The final set of the 16 lowest energy structures is particularly well defined in the region of residues Leu9–Phe20 in 2,2,2-trifluoroethanol, with a mean pairwise root mean square deviation
Trang 1Structure and topology of the transmembrane domain 4 of the
divalent metal transporter in membrane-mimetic environments
Hongyan Li1,2, Fei Li1, Zhong Ming Qian2and Hongzhe Sun1
1
Department of Chemistry and Open Laboratory of Chemical Biology, The University of Hong Kong, China;2Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, China
The divalent metal transporter (DMT1) is a
12-transmem-brane domain protein responsible for dietary iron uptake in
the duodenum and iron acquisition from transferrin in
peripheral tissues The transmembrane domain 4 (TM4)
of DMT1 has been shown to be crucial for its biological
function Here we report the 3D structure and topology of
the DMT1-TM4 peptide by NMR spectroscopy with
simulated annealing calculations in membrane-mimetic
environments, e.g 2,2,2-trifluoroethanol and SDS micelles
The 3D structures of the peptide are similar in both
envi-ronments, with nonordered and flexible N- and C-termini
flanking an ordered helical region The final set of the 16
lowest energy structures is particularly well defined in the
region of residues Leu9–Phe20 in 2,2,2-trifluoroethanol,
with a mean pairwise root mean square deviation of
0.23 ± 0.10 A˚ for the backbone heavy atoms and
0.82 ± 0.17 A˚ for all heavy atoms In SDS micelles, the
length of the helix is dependent on pH values In particular,
the C-terminus becomes well-structured at low pH (4.0), whereas the N-terminal segment (Arg1–Gly7) is flexible and poorly defined at all pH values studied The effects of 12-doxylPtdCho spin-label and paramagnetic metal ions on NMR signal intensities demonstrated that both the N-ter-minus and helical region of the TM4 are embedded into the interior of SDS micelles Unexpectedly, we observed that amide protons exchanged much faster in SDS than in 2,2, 2-trifluoroethanol, indicating that there is possible solvent accessibility in the structure The paramagnetic metal ions broaden NMR signals from residues both situated in aque-ous phase and in the helical region From these results we speculate that DMT1-TM4s may self-assemble to form a channel through which metal ions are likely to be trans-ported These results might provide an insight into the structure-function relationship for the integral DMT1 Keywords: DMT1; membrane; NMR; structure
The divalent metal transporter (DMT1) gene, also known as
Nramp2(natural resistance-associated macrophage
protein-2) and DCT1, was identified recently [1,2] It belongs to a
large family of integral membrane proteins highly conserved
throughout evolution, from bacteria to human beings [3–6]
It is the only known cellular iron importer, and is
responsible for importing iron from the gut into the
entero-cytes and also for transporting iron across the endosomal
membrane in the transferrin cycle [7–9] The DMT1 consists
of 561 amino acids with 12 putative transmembrane
domains [1] The DMT1 gene encodes two messenger
RNAs produced by alternative splicing of two 3¢ exons that
showdifferent 3¢ untranslated regions containing an iron response element (isoform I) and no iron response element (isoform II), as well as distinct C-terminal protein sequences [7–10] Recently, DMT1 mRNA expression has also been detected in the kidney [11]
Direct metal transport studies in Xenopus laevis oocytes have demonstrated that DMT1 (isoform I) is a pH-dependent divalent metal transporter with broad substrate specificity including Fe2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, and toxic metals Cd2+and Pb2+[1] Studies in cultured mammalian cells have also shown that both isoforms of DMT1 are capable of transporting a variety of divalent metal ions across the plasma membrane [12,13] Transport
of these metal ions was shown to occur at pH 5.5, but not at 7.4 [1] The His267/His272 located in the transmembrane domain (TM) 6 has been thought to play an important role
in pH regulation of metal transport by DMT1 [14] However, it is not yet clear how pH regulates DMT1 metal transport The biological importance of this transporter is shown by its involvement in two naturally occurring animal mutants of iron metabolism A mutation (G185R) in TM4
of DMT1 is responsible for microcytic anemia of the mk mice and Belgrade rats, which exhibit severe defects in intestinal iron absorption and erythroid iron utilization [2,7] This suggests that the TM4 of DMT1 may have a unique and important biological function The sequence of this domain is characterized by a high degree of hydrophobicity and is highly conserved among different species [1]
Correspondence to H Sun, Department of Chemistry, The University
of Hong Kong, Pokfulam Road, Hong Kong.
Fax: + 852 2857 1586, Tel.: + 852 2859 8974,
E-mail: hsun@hkucc.hku.hk
Abbreviations: doxylPtdCho, palmitol(doxyl)
stearoyl-phosphatidyl-choline; 12-doxylPtdCho, doxylPtdCho lipids containing the nitroxide
label on C12; DMT1, divalent metal transporter; DMT1-TM4,
transmembrane domain 4 of DMT1; HFIP,
1,1,1,3,3,3,-hexafluoro-2-propanol; TFE, 2,2,2-trifluoroethanol; TM, transmembrane domain.
Note: The coordinate for the 16 lowest energy conformers both in
SDS micelles at pH 6.0 and TFE has been deposited in the protein data
bank (http://www.rcsb.org/pdb/index.html).
