such TMHs can undergo large changes in their degree of tilting during the folding process. The emerging picture is one where the initial membrane-insertion step defines the overall topology of the protein but not the exact positioning of the TMHs relative to the membrane; instead, such fine-tuning results from tertiary interactions among TMHs. J. Mol. Biol. (2014) xx, xxx–xxx
Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
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Table 1. TMH domains analyzed in this study Protein
PDB code
TMH
Orientation
TMH (OPM)
Test segment
Mapped segment
3nmo 2a65 2oar 3b9w
1 3 1 11
Nin Nin Nin Nout
L36-A67 F89-V124 L17-I46 A319-T347
P11-D73 Q72-P143 M1-G62 V315-E367
D29-A52 F89-Y110 A18-T32 I326/T329-A342
ClcA LeuT MscL Rh50
The table includes the membrane orientation in the full-length protein, the membrane-buried segment as annotated in the OPM database, the test segment cloned into the Lep vector, and the membrane-buried segment identified by glycosylation mapping.
individual TMH in the membrane [6–11], analyzed in the absence of its natural partner TMHs, and then compare this to the disposition observed in the intact structure as annotated in the OPM (orientations of proteins in membranes) database [12]. Candidate TMHs were identified by first extracting TMHs that, according to the OPM database, have exceptionally long membrane-buried segments. These TMHs were then analyzed using the ΔG predictor [13] to find examples with predicted
Results Determination of TMH ends by glycosylation mapping Our approach is the same as what was used in the study of perpendicular repositioning of TMHs [5]; that is, we use an established “glycosylation mapping” method to determine the optimal disposition of an
Y
A
Lep TMH1
Lep TMH2
Y
Y
TMH-segment
lumen
Y cytoplasm
Y
Y
TMH-segment
Lep TMH1
Y
Y
Y
lumen
Y cytoplasm
MscL-TMH1
B
G4N
K3N
Lep(G-7N)
o CRM: Percent glycosylated:
-
+ 21
-
+ 52
-
+ 86
Fig. 1. The leader peptidase (Lep) host protein and the glycosylation mapping technique. (A) The test TMHs (white) with adjacent loops were introduced into the C-terminal Lep domain (upper cartoon) and to replace Lep TMH2 (lower cartoon). All constructs contained three Asn-Xaa-Thr glycan acceptor sites, as shown. Since efficient glycosylation is dependent on the distance of an acceptor site from the membrane, the membrane boundaries of the TMH segment can be determined by finding the position of a “reporter” glycan acceptor site (in gray) where it is half-maximally glycosylated. (B) Lep TMH1-TMH2/MscL-TMH1 constructs [cf. upper cartoon (a)] were expressed and labeled with [ 35S]methionine in vitro in the absence (−) or in the presence (+) of column-washed dog pancreas rough microsomes (CRMs). The residue mutated to Asn in the introduced Asn-Ser-Thr glycosylation sequon is indicated. Black dots indicate the number of glycans attached (each adding ~ 2 kDa to the molecular mass of the protein) and the white dot indicates protein not targeted to the CRMs. The degree of glycosylation of the reporter glycan acceptor site is calculated from the relative intensities of the three glycosylated products.
Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
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Large Tilts in Transmembrane Helices
A 100
Degree of glycosylation (%)
Degree of glycosylation (%)
100 80 60 40 20 0
80 60 40 20 0
8
10
12
14
16
18
20
22
61
24
Position in the sequence
62
63
64
65
66
67
68
Position in the sequence
R15 + 14 = D29
B --------------------------------------------------------
N62 - 10 = A52
--------------------------------------------------------
Fig. 2. Glycosylation mapping of ClcA-TMH1. (A) ClcA-TMH1 is annotated in the OPM database as extending from Leu36 to Ala67 (“TMH-structure”; marked in boldface). Half-maximal glycosylation of the reporter acceptor site is seen when the acceptor Asn residues are in positions 15 and 62 (data for two independent experiments are plotted for each construct). The membrane-embedded segment of the isolated TMH1 thus extends from Asp29 to Ala52 (“TMH-mapped”; in boldface. Underlined residues indicate that the point of 50% glycosylation is between these residues). The membrane-embedded segment predicted by the ΔG predictor is also shown (“TMH-predicted”; in boldface. Underlined residues indicate that the predicted free energy of membrane insertion differs by ≤ 0.1 kcal/mol between segment ending at these residues). (B) ClcA-TMH1 shown in the context of the fully folded protein (PDB ID: 1OTS). The membrane-embedded segment determined by glycosylation mapping is in blue, and the additional residues annotated in the OPM database are in yellow (non-polar residues) and pink (polar and charged residues). The broken lines indicate the membrane boundaries as annotated in the OPM database.
transmembrane (TM) segments that are considerably shorter than the OPM-annotated TMH. Finally, the candidate TMHs were inspected in the context of the 3D (3-dimensional) structure to ascertain that they are highly tilted. We focused on four TMHs with particularly high tilt angles: ClcA-TMH1 [14,15], LeuT-TMH3 [16], MscL-TMH1 [17], and Rh50-TMH11 [18]; Table 1. Each of the four tilted TMHs (including 20 upstream and downstream flanking residues or, if shorter, the loops connecting to the neighboring TMHs) were placed within the large extra-membranous P2 domain of host protein leader peptidase (Lep) from Escherichia coli (Fig. 1A), and constructs including either Lep-TMH1 or Lep-(TMH1 + TMH2) were expressed in vitro in the presence of column-washed dog pancreas rough microsomes (CRMs).
Glycosylation mapping can be used to determine the points in the sequence where a TMH enters and exits the membrane, taking advantage of the observation that the distances required for halfmaximal modification of an Asn-X-Thr acceptor site for N-linked glycosylation is 14 residues N-terminally and 10 residues C-terminally from the end of a TMH [7–11]. The N- and C-terminal ends of a TMH, when inserted into the RM membrane in the absence of its natural partner TMHs, can thus be determined by making a series of constructs where the Asn-X-Thr acceptor site is moved progressively closer to the TMH, across the region where the degree of modification drops from ~ 100% to ~ 0% (Fig. 1B). As the addition of a glycan corresponds to an increase in molecular mass of ~ 2 kDa, glycosylated and non-glycosylated forms of the Lep constructs can be
Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
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Large Tilts in Transmembrane Helices
Degree of glycosylation (%)
Degree of glycosylation (%)
A 100 80 60 40 20 0 67
69
71
73
75
77
79
100 80 60 40 20 0 118
119
Position in the sequence
120
121
122
123
124
125
Position in the sequence
G75 + 14 = F89
K121 - 10 = I111
B -------------------------------------
-------------------------------------
Fig. 3. Glycosylation mapping of LeuT-TMH3. (A) LeuT-TMH3 is annotated in the OPM database as extending from Phe89 to Val124 (“TMH-structure”; marked in boldface). Half-maximal glycosylation of the reporter acceptor site is seen when the acceptor Asn residues are in positions 75 and 121. The membrane-embedded segment of the isolated TMH3 thus extends from Phe89 to Ile111 (“TMH-mapped”; in boldface). The membrane-embedded segment predicted by the ΔG predictor is also shown (“TMH-predicted”; in boldface). (B) LeuT-TMH3 shown in the context of the fully folded protein (PDB ID: 2A65). The membrane-embedded segment determined by glycosylation mapping is in blue, and the additional residues annotated in the OPM database are in yellow (non-polar residues) and pink (polar and charged residues). The broken lines indicate the membrane boundaries as annotated in the OPM database.