(Received 29 January 2004, revised 16 March 2004,
accepted 23 March 2004)
Trang 2Although numerous studies have been carried out to
explore the molecular biology aspects of DMT1 since the
discovery of this gene, there has so far been no structural
characterization of either this integral protein or a segment
of it Analysis of the structure of membrane proteins either
by NMR spectroscopy or crystallography has proven
difficult, because the native structures of these integral
proteins are largely dependent on the associated membrane
Recently model peptides, which mimic the sequence of a
segment or a subunit of membrane proteins, have been
widely used to investigate structure and function in several
integral membrane proteins [15–20] This approach has
proved to be very successful in providing qualitative
structural information and in guiding complete structure
determination [21,22] For example, it has enabled 3D
structural models of lactose permease, a 12-transmembrane
helix bundle that transduces free energy, to be derived
recently, based on its transmembrane topology, secondary
structure, and numerous interhelical contacts without using
crystals [23]
We have previously investigated the secondary structure
of the TM4 of DMT1 in various membrane-mimetic
environments, such as 2,2,2-trifluoroethanol (TFE),
deter-gent micelles and phosphate lipids [24] We showed that the
DMT1-TM4 peptide assumed predominately an a-helical
conformation in these environments In the present study,
we have used NMR spectroscopy and a molecular dynamic
simulated annealing approach to characterize the 3D
structures of DMT1-TM4 in both TFE and SDS micelles
at different pH values The topology of the peptide in SDS
micelles was probed by the effects of spin-labels, including
both palmitol(doxyl) stearoyl-phosphatidyl-choline
(doxy-lPtdCho) lipids containing the nitroxide label on C12
(12-doxylPtdCho) and paramagnetic metal ions (Mn2+and
Gd3+), on the intensities of NMR signals The peptide was
found to embed into the interior of SDS micelles The
possibility of formation of a divalent metal channel has been
discussed
Experimental procedures
Materials
The sequence of the peptide (RVPLYGGVLITIADT
FVFLFLDKY) was taken from rat DMT1 and represents
the putative TM4 (residues 179–202) The peptide was
synthesized by a solid-phase method and was purified by
HPLC on a Zorbax SB Phenyl reverse phase column using
0.1% (v/v) trifluoroacetic acid/water and 0.1% (v/v)
trifluoroacetic acid/acetonitrile as solvents (Biopeptide
Co., LLC San Diego, CA, USA) The purity was assessed
by both mass spectrometry and analytical HPLC to be
above 95% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was
obtained from Sigma Deuterated reagents for NMR
sample preparation, e.g 2,2,2-trifluoroethanol-d2 and
2,2,2,-trifluoroethanol-d3 99.94% (TFE), methanol-d4
99.6%, deuterium oxide 99.96%, and sodium dodecyl-d25
sulfate were purchased from Cambridge Isotope
Laborat-ories (Cambridge, MA, USA)
Palmitol(doxyl)-stearoyl-phosphatidylcholine (doxylPtdCho) lipids containing the
nitroxide spin label on C12 were purchased from Avanti
Polar Lipids (Alabaster, AL, USA)
Circular dichroism spectroscopy
CD experiments were performed on a Jasco J-720 spectro-polarimeter at ambient temperature Cells with path lengths
of 0.1 and 1.0 mm were employed for sample solutions containing final peptide concentrations of 6, 12, 23, 47, 94,
188, 375 and 750 lMin TFE Spectra were recorded from
190 to 260 nm at a scan rate of 50 nmÆmin)1with a respond time of 0.25 s, step resolution of 0.1 nm and band width of
1 nm Each spectrum was obtained from the average of four scans Prior to calculation of final ellipiticity, all spectra were corrected by subtraction of background and were smoothed using a fast Fourier transform filter
NMR spectroscopy The samples used for NMR studies were prepared as described previously [24] Briefly, 3 mg of the peptide dissolved in HFIP was mixed with an equal volume of SDS-d25aqueous solution The mixture was further diluted with water, and was subject to lyophilization The resulting powder was then redissolved in 0.6 mL H2O containing 10% (v/v) D2O The concentration of the peptide was approximately 2.0 mM in SDS-d25 (300 mM) Spectra for assignments and structure calculation in the presence of SDS were recorded at 298 K In the presence of TFE, the spectra were recorded at 298 and 305 K to resolve spectral overlap
All spectra were recorded on a Bruker AV600 spectro-meter, operating at a proton frequency of 600.13 MHz Water suppression was carried out using a 3-9-19 watergate-pulse sequence [25,26] The sodium salt of trimethylsilyl-propionate-d4solution was used to reference chemical shifts 1D experiments were acquired using 32 768 data points and processed with 0.3 Hz line broadening The NOESY [27,28] experiments were recorded at mixing times of 50, 150, 200 and 250 ms, and the TOCSY spectra employed the
MLEV-17 pulse sequence [29] with mixing periods of 50–100 ms The relaxation delay was 1.8 s in the TOCSY experiments and 2.0 s in the NOESY experiments Typically, 40–80 transients were collected for each increment of F1 in the NOESY experiments, and 80–120 in the TOCSY experi-ments All 2D experiments were collected using 2048 data points in F2, 256–512 increments in F1 All 2D Spectra were acquired in the phase sensitive mode using States-time-proportional phase incrementation in F1 dimension Spectral data were processed on a computer using standard Bruker software (XWINNMR Version 3.1) Data were zero-filled to 2048 points in F1 dimension and then transformed with a shifted sine-bell squared window function in both dimensions Base line correction was also carried out
Structure calculations Distance constraints were obtained from NOESY spectra recorded with a mixing time of 200 ms in SDS micelles and
150 ms in TFE at 298 and 305 K, respectively In the case
of severe spectral overlap, the corresponding NOEs were excluded from the set used for the structure calculations Both NOE intensities and chemical shifts were extracted using the software [30] and served as an input for
Trang 3the program of CYANA (1.0) [31] On the basis of these
distance constraints obtained using the macro CALIBA, a
systematic analysis of the local conformation around the Ca
atom of each residue, including the dihedral angles /, w, v1
and v2, was performed using the macro GRIDSEARCH as
implemented inCYANA[31] The final nonredundant
upper-limit constraints and the resulting angle constraints were
used in the structural calculations No stereospecific
assign-ments were obtained in any case The 200 randomized
starting structures were energy minimized during 4000 steps
under the NMR constraints, and the 30CYANAconformers
with the lowest target function values were selected for
further energy minimization under the force field of Cornell
et al [32] using a generalized Born solvent model with a
water shell of 8 A˚ inAMBER7 [33,34]
From these calculated structures, 16 conformers with the
lowest energy were selected to represent the NMR
struc-tures The quality of the final structures was accessed using
the program ofPROCHECK-NMR[35] Further analysis and
visualization of the conformers including calculation of root
mean square deviations (rmsds) and identification of
H-bonds was performed using the molecular graphics
programMOLMOL[36]
Paramagnetic broadening experiments
Samples containing 2 mMDMT1-TM4 and 300 mM
SDS-d25in 0.6 mL 90% H2O/10% D2O (v/v) were used in these
experiments The pH was adjusted to either 5.5 or 7.4 by
addition of small aliquots of NaOH Spin-labeled
12-doxylPtdCho was solubilized in methonal-d4, and aliquots
of this solution were then added to the peptide at pH 5.5 to
yield a final concentration of the spin-label of 5 mM This
corresponded to approximately one spin-label per micelle
on the assumption of about 60 molecules per micelle [37]
The TOCSY spectrum (mixing time of 50 ms) was
acquired with a spectral width of 6 p.p.m in F1 dimension,
with 120 transients, and 256 increments in F1 dimension
The pH of the sample was then raised to 7.4 and the
TOCSY spectrum was collected The reference spectrum in
the absence of the spin-label was also recorded under
identical conditions For Gd3+ and Mn2+ broadening
studies, either GdCl3 or MnCl2 were dissolved in H2O
before being added to the sample The experiments were
performed with concentrations of paramagnetic metal ions
of 0.1, 0.2, 0.4 and 1.0 mM The TOCSY spectra were
again recorded in the presence of different amounts of
paramagnetic metal ions at different pH values (e.g 7.4, 5.5
and 4.0)
Hydrogen exchange experiments
In the TFE system, 3 mg of DMT1-TM4 was directly
dissolved in 0.6 mL TFE-d3 Fast exchange amide protons
were monitored by subsequently recording a series of
one-dimensional1H-NMR spectra at 10, 30, 60, 90, 120 and
360 min until no further changes were observed in the
spectra The TOCSY spectrum (mixing time 50 ms) was
then acquired in a total time of 19 h, and those protons
which showed cross-peaks in the Ha–HNregion of TOCSY
spectrum were regarded as slowly exchanging amide
protons
In SDS micelles, 0.6 mL D2O was added to lyophilized samples containing 2 mM peptide and 300 mM SDS-d25 The pD was adjusted to 5.5 by addition of aliquots of NaOD Similarly, the fast exchanging amide protons were monitored by 1D proton NMR spectra recorded at different time intervals from 10 to 120 min The NOESY spectrum (mixing time 200 ms) was then acquired in a total time of 8.5 h, and the protons that appeared in the spectrum were regarded as relatively slowexchanging protons All amide protons were exchanged completely within 12 h
Results
Resonance assignment and secondary structure determination
The DMT1-TM4 peptide is highly hydrophobic, and insol-uble in water and a range of organic solvents We have chosen TFE and SDS to solubilize the peptide and to mimic biological membranes The peptide is stable in these environ-ments for at least a couple of months at room temperature TOCSY and NOESY spectra with a set of mixing times were recorded for DMT1-TM4 in SDS-d2 at different pH values, and reasonably well-resolved spectra were found at a wide range of pH values The spectra recorded in SDS micelles at
pH 6.0 were chosen for sequential assignments and structural calculations, as it is close to the biological function pH ( 5.5)
of its integral protein Moreover, the spectra at this pH were relatively well resolved compared with those at other pH values Figure 1 shows the fingerprint region of the 600 MHz NOESY spectra of DMT1-TM4 in 300 mM SDS-d25 at
pH 6.0 (298 K) and in TFE-d2(305 K) It can be seen that the peptide exhibited sufficient chemical shift dispersions in both environments, allowing unambiguous assignments of most proton frequencies The 1H resonance assignment was straightforward, based on a standard procedure [38] The complete spin systems of the individual amino acid residues were identified using the TOCSY spectra with mixing times of
50 and 100 ms The backbone sequential connectivities were established by following the Ha and HN cross-peaks of adjacent amino acids in the fingerprint and the HN–HNregion
of the TOCSY and NOESY spectra Using this technique it was possible to unambiguously assign almost all the proton resonances including side chains, apart from a fewaromatic protons from phenylalanine residues due to spectral overlap (H Li, F Li, Z M Qian & H Sun, unpublished observation)
We noticed that chemical shift values in TFE and in SDS micelles were similar with the expected exception of amide protons, in particular, the amide protons of N-terminal residues
The chemical shifts of the Haprotons provide informa-tion about secondary structural elements of the peptides Generally, all residues experience a Haupfield shift relative