easily separated and quantitated by SDS-PAGE, as seen in Fig. 1B. ClcA-TMH1 For ClcA-TMH1, the Asn-X-Thr glycan acceptor site was modified to ~ 50% when the Asn residue was placed in positions corresponding to residues Arg15 (N-terminal side) and Asn62 (C-terminal side) in wild-type ClcA (Fig. 2A). Adding 14 residues to the N-terminal position and subtracting 10 residues from the C-terminal position places the membrane-embedded region of the TMH between residues D29 and A52 (“TMH-mapped” in Fig. 2A). This corresponds closely to the TMH segment predicted by the ΔG predictor [13] (“TMH-predicted” in Fig. 2A) but does not match the ends of the highly tilted TMH1 (L36-A67) as deduced from the 3D structure of the native protein (“TMH-structure” in Fig. 2A). The C-terminal membrane-embedded part of ClcA-TMH1 is extended by 15 residues compared
to the glycosylation mapping result, while the N-terminal end of TMH1 is shifted by 7 residues from the mapped segment. The C-terminal part of TMH1 is overall polar in character, and the polar residues are mostly buried in the interface between TMH1 and other parts of the protein or exposed to the lipid headgroups (Fig. 2B). It thus appears that four helical turns at the C-terminal end of ClcA-TMH1 are pulled into the membrane and that two helical turns at the N-terminal end are pushed out of the membrane when the protein folds into its final structure. LeuT-TMH3 According to both the glycosylation mapping data and the ΔG predictor, the initially membrane-embedded part of LeuT-TMH3 (F89-I111) is considerably shorter than what is observed in the crystal structure (F89-V124) (Fig. 3A), and a 13-residuelong C-terminal part is pulled into the membrane
Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
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Large Tilts in Transmembrane Helices
100
Degree of glycosylation (%)
Degree of glycosylation (%)
A 80 60 40 20 0 -8
-6
-4
-2
0
2
4
100 80 60 40 20 0 40
42
Position in the sequence
B
K3 + 14 = L17
44
46
48
50
Position in the sequence
L42 - 10 = T32
-------------------------------------
-------------------------------------
Fig. 4. Glycosylation mapping of MscL-TMH1. (A) MscL-TMH1 is annotated in the OPM database as extending from Leu17 to Gly47 (“TMH-structure”; marked in boldface). Half-maximal glycosylation of the reporter acceptor site is seen when the acceptor Asn residues are in positions 3 and 42. The membrane-embedded segment of the isolated TMH1 thus extends from Leu17 to Thr32 (“TMH-mapped”; in boldface). The membrane-embedded segment predicted by the ΔG predictor is also shown (“TMH-predicted”; in boldface). (B) MscL-TMH1 shown in the context of the fully folded protein (PDB ID: 2OAR). The membrane-embedded segment determined by glycosylation mapping is in blue, and the additional residues annotated in the OPM database are in yellow (non-polar residues) and pink (polar and charged residues). The broken lines indicate the membrane boundaries as annotated in the OPM database.
during folding. The polar residues in the C-terminal extension are buried within the structure and apolar residues are lipid exposed (Fig. 3B). MscL-TMH1 Glycosylation mapping identifies residues L17-T32 as the initially membrane-embedded segment (Fig. 4A). The N-terminal end coincides with the N-terminal end of the TMH in the X-ray structure, while the C-terminal end of the TMH is extended by 15 residues in the structure. The extension contains mostly polar and charged residues that are buried in the protein (except for one lysine that snorkels into the lipid headgroup region [19]) and a few apolar residues that are exposed to the surrounding lipid (Fig. 4B). In this case, the ΔG predictor mispredicts the N-terminal end of the TMH by 9 residues relative to both the mapped end and the end seen in the structure but predicts the C-terminal end only 3 residues away from the mapped end.
Rh50-TMH11 The C-terminal end of initially membrane-embedded part of Rh50-TMH11 is well-defined by glycosylation mapping data and is 5 residues away from the end of the TMH in the X-ray structure (Fig. 5A). Both of the two polar residues in this segment, Lys345 and Thr347, are located in the lipid headgroup region in the folded structure. The N-terminal end is difficult to assign by glycosylation mapping, as the constructs with the acceptor Asn in positions 312 and 315 are equally well modified (~ 50% glycosylation). This places the N-terminal end somewhere between Ile326 and Thr329. This uncertainty in the mapping data may be because the N-terminal end of the TMH has a mildly apolar character and may fluctuate in location relative to the membrane when inserted by itself. In any case, an additional ~ 2 helical turns at the N-terminal end of TMH11 are seen in the X-ray structure (Fig. 5B). Rh50-TMH11 thus tilts by bringing
Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
6
Large Tilts in Transmembrane Helices
Degree of glycosylation (%)
Degree of glycosylation (%)
A 100 80 60 40 20 0 308
310
312
314
316
318
Position in the sequence
B
V315 + 14 = T329
100 80 60 40 20 0 349
351
353
355
357
359
361
Position in the sequence
Q352 - 10 = A342
-------------------------------------
------------------------------------Fig. 5. Glycosylation mapping of Rh50-TMH11. (A) Rh50-TMH11 is annotated in the OPM database as extending from Val320 to Thr347 (“TMH-structure”; marked in boldface). Half-maximal glycosylation of the reporter acceptor site is seen when the acceptor Asn residues are in positions 315 and 352. The membrane-embedded segment of the isolated TMH11 thus extends from Thr329 to Ala342 (“TMH-mapped”; in boldface). The membrane-embedded segment predicted by the ΔG predictor is also shown (“TMH-predicted”; in boldface). (B) Rh50-TMH11 shown in the context of the fully folded protein (PDB ID: 3B9W). The membrane-embedded segment determined by glycosylation mapping is in blue, and the additional residues annotated in the OPM database are in yellow (non-polar residues) and pink (polar and charged residues). The broken lines indicate the membrane boundaries as annotated in the OPM database.
additional segments from both the N-terminus and the C-terminus into the membrane. The segment predicted by the ΔG predictor is closer to the TMH seen in the structure than to the mapped segment, especially at the C-terminal end.