to the random-coil value when adopting a helical confor-mation and a downfield shift when found in an extended or b-strand structure [39] The peptide was predicted to adopt a helical conformation in the segment of Leu9–Phe20 in SDS micelles and Gly6–Lys23 in TFE from the chemical shift index method [40] (Fig 2) Further investigation by exam-ining the observed NOE connectivities produced similar results All HNresonances from Tyr5 to Phe20 were found
to be connected by (i, i+1) connectivities except for Phe16
Trang 4and Val17, which are overlapped together in TFE (Fig 2),
indicative of a helical conformation in this region This is
in agreement with our previous CD studies, which
demon-strated high helical contents in the DMT1-TM4 peptide
[24] Furthermore, we also observed that the chemical shift
for threonine Hbis greater than that of Hafor both Thr11
and Thr15, indicating that both threonines are situated in
the helical region Evaluation of the secondary structure
from backbone coupling constants was hampered due to
extensive line broadening both in the TFE and SDS micelle
environments, which retards determination of these
coup-ling constants
Structure calculations and description
Distance constraints were obtained from NOESY spectra
recorded with a mixing time of 200 ms measured in 90%
HO/10% DO (v/v) containing 2 mMpeptide and 300 mM
SDS-d25 at pH 6.0, and 150 ms in TFE-d2 The NOE connectivities and numbers of NOEs per constraints for DMT1-TM4 in both solvents are summarized in Fig 2 Except for unresolved cross-peaks between the residue pairs Leu21/Asp22 in SDS, and Phe16/Val17 and Phe20/Leu21 in TFE, almost all of the possible HNi =HNiþ1[38], and sequential NOEs were observed in the segment of Tyr5–Phe20 In addition, the presence of medium-range connectivities [38], such as Hai=HNiþ3, Hai=Hbiþ3and Hai=HNiþ4was also observed for Val8–Phe20 in SDS and Val8–Lys23 in TFE, indicative
of a well-structured peptide in helical conformation over each span [41,42] The absence of medium-range NOEs
at the N-terminus suggested no defined structure in this segment However, in the C-terminal segment, NOEs between Ha
i=HN iþ2 and HN
i=HN iþ2, which are characteristic
of 310-helix [38], were also detected in TFE No long-range NOEs were observed over the full peptide, indicating that the peptide does not form tertiary folds
Fig 1 Fingerprint region of the 600 MHz
NOESY spectra of DMT1-TM4 (A) 200 ms
NOE spectrum of 2 m M DMT1-TM4 in
300 m M SDS-d 25 at pH 6.0, 298 K (B) 150 ms
NOE spectrum of 2 m M peptide in TFE-d 2 ,
305 K The sequential assignment of all
resi-dues is indicated.
Trang 5Totals of 241 and 265 meaningful upper-limit distance
constraints were obtained based on totals of 358 and 378
assigned NOE cross-peaks for DMT1-TM4 in SDS at
pH 6.0 and in TFE, respectively A total of 79 dihedral
angle constraints for 50 angles in SDS vs 85 constraints for
55 angles in TFE, derived using the macroGRIDSEARCHas
implemented in CYANA [31], were also included in the
structure calculations In no case could stereospecific
assignment be achieved The structures were calculated by
molecular dynamics in torsion angle space using a simulated
annealing protocol as implemented in the programCYANA
[31] Under this protocol, 200 randomized starting
struc-tures were energy minimized under the NMR constraints
and the 30 structures with no violations > 0.2 A˚ for the
distance constraint and > 5 for the angle constraint, as well
as with the lowest target function were selected in either
SDS or TFE for further energy minimization The structural
statistics showed that the structures of DMT1-TM4 in both
membrane-mimetic environments were well defined by
NMR data, as indicated by the lowvalues of the target
function (Table 1) The backbone / and w dihedral angles
were also uniformly well-defined, as judged from an angular
order parameter of 1.0 in the span of Leu9–Phe20 [43]
These structures were subjected to an energy minimization
using the programAMBER7 [33,34] in theAMBERforce field
[32] The final 16 lowest energy structures of DMT1-TM4 in
both SDS (pH 6.0) and TFE were chosen to represent the solution structures of the peptide, as shown in Fig 3 The quality of the final structures was assessed using the programPROCHECK-NMR[35] In the range of well-defined residues, i.e Leu9–Phe18 in SDS (pH 6.0) and Leu9–Phe20
in TFE, 99.4% and 91.7% occupy the most favored regions
of the Ramachandran space in SDS and TFE, respectively, and none are found in the disallowed regions (Table 1) The overall structure of DMT1-TM4 in SDS micelles is similar to that in TFE The mean structures obtained from MOLMOLshowed that the peptide folded into an a-helical conformation for Leu9–Phe18 in SDS and Leu9–Phe20 in TFE The pairwise rmsds between the minimized structures and the mean structure in SDS at pH 6.0 were 0.18 ± 0.06 and 0.85 ± 0.17 A˚ for the backbone and all heavy atoms, respectively, in the segment Leu9–Phe18, vs 0.20 ± 0.06 and 0.89 ± 0.16 A˚ in the segment Leu9–Phe20 (Table 1) The pairwise rmsds between the minimized structures and mean structure in TFE were 0.23 ± 0.10 and 0.82 ± 0.17 A˚ for the backbone and all heavy atoms, respectively, in the segment Leu9–Phe20, vs 0.26 ± 0.12 and 0.87 ± 0.17 A˚ in the segment Leu9–Lys23 This suggested that the structures of the peptide from the residue Leu9 towards the C-terminal residues were well-defined by NMR constraints, which is consistent with the observed pattern of sequential and medium-range NOEs and the
Fig 2 Summary of NMR spectroscopy data for secondary structure prediction for DMT1-TM4peptide (A) In SDS micelles at pH 6.0, 298 K and (B) in TFE at 305 K The NOE connectivities, amide proton exchange rates, chemical shift index values as well as numbers of NOE constraints per residue for DMT1-TM4 are shown Slowly and rapidly exchanging amide protons are represented as filled and open circles, respectively The NOEs
of intra, sequential and medium range are indicated as white, light gray and dark gray bars, respectively.