The majority of long, tilted TMHs have long hydrophobic segments Although the correspondence between the membrane-embedded segments determined by
Fig. 6. The majority of long, tilted TMHs contain long hydrophobic segments. (A) Fraction of TMHs with a hydrophobic segment of length N residues as identified by the ΔG predictor in a data set composed of TMHs with long membrane-embedded segments (L ≥ 29 residues) extracted from the OPM database. (B) TMH1 in the yeast mitochondrial ADP/ATP carrier isoform 3 (PDB ID: 4C9Q) provides an example of a TMH that has a much shorter hydrophobic segment (N = 19 residues) than the membrane-embedded segment annotated in OPM (L = 31 residues). The membrane-embedded segment predicted by the ΔG predictor is in blue, and the additional residues annotated in the OPM database are in yellow (non-polar residues) and pink (polar and charged residues). The broken lines indicate the membrane boundaries as annotated in the OPM database. Note that the polar part of the TMH is almost completely buried in the structure. (C) TMH4 in subunit F of the MalFGK maltose transporter complex (PDB ID: 3PUW) provides an example of a TMH that has an almost equally long hydrophobic segment (N = 30 residues) as the membrane-embedded segment annotated in OPM (L = 31 residues). The membrane-embedded segment predicted by the ΔG predictor is in blue; the additional polar residue annotated in the OPM database is buried in the structure. The broken lines indicate the membrane boundaries as annotated in the OPM database.
Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
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Large Tilts in Transmembrane Helices
glycosylation mapping of isolated TMHs and the segments predicted by the ΔG predictor is not perfect, there is nevertheless a reasonable agreement between the two measures [5]. We therefore used the ΔG predictor as a proxy for glycosylation mapping and compared the length of the segment
A
identified by the ΔG predictor with the length of the membrane-embedded parts of all TMHs in 188 non-redundant high-resolution protein structures, as annotated in the OPM database [12]. We focused on long TMHs with membrane-embedded parts of length L ≥ 29 residues (as annotated in OPM). As
0.25
Fraction
0.20
0.15
0.10
0.05
0.00 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Length of hydrophobic segment (N)
B
-------------------------------------
-------------------------------------
C
-------------------------------------------------------------------------
Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
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Large Tilts in Transmembrane Helices
seen in Fig. 6A, the segment predicted by the ΔG predictor was considerably shorter (≤ 24 residues) than the OPM segment in 4 out of 20 TMHs with L ≥ 29 residues and was of comparable length to the OPM segment in 16 out of 20 cases. An example of each kind is shown in Fig. 6B and C. Thus, the majority of the long, tilted TMHs in currently known structures are hydrophobic throughout their lengths and hence probably tilt in the membrane independently of tertiary interactions. Nevertheless, a sizeable fraction of the TMHs seem to behave according to a “tilting-during-folding” paradigm.
Discussion TMHs with a high degree of tilt relative to the plane of the membrane are not uncommon in multispanning membrane proteins [20], raising the question whether such TMHs have an inherent tendency to tilt when inserted into the membrane or if tilting is induced during folding of the protein. Using a glycosylation mapping technique, we have determined the segment that becomes embedded in the membrane of four highly titled TMHs engineered into an unrelated host protein, that is, when they do not engage in tertiary interactions within a folded multispanning protein. In all four cases, the embedded segment is considerably shorter than the part of the TMH that resides within the plane of the membrane in the native structure. In three out of the four cases, C-terminal rather polar helical segments of 13–15 residues are apparently pulled into the membrane during folding; in the fourth case, the membrane-embedded TMH is extended by approximately five residues at each end in the native structure. In all cases, the polar residues in the helical extensions tend to pack against other parts of the protein or snorkel into the lipid headgroup region, while apolar residues turn toward the surrounding membrane. While these results pertain to rather extreme cases —the majority of long, tilted TMHs are hydrophobic throughout their lengths—they nevertheless show that TMHs not only can be repositioned perpendicular to the membrane plane during folding [5] but also can undergo large tilting motions. The TM segments predicted by the ΔG predictor generally match those identified by glycosylation mapping much better than they match the TMHs seen in the structures. This is not surprising, given that the ΔG predictor is based on free energies of membrane integration determined for model TMHs that do not interact with other TMHs [13]. It does illustrate, however, that we should not expect that prediction of the precise ends of the membrane-embedded parts of TMHs in multi-spanning proteins can be achieved by methods that score only the local hydrophobicity along the chain but will require modeling of the full 3D structure.