Trang 6prediction based on the chemical shift index However, the
pairwise rmsds between these structures and mean
struc-tures in the range Arg1–Tyr24 were significantly increased
for the backbone and all heavy atoms in both SDS and TFE
(Table 1), which suggested that the N-terminus was poorly
defined compared with the C-terminus in both SDS and
TFE, consistent with the fewer medium range NOEs
observed in this region This is probably due to some
flexibility in this region Although the C-terminal region
(Leu21–Tyr24) does not fold into a typical helical structure, it
is relatively ordered compared with the N-terminal region In
particular, it is extremely close to a-helical folding in TFE,
judging from both angular order parameters ( 1.0) for
backbone / and w dihedral angles and Ramachandran space
analysis from PROCHECK-NMR When the structures of
DMT1-TM4 in both SDS and TFE were superimposed
over the backbone atoms of Leu9–Phe18 for a best fit, we
noticed that the lower part of the helices and the C-terminus
were differently oriented in SDS compared with that in TFE
The C-terminus was bent towards the helical core in SDS
micelles at pH 6.0 The aromatic rings of Phe16 and Phe20
were oriented more or less in a plane parallel to each other in
TFE (Fig 3B) In contrast, the rings of Phe16 and Phe20
were almost perpendicular to each other in SDS (Fig 3A)
In most structures, HN
iþ4! COi hydrogen bonds were present in the helical region comprising residues Leu9–
Phe20 in SDS micelles and TFE, with the exception of the
missing H-bonds between Phe16 and Phe20 in both SDS
and TFE Additionally, a HNiþ3! COihydrogen bond was present between Val17 and Phe20 in both SDS and TFE
pH effects on the structures The effects of pH on peptide conformation in SDS micelles were investigated by acquiring a series of NOESY spectra over the pH range of 4.0–7.5 at 298 K Changes in conformation over this range were assessed by analyzing the differences of Ha chemical shifts from random coil values (data not shown) Generally, chemical shifts were closer to random coil values at a higher pH, implying that the peptide becomes less structured as pH values increase However, residue Leu19 gave rise to a different pattern Figure 4 shows a summary of the intra- and inter-residual NOE connectivities for the peptide in SDS micelles at
pH 4.0 and 7.5 From the pattern of NOEs, a well-defined a-helical region comprised of residues Val8–Lys23, and Gly7–Phe18 at pH 4.0 and 7.5, respectively, was proposed, while the N-terminus was probably in an extended confor-mation at both pH values The structures of DMT1-TM4 at both pH 4.0 and 7.5 were calculated subsequently using molecular dynamics in torsion angle space, using a simu-lated annealing protocol as described above
From a total of 328 (pH 4.0) and 340 (pH 7.5) NOE assignments, 222 (31 medium range, 103 intraresidue and 88 sequential NOEs, pH 4.0) and 232 (39 medium range, 103 intraresidue and 90 sequential NOEs, pH 7.5) nonredun-dant upper-limit constraints were obtained for the structural calculations Sixteen structures with lowest target functions were selected for each pH value, and were superimposed over the backbone atoms of Ile10–Val17 (Fig 5) The structures of DMT1-TM4 at both pH 4.0 and 7.5 were well characterized by NMR data with no distance violations larger than 0.2 A˚ At pH 4.0, the pairwise backbone rmsds over residues Leu9–Lys23 were 0.67 ± 0.20 and 1.52 ± 0.30 A˚ for backbone atoms and all heavy atoms, respectively; while at pH 7.5, the pairwise backbone rmsds over residues Leu9–Val17 were 0.31 ± 0.11 and 0.89 ± 0.21 A˚ for backbone atoms and all heavy atoms, respectively
To highlight the structural difference between the conformations at both pH values, the structures were superimposed over the backbone atoms of residues Ile10– Val17 for a best fit (Fig 5A) In general, the N-terminus of the peptide was highly flexible and mostly unstructured at both pH values, consistent with the lack of medium-range NOEs, e.g Hai=HNiþ3, Hai=Hbiþ3 and Hai=HNiþ4 (Fig 4) However, a longer helix was formed at lower pH, e.g Leu9– Lys23 at pH 4.0 vs Leu9–Val17 at pH 7.5, indicating that the C-terminal end is more susceptible to unfolding as the
pH value increases At pH 4.0, in the segment of Leu9– Lys23, HNiþ4! COihydrogen bonds indicative of a-helices were observed for the majority of structures out of the 16 conformers However, H-bonds between Val17 and Leu21 were not detected in any of the 16 conformers, while the H-bonds between Asp14 and Phe18 were missing in the majority of the 16 conformers, indicating that the structures may be distorted in this region Similarly, H-bonds charac-teristic of a-helices were also found in most of the structures
of the 16 conformers at pH 7.5 in the segment of Leu9– Val17, except Ile12 and Phe16 in some of the conformers
Table 1 Structural statistics for DMT1-TM4in the presence of SDS at
pH 6.0 and in TFE.