Materials and Methods Enzymes and chemicals Unless otherwise stated, all chemicals were from Sigma-Aldrich (St. Louis, MO, USA) and oligonucleotides were from MWG Biotech AG (Ebersberg, Germany). All enzymes were from Fermentas (Burlington, Ontario, Canada), except Phusion DNA polymerase from Finnzymes OY (Espoo, Finland). The plasmid pGEM-1 and the TNT SP6 Quick Coupled Transcription/Translation System were from Promega Biotech AB (Madison, WI, USA). [ 35S] Methionine was from Perkin Elmer (Boston, MA, USA) and the column-washed dog pancreas rough microsomes (CRMs) were from tRNAprobes (College Station, TX, USA). For all gel extractions, the E.Z.N.A. Gel Extraction Kit was used. The Qiaprep Miniprep Plasmid Purification Kits from QIAGEN (Hilden, Germany) were used for plasmid purifications. E.Z.N.A. Cycle Pure and Gel Extraction Kits from Omega Bio-Tek (Norcross, GA, USA) were used during post-PCR manipulation. All constructs were sent for sequencing of the plasmid DNA at Eurofins MWG Operon. DNA manipulations The lepB gene had previously been introduced into the pGEM-1 vector under the control of the SP6 promoter [21] and with the context 5′ of the initiator codon changed to a Kozak consensus sequence [21,22]. To allow Lep to “host” protein segments, we had introduced SpeI and KpnI restriction recognition sites in the sequence in the middle of the P2 domain or to replace the second Lep TMH. One glycosylation site was placed on each side of the TMH segment to be tested (see Fig. 1). Double-stranded oligonucleotides encoding the TM region of interest (see Table 1) were introduced into the lepB gene as SpeI-KpnI-restricted amplified PCR fragments using primers complementary to the 5′ and 3′ ends of the selected part of the gene. Both the vector and the PCR fragments were digested with SpeI and KpnI, separated on agarose gel, and fragments of correct size were excised from gel and purified. PCR fragments were ligated to the vector carrying the lepB gene using Rapid DNA Ligation Kit. Using these constructs as templates, we performed PCR to introduce a third glycosylation site at various positions relative to the test TMH segment. We used the Asn-Ser-Thr glycosylation site sequon in all constructs to prevent fluctuations in glycosylation efficiency based on changes at the second position [23] or due to variation between Ser and Thr at the third positions [24]. Since the presence of proline residues either in the middle of or next to the sequon has a negative influence on the glycosylation efficiency, any such proline residues were changed into glutamines [25]. Expression in vitro Constructs in pGEM-1 were transcribed and translated in the TNT SP6 Quick Coupled System from Promega. A master mix, containing [ 35S]methionine (5 μCi), and lysate were mixed together in such a way that the amount of lysate was 10 times the volume of [ 35S]methionine. We
Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
9
Large Tilts in Transmembrane Helices then added 5.5 μl of this master mix to 100 ng DNA. For the glycosylation experiments, the master mix was supplemented with dog pancreas column-washed rough microsomes, in an amount that would yield N 80% properly targeted protein (i.e., b 20% non-glycosylated product). Samples were incubated at 30 °C for 90 min. Duplicates were made for all constructs. Translated proteins were separated by SDS-PAGE and visualized in a Fuji FLA-3000 phosphoimager (Fujifilm, Tokyo, Japan) with the ImageReader V1.8J/Image Gauge V 3.45 software (Fujifilm). The MultiGauge software was used to create one-dimensional intensity profiles for each lane on the gels, where the triply glycosylated proteins have the highest molecular mass of + 6 kDa; doubly glycosylated proteins, + 4 kDa; and singly glycosylated proteins, + 2 kDa relative to the non-glycosylated protein. These profiles were then analyzed using the multi-Gaussian fit program from the Qtiplot software package †, and the peak areas of the glycosylated protein bands in the profile were obtained. Finally, the fractions of singly, doubly, and triply glycosylated protein were calculated. Non-glycosylated protein was not included in the calculation, as this represents molecules not properly targeted to the microsomal membranes. Bioinformatics analysis of tilted TMHs A data set of non-redundant TM proteins was retrieved from the OPM database [12]. We first obtained the definition of all TM subunits in the OPM ‡ and then extracted those entries that are α-helical TM proteins. For all the extracted α-helical TM proteins, we obtained their amino acid sequences from the SEQRES record in the PDB files. Since the definition of TM subunits in OPM is based on the sequence in the ATOM record in PDB files, we then mapped the sequence indices from the ATOM record to the SEQRES record by alignment. The orientation of TM proteins was determined based on the annotation of location of the N-terminus for each entry in the OPM. This procedure resulted in a data set of 615 unique TM proteins. We further reduced the redundancy to b 20% sequence identity by PISCES [26], to achieve a final, non-redundant data set of 188 TM subunits. For each TMH in the data set (including the upstream and downstream flanking loops), we used the ΔG predictor to identify the most hydrophobic region and its ΔG value, trying all window sizes from 15 to 39 residues. TMHs with an OPM-annotated length of less than 15 residues were excluded in order to remove TM β-strands and a few other strangely annotated segments.
Acknowledgements This work was supported by grants from the Swedish Cancer Foundation, the Swedish Research Council, the Swedish Foundation for Strategic Research, the Knut and Alice Wallenberg Foundation, and the European Research Council (ERC-2008-AdG 232648) to G.v.H.; from the Swedish Research Council (no. 2009-5072) to A.E.
and M.V.; and from Magnus Bergvalls Stiftelse, Carl Tryggers Stiftelse, and the Swedish Foundation for International Cooperation in Research and Higher Education to I.M.N. Received 14 February 2014; Received in revised form 19 April 2014; Accepted 22 April 2014 Available online xxxx Keywords: transmembrane helix; membrane protein folding; translocon †http://www.qtiplot.ro/ ‡downloaded from http://opm.phar.umich.edu/ subunits.php on 2014-04-03 Abbreviations used: TMH, transmembrane helix; TM, transmembrane.
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Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
10 [12] Lomize MA, Lomize AL, Pogozheva ID, Mosberg HI. OPM: orientations of proteins in membranes database. Bioinformatics 2006;22:623–5. [13] Hessa T, Meindl-Beinker NM, Bernsel A, Kim H, Sato Y, Lerch-Bader M, et al. Molecular code for transmembranehelix recognition by the Sec61 translocon. Nature 2007;450:1026–30. [14] Dutzler R, Campbell EB, MacKinnon R. Gating the selectivity filter in ClC chloride channels. Science 2003;300:108–12. [15] Robertson JL, Kolmakova-Partensky L, Miller C. Design, function and structure of a monomeric ClC transporter. Nature 2010;468:844–7. [16] Yamashita A, Singh SK, Kawate T, Jin Y, Gouaux E. Crystal structure of a bacterial homologue of Na+/Cl−-dependent neurotransmitter transporters. Nature 2005;437:215–23. [17] Chang G, Spencer R, Lee A, Barclay M, Rees D. Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel. Science 1998;282:2220–6. [18] Lupo D, Li XD, Durand A, Tomizaki T, Cherif-Zahar B, Matassi G, et al. The 1.3-Ǻ resolution structure of Nitrosomonas europaea Rh50 and mechanistic implications for NH3 transport by Rhesus family proteins. Proc Natl Acad Sci U S A 2007;104:19303–8. [19] Monné M, Nilsson I, Johansson M, Elmhed N, von Heijne G. Positively and negatively charged residues have different
Large Tilts in Transmembrane Helices
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YJMBI-64435; No. of pages: 10; 4C: 3, 4, 5, 6, 7
Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation Minttu Virkki 1, 2 , Carolina Boekel 1 , Kristoffer Illergård 1, 2 , Christoph Peters 1, 2 , Nanjiang Shu 1, 2 , Konstantinos D. Tsirigos 1, 2 , Arne Elofsson 1, 2 , Gunnar von Heijne 1, 2 and IngMarie Nilsson 1 1 - Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden 2 - Science for Life Laboratory, Stockholm University, SE-17177 Solna, Sweden
Correspondence to Gunnar von Heijne and IngMarie Nilsson: G. von Heijne is to be contacted at: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden. [email protected]; [email protected]. http://dx.doi.org/10.1016/j.jmb.2014.04.020 Edited by J. Bowie
Abstract While early structural models of helix-bundle integral membrane proteins posited that the transmembrane α-helices [transmembrane helices (TMHs)] were orientated more or less perpendicular to the membrane plane, there is now ample evidence from high-resolution structures that many TMHs have significant tilt angles relative to the membrane. Here, we address the question whether the tilt is an intrinsic property of the TMH in question or if it is imparted on the TMH during folding of the protein. Using a glycosylation mapping technique, we show that four highly tilted helices found in multi-spanning membrane proteins all have much shorter membrane-embedded segments when inserted by themselves into the membrane than seen in the high-resolution structures. This suggests that tilting can be induced by tertiary packing interactions within the protein, subsequent to the initial membrane-insertion step. © 2014 Elsevier Ltd. All rights reserved.