Parameter
SDS (pH 6.0, 298 K)
TFE (305 K) Target function (A˚ 2 ) 0.03 ± 0.01 0.12 ± 0.02
Experimental NMR constraints
No of distance constrains 241 265
Intraresidues 111 118
Sequential (i ¼ 1) 89 75
Medium range
(1 < 1/2i-j1/2 £ 4)
Long range (1/2i-j1/2 > 4) 0 0
NOE constraint violations (A˚)
Sum 0.30 ± 0.10 1.10 ± 0.20
Maximum 0.08 ± 0.02 0.18 ± 0.02
AMBER energy (kcalÆmol)1) )856.7 ± 6.2 )857.2 ± 5.1
rmsd from the mean structure (A˚)
Residues 1–24
Backbone atoms 4.07 ± 1.28 2.84 ± 0.90
All heavy atoms 5.37 ± 1.36 4.00 ± 1.05
Residues 9–20
Backbone atoms 0.20 ± 0.06 0.23 ± 0.10
All heavy atoms 0.89 ± 0.16 0.82 ± 0.17
Ramachandran statistics
(residues 9–20)a(%)
The most favored regions 99.4 91.7
Additional allowed regions 8.9 8.3
Generously allowed regions 0 0
Disallowed regions 0 0
a
Analyzed using PROCHECK - NMR
Trang 7Moreover, some structures displayed HN
iþ3! COihydrogen bonds indicative of 310helices between Ala13 and Phe16,
and also between Ile12 and Thr15
The structures of DMT1-TM4 in TFE were
super-imposed with those in SDS micelles at pH 4.0 over the
backbone atoms of Leu9–Phe20 for comparison (Fig 5B)
We noticed no significant difference between the structures
in these environments, and even the orientations of the side
chains were remarkably similar
Amide proton exchange
Backbone proton-deuterium exchange experiments have
long been used to verify whether amide protons are involved
in hydrogen bonds or are largely shielded from solvent
access [44] These experiments have also been used recently
to determine the residues which are involved in the binding
of peptides to membranes [37,45,46] The exchange rate of the amide protons of DMT1-TM4 in both TFE and SDS micelles was monitored by both 1D and 2D 1H NMR spectroscopy (e.g TOCSY and NOESY) and was analyzed semiquantitatively in terms of either a rapid or slow exchange of the various residues (Fig 2) In the presence
of TFE-d3, both N-terminal (Val2, Leu4, Tyr5, Gly6 and Gly7) and C-terminal residues (Asp22, Lys23 and Tyr24) decreased their intensities rapidly and almost completely disappeared within 2 h (except Gly6, which disappeared 6 h later) However, the residues involved in the formation of the helix comprising of Val8–Leu21 retained their intensities
in this period A TOCSY spectrum was then recorded that
Fig 3 Stereoview of NMR structures of DMT1-TM4in SDS micelles and TFE (A) All atoms of 16 final structures of DMT1-TM4 in SDS micelles at pH 6.0 with superimposition over the backbone atoms of residues Leu9– Phe18 (B) All atoms of 16 final structures of DMT1-TM4 in TFE overlaid over the back-bone atoms of Leu9–Phe20.