Introduction Nearly all helix-bundle-type integral membrane proteins are inserted co-translationally into their target membrane via a translocon, that is, a protein-conducting channel that can mediate both translocation of polar protein segments across the membrane and lateral insertion of transmembrane helices (TMHs) into the membrane [1]. While the general principles of translocon-mediated insertion of TMHs are fairly well understood [2], much less is known about the ensuing steps of folding and oligomerization in the membrane. One illustration of this lack of knowledge is that current bioinformatics algorithms can predict the number and orientation of TMHs in a multi-spanning protein quite accurately but per0022-2836/© 2014 Elsevier Ltd. All rights reserved.
form much less well in predicting the precise ends of the TMHs and their degree of tilting in the membrane [3,4]. In a recent study, we showed that TMHs can undergo rather large repositionings perpendicular to the membrane during folding and oligomerization [5]. We now report a similar study on TMHs that are highly tilted in the final folded structure and show, in analogy to the results mentioned above, that such TMHs can undergo large changes in their degree of tilting during the folding process. The emerging picture is one where the initial membrane-insertion step defines the overall topology of the protein but not the exact positioning of the TMHs relative to the membrane; instead, such fine-tuning results from tertiary interactions among TMHs. J. Mol. Biol. (2014) xx, xxx–xxx
Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
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Table 1. TMH domains analyzed in this study Protein
PDB code
TMH
Orientation
TMH (OPM)
Test segment
Mapped segment
3nmo 2a65 2oar 3b9w
1 3 1 11
Nin Nin Nin Nout
L36-A67 F89-V124 L17-I46 A319-T347
P11-D73 Q72-P143 M1-G62 V315-E367
D29-A52 F89-Y110 A18-T32 I326/T329-A342
ClcA LeuT MscL Rh50
The table includes the membrane orientation in the full-length protein, the membrane-buried segment as annotated in the OPM database, the test segment cloned into the Lep vector, and the membrane-buried segment identified by glycosylation mapping.
individual TMH in the membrane [6–11], analyzed in the absence of its natural partner TMHs, and then compare this to the disposition observed in the intact structure as annotated in the OPM (orientations of proteins in membranes) database [12]. Candidate TMHs were identified by first extracting TMHs that, according to the OPM database, have exceptionally long membrane-buried segments. These TMHs were then analyzed using the ΔG predictor [13] to find examples with predicted
Results Determination of TMH ends by glycosylation mapping Our approach is the same as what was used in the study of perpendicular repositioning of TMHs [5]; that is, we use an established “glycosylation mapping” method to determine the optimal disposition of an
Y
A
Lep TMH1
Lep TMH2
Y
Y
TMH-segment
lumen
Y cytoplasm
Y
Y
TMH-segment
Lep TMH1
Y
Y
Y
lumen
Y cytoplasm
MscL-TMH1
B
G4N
K3N
Lep(G-7N)
o CRM: Percent glycosylated:
-
+ 21
-
+ 52
-
+ 86
Fig. 1. The leader peptidase (Lep) host protein and the glycosylation mapping technique. (A) The test TMHs (white) with adjacent loops were introduced into the C-terminal Lep domain (upper cartoon) and to replace Lep TMH2 (lower cartoon). All constructs contained three Asn-Xaa-Thr glycan acceptor sites, as shown. Since efficient glycosylation is dependent on the distance of an acceptor site from the membrane, the membrane boundaries of the TMH segment can be determined by finding the position of a “reporter” glycan acceptor site (in gray) where it is half-maximally glycosylated. (B) Lep TMH1-TMH2/MscL-TMH1 constructs [cf. upper cartoon (a)] were expressed and labeled with [ 35S]methionine in vitro in the absence (−) or in the presence (+) of column-washed dog pancreas rough microsomes (CRMs). The residue mutated to Asn in the introduced Asn-Ser-Thr glycosylation sequon is indicated. Black dots indicate the number of glycans attached (each adding ~ 2 kDa to the molecular mass of the protein) and the white dot indicates protein not targeted to the CRMs. The degree of glycosylation of the reporter glycan acceptor site is calculated from the relative intensities of the three glycosylated products.
Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
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Large Tilts in Transmembrane Helices
A 100
Degree of glycosylation (%)
Degree of glycosylation (%)
100 80 60 40 20 0
80 60 40 20 0
8
10
12
14
16
18
20
22
61
24
Position in the sequence
62
63
64
65
66
67
68
Position in the sequence
R15 + 14 = D29
B --------------------------------------------------------
N62 - 10 = A52
--------------------------------------------------------
Fig. 2. Glycosylation mapping of ClcA-TMH1. (A) ClcA-TMH1 is annotated in the OPM database as extending from Leu36 to Ala67 (“TMH-structure”; marked in boldface). Half-maximal glycosylation of the reporter acceptor site is seen when the acceptor Asn residues are in positions 15 and 62 (data for two independent experiments are plotted for each construct). The membrane-embedded segment of the isolated TMH1 thus extends from Asp29 to Ala52 (“TMH-mapped”; in boldface. Underlined residues indicate that the point of 50% glycosylation is between these residues). The membrane-embedded segment predicted by the ΔG predictor is also shown (“TMH-predicted”; in boldface. Underlined residues indicate that the predicted free energy of membrane insertion differs by ≤ 0.1 kcal/mol between segment ending at these residues). (B) ClcA-TMH1 shown in the context of the fully folded protein (PDB ID: 1OTS). The membrane-embedded segment determined by glycosylation mapping is in blue, and the additional residues annotated in the OPM database are in yellow (non-polar residues) and pink (polar and charged residues). The broken lines indicate the membrane boundaries as annotated in the OPM database.
transmembrane (TM) segments that are considerably shorter than the OPM-annotated TMH. Finally, the candidate TMHs were inspected in the context of the 3D (3-dimensional) structure to ascertain that they are highly tilted. We focused on four TMHs with particularly high tilt angles: ClcA-TMH1 [14,15], LeuT-TMH3 [16], MscL-TMH1 [17], and Rh50-TMH11 [18]; Table 1. Each of the four tilted TMHs (including 20 upstream and downstream flanking residues or, if shorter, the loops connecting to the neighboring TMHs) were placed within the large extra-membranous P2 domain of host protein leader peptidase (Lep) from Escherichia coli (Fig. 1A), and constructs including either Lep-TMH1 or Lep-(TMH1 + TMH2) were expressed in vitro in the presence of column-washed dog pancreas rough microsomes (CRMs).
Glycosylation mapping can be used to determine the points in the sequence where a TMH enters and exits the membrane, taking advantage of the observation that the distances required for halfmaximal modification of an Asn-X-Thr acceptor site for N-linked glycosylation is 14 residues N-terminally and 10 residues C-terminally from the end of a TMH [7–11]. The N- and C-terminal ends of a TMH, when inserted into the RM membrane in the absence of its natural partner TMHs, can thus be determined by making a series of constructs where the Asn-X-Thr acceptor site is moved progressively closer to the TMH, across the region where the degree of modification drops from ~ 100% to ~ 0% (Fig. 1B). As the addition of a glycan corresponds to an increase in molecular mass of ~ 2 kDa, glycosylated and non-glycosylated forms of the Lep constructs can be
Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
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Large Tilts in Transmembrane Helices
Degree of glycosylation (%)
Degree of glycosylation (%)
A 100 80 60 40 20 0 67
69
71
73
75
77
79
100 80 60 40 20 0 118
119
Position in the sequence
120
121
122
123
124
125
Position in the sequence
G75 + 14 = F89
K121 - 10 = I111
B -------------------------------------
-------------------------------------
Fig. 3. Glycosylation mapping of LeuT-TMH3. (A) LeuT-TMH3 is annotated in the OPM database as extending from Phe89 to Val124 (“TMH-structure”; marked in boldface). Half-maximal glycosylation of the reporter acceptor site is seen when the acceptor Asn residues are in positions 75 and 121. The membrane-embedded segment of the isolated TMH3 thus extends from Phe89 to Ile111 (“TMH-mapped”; in boldface). The membrane-embedded segment predicted by the ΔG predictor is also shown (“TMH-predicted”; in boldface). (B) LeuT-TMH3 shown in the context of the fully folded protein (PDB ID: 2A65). The membrane-embedded segment determined by glycosylation mapping is in blue, and the additional residues annotated in the OPM database are in yellow (non-polar residues) and pink (polar and charged residues). The broken lines indicate the membrane boundaries as annotated in the OPM database.
easily separated and quantitated by SDS-PAGE, as seen in Fig. 1B. ClcA-TMH1 For ClcA-TMH1, the Asn-X-Thr glycan acceptor site was modified to ~ 50% when the Asn residue was placed in positions corresponding to residues Arg15 (N-terminal side) and Asn62 (C-terminal side) in wild-type ClcA (Fig. 2A). Adding 14 residues to the N-terminal position and subtracting 10 residues from the C-terminal position places the membrane-embedded region of the TMH between residues D29 and A52 (“TMH-mapped” in Fig. 2A). This corresponds closely to the TMH segment predicted by the ΔG predictor [13] (“TMH-predicted” in Fig. 2A) but does not match the ends of the highly tilted TMH1 (L36-A67) as deduced from the 3D structure of the native protein (“TMH-structure” in Fig. 2A). The C-terminal membrane-embedded part of ClcA-TMH1 is extended by 15 residues compared
to the glycosylation mapping result, while the N-terminal end of TMH1 is shifted by 7 residues from the mapped segment. The C-terminal part of TMH1 is overall polar in character, and the polar residues are mostly buried in the interface between TMH1 and other parts of the protein or exposed to the lipid headgroups (Fig. 2B). It thus appears that four helical turns at the C-terminal end of ClcA-TMH1 are pulled into the membrane and that two helical turns at the N-terminal end are pushed out of the membrane when the protein folds into its final structure. LeuT-TMH3 According to both the glycosylation mapping data and the ΔG predictor, the initially membrane-embedded part of LeuT-TMH3 (F89-I111) is considerably shorter than what is observed in the crystal structure (F89-V124) (Fig. 3A), and a 13-residuelong C-terminal part is pulled into the membrane
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5
Large Tilts in Transmembrane Helices
100
Degree of glycosylation (%)
Degree of glycosylation (%)
A 80 60 40 20 0 -8
-6
-4
-2
0
2
4
100 80 60 40 20 0 40
42
Position in the sequence
B
K3 + 14 = L17
44
46
48
50
Position in the sequence
L42 - 10 = T32
-------------------------------------
-------------------------------------
Fig. 4. Glycosylation mapping of MscL-TMH1. (A) MscL-TMH1 is annotated in the OPM database as extending from Leu17 to Gly47 (“TMH-structure”; marked in boldface). Half-maximal glycosylation of the reporter acceptor site is seen when the acceptor Asn residues are in positions 3 and 42. The membrane-embedded segment of the isolated TMH1 thus extends from Leu17 to Thr32 (“TMH-mapped”; in boldface). The membrane-embedded segment predicted by the ΔG predictor is also shown (“TMH-predicted”; in boldface). (B) MscL-TMH1 shown in the context of the fully folded protein (PDB ID: 2OAR). The membrane-embedded segment determined by glycosylation mapping is in blue, and the additional residues annotated in the OPM database are in yellow (non-polar residues) and pink (polar and charged residues). The broken lines indicate the membrane boundaries as annotated in the OPM database.
during folding. The polar residues in the C-terminal extension are buried within the structure and apolar residues are lipid exposed (Fig. 3B). MscL-TMH1 Glycosylation mapping identifies residues L17-T32 as the initially membrane-embedded segment (Fig. 4A). The N-terminal end coincides with the N-terminal end of the TMH in the X-ray structure, while the C-terminal end of the TMH is extended by 15 residues in the structure. The extension contains mostly polar and charged residues that are buried in the protein (except for one lysine that snorkels into the lipid headgroup region [19]) and a few apolar residues that are exposed to the surrounding lipid (Fig. 4B). In this case, the ΔG predictor mispredicts the N-terminal end of the TMH by 9 residues relative to both the mapped end and the end seen in the structure but predicts the C-terminal end only 3 residues away from the mapped end.