Trang 8showed that residues Val8–Leu21 were still observable after
24 h, and thus considered as slowly exchanging protons
(data not shown), which is consistent with the formation of
H-bonds Similarly, both the N- and C-terminal residues
lost their intensities within 30 min after addition of D2O to a
lyophilized sample containing SDS-d25at pD 5.5 Residues involved in the formation of the helix mostly retained their intensities in the first 2 h, except Thr11 and Asp14 A NOESY spectrum recorded after 8 h showed that residues Ile10, Ile12, Asp13, Phe16, Val17 and Leu19–Leu21 were observable in this period of time (H Li, F Li, Z M Qian &
H Sun, unpublished observation), indicative of slowly exchanging protons (Fig 2) Nearly all cross-peaks in the NOESY spectrum vanished after 12 h and no amide protons were observable in 1D 1H spectrum after 20 h (data not shown) The amide proton exchange experiments presented here suggest that hydrogen bonds play an important role in the stabilization of DMT1-TM4 confor-mations both in TFE and SDS micelles The faster exchange
of amide protons of Thr11 and Asp14 in SDS micelles indicates that there is probably some solvent accessibility in the peptide between the micellar and aqueous environments
Paramagnetic broadening studies 12-DoxylPtdCho Information from 1H line-broadening caused by the lipid-soluble spin-labeled compound 12-doxylPtdCho was used to determine the position of the peptide relative to the micelle surface We used the doxylPtdCho lipids containing doxyl groups at C12 atoms
of the stearoyl side-chain, which have been demonstrated previously to be located near the center of the micelles [47,48] Therefore, peptide protons located in the center of the micelles would be the most affected, whereas those located at the micelle surface or outside of micelles would be the least affected The specific broadening of proton signals was monitored using TOCSY spectra at the SDS/12-doxylPtdCho molar ratio of 60 : 1, i.e approximately one spin-label per micelle The presence of 12-doxylPtdCho caused the complete disappearance of the cross-peaks of Ala13 in the TOCSY spectrum (Fig 6B), a significant reduction in intensities of the cross-peaks of Leu9 and Phe16, and a moderate reduction in intensities of the
cross-Fig 4 Effects of pH on secondary structures of DMT1-TM4 in SDS
micelles The NOE connectivities of DMT1-TM4 in SDS micelles at
pH 4.0 (top) and pH 7.4 (bottom) are summarized.
Fig 5 Comparison of NMR structures of
DMT-TM4at different pH values as well as in
different membrane-mimetic environments (A)
Backbone atoms of 16 structures of DMT1 in
SDS micelles at pH 4.0 (green) are overlaid
with those at pH 7.5 (blue) The structures are
superimposed over the backbone atoms of
Ile10–Val17 for a best fit (B) Backbone atoms
of 16 structures of DMT1 in SDS pH 4.0
(green) are superimposed with those in TFE
(red) over the backbone atoms of Leu9–
Phe20.
Trang 9peaks of Ile10, Ile12 and Val17 This suggested that the
peptide was inserted in the interior of micelles However,
little effect was observed for Asp22, Lys23 and Tyr24,
indicating that the C-terminus is probably exposed outside
the micelles The N-terminus residues (Val2, Leu4 and Tyr5)
surprisingly decreased their intensities significantly in the
presence of 12-doxylPtdCho, implying that the N-terminus
may also be located inside the micelles
Mn2+ and Gd3+ broadening It has been shown
previ-ously that paramagnetic metal ions particularly affect
resonances of water and the surface of SDS micelles [49]
The paramagnetic broadening effects of Mn2+and Gd3+
on the peptide resonances were studied by comparing 1D
1H and 2D TOCSY or NOESY spectra in the presence and
absence of the paramagnetic metal ions The amplitudes of
the spectra in the presence of the paramagnetic metal ions
were normalized to the least affected cross-peaks The
paramagnetic metal ions were titrated into the samples
containing 2 mM DMT1-TM4 in 300 mM SDS-d25, and
some of the results are shown in Figs 6 and 7
Addition of 0.1 mMMn2+to DMT1 in SDS micelles at
pH 5.5 led to the complete disappearance of cross-peaks of
the C-terminal residues in the 2D TOCSY spectrum of
Phe18–Tyr24, indicating that these residues might locate
outside SDS micelles A 3–4 periodic residue broadening
was noticed from residues of Ile10–Phe18 The intensities of the Ha-HN cross-peaks of Thr11, Ile12 and Thr15 were drastically reduced, and Asp14 completely disappeared in
Fig 6 Effects of paramagnetic agents on the TOCSY spectra (HN-Haregion) of 2 m M DMT1-TM4in 300 m M SDS- d25 at 298 K and pH 5.5 (A) In the absence of paramagnetic agents (B) In the presence of 5 m M 12-doxylPtdCho (12-doxylPC) (C) In the presence of 0.2 m M Mn 2+ (D) In the presence of 0.1 m M Gd3+.
Fig 7 pH-regulated location of the C-terminus in SDS micelles Residual relative intensities of H a -H N 2D cross-peaks of DMT1-TM4
in SDS micelles in the presence of 0.2 m M Mn2+at pH 5.5 (j) and 4.0 (d) Those calculated from H a -H b cross-peaks are represented as s.