Rh50-TMH11 The C-terminal end of initially membrane-embedded part of Rh50-TMH11 is well-defined by glycosylation mapping data and is 5 residues away from the end of the TMH in the X-ray structure (Fig. 5A). Both of the two polar residues in this segment, Lys345 and Thr347, are located in the lipid headgroup region in the folded structure. The N-terminal end is difficult to assign by glycosylation mapping, as the constructs with the acceptor Asn in positions 312 and 315 are equally well modified (~ 50% glycosylation). This places the N-terminal end somewhere between Ile326 and Thr329. This uncertainty in the mapping data may be because the N-terminal end of the TMH has a mildly apolar character and may fluctuate in location relative to the membrane when inserted by itself. In any case, an additional ~ 2 helical turns at the N-terminal end of TMH11 are seen in the X-ray structure (Fig. 5B). Rh50-TMH11 thus tilts by bringing
Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
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Degree of glycosylation (%)
Degree of glycosylation (%)
A 100 80 60 40 20 0 308
310
312
314
316
318
Position in the sequence
B
V315 + 14 = T329
100 80 60 40 20 0 349
351
353
355
357
359
361
Position in the sequence
Q352 - 10 = A342
-------------------------------------
------------------------------------Fig. 5. Glycosylation mapping of Rh50-TMH11. (A) Rh50-TMH11 is annotated in the OPM database as extending from Val320 to Thr347 (“TMH-structure”; marked in boldface). Half-maximal glycosylation of the reporter acceptor site is seen when the acceptor Asn residues are in positions 315 and 352. The membrane-embedded segment of the isolated TMH11 thus extends from Thr329 to Ala342 (“TMH-mapped”; in boldface). The membrane-embedded segment predicted by the ΔG predictor is also shown (“TMH-predicted”; in boldface). (B) Rh50-TMH11 shown in the context of the fully folded protein (PDB ID: 3B9W). The membrane-embedded segment determined by glycosylation mapping is in blue, and the additional residues annotated in the OPM database are in yellow (non-polar residues) and pink (polar and charged residues). The broken lines indicate the membrane boundaries as annotated in the OPM database.
additional segments from both the N-terminus and the C-terminus into the membrane. The segment predicted by the ΔG predictor is closer to the TMH seen in the structure than to the mapped segment, especially at the C-terminal end.
The majority of long, tilted TMHs have long hydrophobic segments Although the correspondence between the membrane-embedded segments determined by
Fig. 6. The majority of long, tilted TMHs contain long hydrophobic segments. (A) Fraction of TMHs with a hydrophobic segment of length N residues as identified by the ΔG predictor in a data set composed of TMHs with long membrane-embedded segments (L ≥ 29 residues) extracted from the OPM database. (B) TMH1 in the yeast mitochondrial ADP/ATP carrier isoform 3 (PDB ID: 4C9Q) provides an example of a TMH that has a much shorter hydrophobic segment (N = 19 residues) than the membrane-embedded segment annotated in OPM (L = 31 residues). The membrane-embedded segment predicted by the ΔG predictor is in blue, and the additional residues annotated in the OPM database are in yellow (non-polar residues) and pink (polar and charged residues). The broken lines indicate the membrane boundaries as annotated in the OPM database. Note that the polar part of the TMH is almost completely buried in the structure. (C) TMH4 in subunit F of the MalFGK maltose transporter complex (PDB ID: 3PUW) provides an example of a TMH that has an almost equally long hydrophobic segment (N = 30 residues) as the membrane-embedded segment annotated in OPM (L = 31 residues). The membrane-embedded segment predicted by the ΔG predictor is in blue; the additional polar residue annotated in the OPM database is buried in the structure. The broken lines indicate the membrane boundaries as annotated in the OPM database.
Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
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Large Tilts in Transmembrane Helices
glycosylation mapping of isolated TMHs and the segments predicted by the ΔG predictor is not perfect, there is nevertheless a reasonable agreement between the two measures [5]. We therefore used the ΔG predictor as a proxy for glycosylation mapping and compared the length of the segment
A
identified by the ΔG predictor with the length of the membrane-embedded parts of all TMHs in 188 non-redundant high-resolution protein structures, as annotated in the OPM database [12]. We focused on long TMHs with membrane-embedded parts of length L ≥ 29 residues (as annotated in OPM). As
0.25
Fraction
0.20
0.15
0.10
0.05
0.00 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Length of hydrophobic segment (N)
B
-------------------------------------
-------------------------------------
C
-------------------------------------------------------------------------
Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
8
Large Tilts in Transmembrane Helices
seen in Fig. 6A, the segment predicted by the ΔG predictor was considerably shorter (≤ 24 residues) than the OPM segment in 4 out of 20 TMHs with L ≥ 29 residues and was of comparable length to the OPM segment in 16 out of 20 cases. An example of each kind is shown in Fig. 6B and C. Thus, the majority of the long, tilted TMHs in currently known structures are hydrophobic throughout their lengths and hence probably tilt in the membrane independently of tertiary interactions. Nevertheless, a sizeable fraction of the TMHs seem to behave according to a “tilting-during-folding” paradigm.
Discussion TMHs with a high degree of tilt relative to the plane of the membrane are not uncommon in multispanning membrane proteins [20], raising the question whether such TMHs have an inherent tendency to tilt when inserted into the membrane or if tilting is induced during folding of the protein. Using a glycosylation mapping technique, we have determined the segment that becomes embedded in the membrane of four highly titled TMHs engineered into an unrelated host protein, that is, when they do not engage in tertiary interactions within a folded multispanning protein. In all four cases, the embedded segment is considerably shorter than the part of the TMH that resides within the plane of the membrane in the native structure. In three out of the four cases, C-terminal rather polar helical segments of 13–15 residues are apparently pulled into the membrane during folding; in the fourth case, the membrane-embedded TMH is extended by approximately five residues at each end in the native structure. In all cases, the polar residues in the helical extensions tend to pack against other parts of the protein or snorkel into the lipid headgroup region, while apolar residues turn toward the surrounding membrane. While these results pertain to rather extreme cases —the majority of long, tilted TMHs are hydrophobic throughout their lengths—they nevertheless show that TMHs not only can be repositioned perpendicular to the membrane plane during folding [5] but also can undergo large tilting motions. The TM segments predicted by the ΔG predictor generally match those identified by glycosylation mapping much better than they match the TMHs seen in the structures. This is not surprising, given that the ΔG predictor is based on free energies of membrane integration determined for model TMHs that do not interact with other TMHs [13]. It does illustrate, however, that we should not expect that prediction of the precise ends of the membrane-embedded parts of TMHs in multi-spanning proteins can be achieved by methods that score only the local hydrophobicity along the chain but will require modeling of the full 3D structure.
Materials and Methods Enzymes and chemicals Unless otherwise stated, all chemicals were from Sigma-Aldrich (St. Louis, MO, USA) and oligonucleotides were from MWG Biotech AG (Ebersberg, Germany). All enzymes were from Fermentas (Burlington, Ontario, Canada), except Phusion DNA polymerase from Finnzymes OY (Espoo, Finland). The plasmid pGEM-1 and the TNT SP6 Quick Coupled Transcription/Translation System were from Promega Biotech AB (Madison, WI, USA). [ 35S] Methionine was from Perkin Elmer (Boston, MA, USA) and the column-washed dog pancreas rough microsomes (CRMs) were from tRNAprobes (College Station, TX, USA). For all gel extractions, the E.Z.N.A. Gel Extraction Kit was used. The Qiaprep Miniprep Plasmid Purification Kits from QIAGEN (Hilden, Germany) were used for plasmid purifications. E.Z.N.A. Cycle Pure and Gel Extraction Kits from Omega Bio-Tek (Norcross, GA, USA) were used during post-PCR manipulation. All constructs were sent for sequencing of the plasmid DNA at Eurofins MWG Operon. DNA manipulations The lepB gene had previously been introduced into the pGEM-1 vector under the control of the SP6 promoter [21] and with the context 5′ of the initiator codon changed to a Kozak consensus sequence [21,22]. To allow Lep to “host” protein segments, we had introduced SpeI and KpnI restriction recognition sites in the sequence in the middle of the P2 domain or to replace the second Lep TMH. One glycosylation site was placed on each side of the TMH segment to be tested (see Fig. 1). Double-stranded oligonucleotides encoding the TM region of interest (see Table 1) were introduced into the lepB gene as SpeI-KpnI-restricted amplified PCR fragments using primers complementary to the 5′ and 3′ ends of the selected part of the gene. Both the vector and the PCR fragments were digested with SpeI and KpnI, separated on agarose gel, and fragments of correct size were excised from gel and purified. PCR fragments were ligated to the vector carrying the lepB gene using Rapid DNA Ligation Kit. Using these constructs as templates, we performed PCR to introduce a third glycosylation site at various positions relative to the test TMH segment. We used the Asn-Ser-Thr glycosylation site sequon in all constructs to prevent fluctuations in glycosylation efficiency based on changes at the second position [23] or due to variation between Ser and Thr at the third positions [24]. Since the presence of proline residues either in the middle of or next to the sequon has a negative influence on the glycosylation efficiency, any such proline residues were changed into glutamines [25]. Expression in vitro Constructs in pGEM-1 were transcribed and translated in the TNT SP6 Quick Coupled System from Promega. A master mix, containing [ 35S]methionine (5 μCi), and lysate were mixed together in such a way that the amount of lysate was 10 times the volume of [ 35S]methionine. We
Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020
9
Large Tilts in Transmembrane Helices then added 5.5 μl of this master mix to 100 ng DNA. For the glycosylation experiments, the master mix was supplemented with dog pancreas column-washed rough microsomes, in an amount that would yield N 80% properly targeted protein (i.e., b 20% non-glycosylated product). Samples were incubated at 30 °C for 90 min. Duplicates were made for all constructs. Translated proteins were separated by SDS-PAGE and visualized in a Fuji FLA-3000 phosphoimager (Fujifilm, Tokyo, Japan) with the ImageReader V1.8J/Image Gauge V 3.45 software (Fujifilm). The MultiGauge software was used to create one-dimensional intensity profiles for each lane on the gels, where the triply glycosylated proteins have the highest molecular mass of + 6 kDa; doubly glycosylated proteins, + 4 kDa; and singly glycosylated proteins, + 2 kDa relative to the non-glycosylated protein. These profiles were then analyzed using the multi-Gaussian fit program from the Qtiplot software package †, and the peak areas of the glycosylated protein bands in the profile were obtained. Finally, the fractions of singly, doubly, and triply glycosylated protein were calculated. Non-glycosylated protein was not included in the calculation, as this represents molecules not properly targeted to the microsomal membranes. Bioinformatics analysis of tilted TMHs A data set of non-redundant TM proteins was retrieved from the OPM database [12]. We first obtained the definition of all TM subunits in the OPM ‡ and then extracted those entries that are α-helical TM proteins. For all the extracted α-helical TM proteins, we obtained their amino acid sequences from the SEQRES record in the PDB files. Since the definition of TM subunits in OPM is based on the sequence in the ATOM record in PDB files, we then mapped the sequence indices from the ATOM record to the SEQRES record by alignment. The orientation of TM proteins was determined based on the annotation of location of the N-terminus for each entry in the OPM. This procedure resulted in a data set of 615 unique TM proteins. We further reduced the redundancy to b 20% sequence identity by PISCES [26], to achieve a final, non-redundant data set of 188 TM subunits. For each TMH in the data set (including the upstream and downstream flanking loops), we used the ΔG predictor to identify the most hydrophobic region and its ΔG value, trying all window sizes from 15 to 39 residues. TMHs with an OPM-annotated length of less than 15 residues were excluded in order to remove TM β-strands and a few other strangely annotated segments.
Acknowledgements This work was supported by grants from the Swedish Cancer Foundation, the Swedish Research Council, the Swedish Foundation for Strategic Research, the Knut and Alice Wallenberg Foundation, and the European Research Council (ERC-2008-AdG 232648) to G.v.H.; from the Swedish Research Council (no. 2009-5072) to A.E.
and M.V.; and from Magnus Bergvalls Stiftelse, Carl Tryggers Stiftelse, and the Swedish Foundation for International Cooperation in Research and Higher Education to I.M.N. Received 14 February 2014; Received in revised form 19 April 2014; Accepted 22 April 2014 Available online xxxx Keywords: transmembrane helix; membrane protein folding; translocon †http://www.qtiplot.ro/ ‡downloaded from http://opm.phar.umich.edu/ subunits.php on 2014-04-03 Abbreviations used: TMH, transmembrane helix; TM, transmembrane.
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Please cite this article as: Virkki Minttu, et al, Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.020