Trang 10the presence of 0.2 mMMn2+(Figs 6 and 7) This suggests
that there is possible solvent accessibility in the structure,
presumably due to formation of a channel through self
association of peptide monomers In contrast, the
N-terminal residues (Val2 and Leu4–Leu9) almost retained
their intensities, indicating that the N-terminus was
embed-ded in the SDS micelles and was solvent inaccessible When
Mn2+ concentration was increased to 1 mM, only the
N-terminal residues were observable in the TOCSY
spec-trum at pH 5.5 (H Li, F Li, Z M Qian & H Sun,
unpublished observation) Similar effects of Mn2+on the
resonances of DMT1 in SDS micelles were observed at
pH 7.4 However, the effects were more pronounced at this
pH value than at pH 5.5, in particular for the residues of
Ile10, Ile12, Ala13, Phe16 and Val17, which either
signifi-cantly reduced the intensities or completely disappeared
from the TOCSY spectrum in the presence of 0.2 mM
Mn2+(H Li, F Li, Z M Qian & H Sun, unpublished
observation)
Titration of Gd3+ (0.1, 0.2 and 0.4 mM) into 2 mM
DMT1-TM4 containing 300 mM SDS-d25 at pH 5.5 was
also made It was noticed that the N-terminal residues
(Val2–Leu9) almost retained their intensities, whereas the
C-terminal residues and those involved in the formation of
helix completely disappeared from the TOCSY spectrum
(Fig 6D) in the presence of 0.1 mMGd3+at pH 5.5 This is
in agreement with the Mn2+titrations, but the
paramag-netic effects were more evident in the presence of Gd3+
Further addition of Gd3+ (0.4 mM) led to only a slight
decrease in the intensities of the remaining cross-peaks
(H Li, F Li, Z M Qian & H Sun, unpublished
observation)
Interestingly, when the pH was lowered from 5.5 to 4.0 in
the presence of paramagnetic metal ions (0.2 mMMn2+or
Gd3+), we unexpectedly observed that the resonances not
observable at pH 5.5 regained their intensities at pH 4.0 in
both 1D and 2D NMR spectra Figure 7 shows the
normalized Ha-HN cross-peak intensities for the peptide
in the presence of 0.2 mM Mn2+at pH 4.0 and 5.5 The
residual intensities for Leu19 and Tyr24 were calculated
from the cross-peaks of Ha-Hb, as the amide protons of
Tyr24 and Leu19 overlapped with Asp22 and Thr15,
respectively, in the presence of Mn2+ at pH 4.0 As
illustrated in Fig 7, residues Leu9–Phe20 almost completely
regained their intensities; while residues Leu21–Tyr24
regained only 40% of their intensities This was still the
case even in the presence of 1.0 mM Mn2+, although the
spectra were considerably broader (data not shown)
Simi-larly, the intensities of the cross-peaks were also recovered in
the presence of Gd3+at pH 4.0, but the degree to which the
intensities recovered was lower in the presence of Gd3+,
particularly for the C-terminal residues, than in the presence
of the same amounts of Mn2+(H Li, F Li, Z M Qian &
H Sun, unpublished observation) This indicated that the
peptide is probably embedded completely inside the SDS
micelles by shrinking the C-terminus towards the micelles,
thus the entrance of the Mn2+is probably blocked
Discussion
In the present study, we have characterized the 3D
structures of DMT1-TM4 peptide in membrane-mimetic
environments, e.g TFE and SDS micelles at different pH values by NMR spectroscopy and simulated annealing calculations The solution structures of DMT1-TM4 in both environments are remarkably similar, consistent with our previous CD studies [24] The N-terminal segment is unstructured and highly flexible, and consists of residues Arg1–Gly7 in both environments The best-defined helical region in TFE involves residues Leu9–Phe20, with a rmsd value of 0.23 A˚ for the backbone atoms, which is further supported by the slowamide proton exchange of these residues (Fig 2B); while the C-terminal segment (Leu21– Tyr24) folded into a conformation which is extremely close
to helical folding, based upon both Ramachandran plot and angular order parameters for backbone / and w dihedral angles ( 1.0) Similarly, the peptide adopts an a-helical conformation in SDS micelles However, the folding of the peptide is sensitive to the pH, and particularly for the C-terminus It forms a helix comprising residues Leu9– Lys23 at pH 4.0 vs Leu9–Val17 at pH 7.4 (Fig 5) Interestingly, the C-terminal part became well folded only
at lowpH values (e.g pH 4.0) This is probably due to the protonation of Asp22 (pKa) which consequently has less repulsion with the anionic head group of SDS molecules Whether the C-terminus has a regulative role in metal transport remains to be investigated further
It is of interest to investigate whether the peptide is inserted into the micelles or lies along the micelle surface Relaxation probes have been widely used to determine micelle-embedded [50,51] or water exposed fragments of polypeptides [52] In the present study, paramagnetic broadening effects on the peptide resonances were used to investigate the topology of the peptide relative to the SDS micelle surface This includes 12-doxylPtdCho and para-magnetic metal ions (Mn2+and Gd3+) Although we have previously used 5- and 16-doxyl-stearic acids to probe the location of the peptide relative to the micellar surface [24], the N-terminus was found to be affected by both spin-labels, and it is therefore hard to drawa firm conclusion for its location In addition, we could not exclude the possibility that the positively charged N-terminus interacts directly with the negatively charged carboxyl-function from the stearic acids, thus forcing the N-terminus into spatial proximity with the spin-labels In order to avoid this effect,
we therefore used 12-doxylPtdCho to study the topology of the peptide in SDS micelles The doxylPtdCho does not perturb the peptide structures as a NOESY spectrum recorded after addition of the spin label a week later, a period during which the free-radicals are normally quenched, shows almost identical NOE cross-peaks com-pared with those in the absence of the spin label (H Li,
F Li, Z M Qian & H Sun, unpublished observation) The presence of 12-doxylPtdCho, similar to 16-doxyl-stearic acids, caused complete disappearance of Ala13 and signi-ficant reduction intensities of Ile12 and Phe16 However, there were no changes on the C-terminal residues, which clearly suggested that the peptide was inserted into the interior of SDS micelles with the C-terminal exposed outside the micelles 12-doxylPtdCho again reduced dramatically the intensities for the cross-peaks of the N-terminal (Val2, Leu4, Tyr5) residues (Fig 6)
Although spin-label broadening is an effective approach
to study the membrane location of peptides, it is difficult to