Báo cáo khoa học: Structure and topology of the transmembrane domain 4 of the divalent metal transporter in membrane-mimetic environments docx

Eur J Biochem 271, 1938–1951 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04104.x Structure and topology of the transmembrane domain of the divalent metal transporter in membrane-mimetic environments Hongyan Li1,2, Fei Li1, Zhong Ming Qian2 and Hongzhe Sun1 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-transmembrane domain protein responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues The transmembrane domain (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 environments, 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-terminus 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 aqueous 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 transported These results might provide an insight into the structure-function relationship for the integral DMT1 The divalent metal transporter (DMT1) gene, also known as Nramp2 (natural resistance-associated macrophage protein2) 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 enterocytes 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 show different 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 pHdependent 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) 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-phosphatidylcholine; 12-doxylPtdCho, doxylPtdCho lipids containing the nitroxide label on C12; DMT1, divalent metal transporter; DMT1-TM4, transmembrane domain of DMT1; HFIP, 1,1,1,3,3,3,-hexafluoro-2propanol; 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) Keywords: DMT1; membrane; NMR; structure Ó FEBS 2004 Structure and topology of TM4 of DMT1 (Eur J Biochem 271) 1939 Although 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), detergent 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 (doxylPtdCho) 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 Laboratories (Cambridge, MA, USA) Palmitol(doxyl)-stearoylphosphatidylcholine (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 spectropolarimeter 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 lM in TFE Spectra were recorded from 190 to 260 nm at a scan rate of 50 nmỈmin)1 with a respond time of 0.25 s, step resolution of 0.1 nm and band width of 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,  mg of the peptide dissolved in HFIP was mixed with an equal volume of SDS-d25 aqueous 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 spectrometer, operating at a proton frequency of 600.13 MHz Water suppression was carried out using a 3-9-19 watergatepulse sequence [25,26] The sodium salt of trimethylsilylpropionate-d4 solution 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 MLEV17 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 experiments 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-timeproportional 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 SPARKY software [30] and served as an input for Ó FEBS 2004 1940 H Li et al (Eur J Biochem 271) the 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 in CYANA [31] The final nonredundant upperlimit constraints and the resulting angle constraints were used in the structural calculations No stereospecific assignments were obtained in any case The 200 randomized starting structures were energy minimized during 4000 steps under the NMR constraints, and the 30 CYANA conformers 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 A in AMBER7 [33,34] From these calculated structures, 16 conformers with the lowest energy were selected to represent the NMR structures The quality of the final structures was accessed using the program of PROCHECK-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 program MOLMOL [36] Paramagnetic broadening experiments Samples containing mM DMT1-TM4 and 300 mM SDSd25 in 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 12doxylPtdCho 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 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 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, 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 onedimensional 1H-NMR spectra at 10, 30, 60, 90, 120 and 360 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–HN region of TOCSY spectrum were regarded as slowly exchanging amide protons In SDS micelles, 0.6 mL D2O was added to lyophilized samples containing 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 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 slow exchanging 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 insoluble 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 environments 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-d25 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 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–HN region 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 few aromatic 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 Ha protons provide information about secondary structural elements of the peptides Generally, all residues experience a Ha upfield shift relative to the random-coil value when adopting a helical conformation 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 examining the observed NOE connectivities produced similar results All HN resonances from Tyr5 to Phe20 were found to be connected by (i, i+1) connectivities except for Phe16 Ó FEBS 2004 Structure and topology of TM4 of DMT1 (Eur J Biochem 271) 1941 Fig Fingerprint region of the 600 MHz NOESY spectra of DMT1-TM4 (A) 200 ms NOE spectrum of mM DMT1-TM4 in 300 mM SDS-d25 at pH 6.0, 298 K (B) 150 ms NOE spectrum of mM peptide in TFE-d2, 305 K The sequential assignment of all residues is indicated and 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 demonstrated high helical contents in the DMT1-TM4 peptide [24] Furthermore, we also observed that the chemical shift for threonine Hb is greater than that of Ha for 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 coupling constants Structure calculations and description Distance constraints were obtained from NOESY spectra recorded with a mixing time of 200 ms measured in 90% H2O/10% D2O (v/v) containing mM peptide 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 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 HN =HN [38], and sequential i iỵ1 NOEs were observed in the segment of Tyr5Phe20 In addition, the presence of medium-range connectivities [38], such as Ha =HN , Ha =Hb and Ha =HN was also observed i iỵ3 i i iỵ4 iỵ3 for Val8Phe20 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 =HN and HN =HN , which are characteristic i iỵ2 i iỵ2 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 1942 H Li et al (Eur J Biochem 271) Ó FEBS 2004 Fig Summary of NMR spectroscopy data for secondary structure prediction for DMT1-TM4 peptide (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 Totals 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 macro GRIDSEARCH as 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 program CYANA [31] Under this protocol, 200 randomized starting structures 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 low values 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 program AMBER7 [33,34] in the AMBER force 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 The quality of the final structures was assessed using the program PROCHECK-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 MOLMOL showed 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 Ó FEBS 2004 Structure and topology of TM4 of DMT1 (Eur J Biochem 271) 1943 Table Structural statistics for DMT1-TM4 in the presence of SDS at pH 6.0 and in TFE SDS TFE (pH 6.0, 298 K) (305 K) Parameter ˚ Target function (A2) Experimental NMR constraints No of distance constrains Intraresidues Sequential (i ¼ 1) Medium range (1 < 1/2i-j1/2 £ 4) Long range (1/2i-j1/2 > 4) ˚ NOE constraint violations (A) Sum Maximum 0.03 ± 0.01 241 111 89 41 265 118 75 72 0 0.30 ± 0.10 0.08 ± 0.02 1.10 ± 0.20 0.18 ± 0.02 AMBER energy (kcalỈmol)1) )856.7 ± 6.2 ˚) rmsd from the mean structure (A Residues 1–24 Backbone atoms 4.07 ± 1.28 All heavy atoms 5.37 ± 1.36 Residues 9–20 Backbone atoms 0.20 ± 0.06 All heavy atoms 0.89 ± 0.16 Ramachandran statistics (residues 9–20)a (%) The most favored regions 99.4 Additional allowed regions 8.9 Generously allowed regions Disallowed regions a Analyzed using 0.12 ± 0.02 )857.2 ± 5.1 2.84 ± 0.90 4.00 ± 1.05 0.23 ± 0.10 0.82 ± 0.17 91.7 8.3 0 PROCHECK-NMR prediction based on the chemical shift index However, the pairwise rmsds between these structures and mean structures 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 ! COi hydrogen bonds were iỵ4 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 HN ! COi hydrogen bond was iỵ3 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 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 conformation 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 simulated 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) nonredundant 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 Ha =HN , Ha =Hb and Ha =HN (Fig 4) i iỵ3 i i iỵ4 iỵ3 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, HN ! COi hydrogen bonds indicative of a-helices iỵ4 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 characteristic 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 1944 H Li et al (Eur J Biochem 271) Ó FEBS 2004 Fig Stereoview of NMR structures of DMT1-TM4 in 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 backbone atoms of Leu9Phe20 Moreover, some structures displayed HN ! COi hydrogen iỵ3 bonds indicative of 310 helices between Ala13 and Phe16, and also between Ile12 and Thr15 The structures of DMT1-TM4 in TFE were superimposed 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 h (except Gly6, which disappeared 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 Ó FEBS 2004 Structure and topology of TM4 of DMT1 (Eur J Biochem 271) 1945 lyophilized sample containing SDS-d25 at pD 5.5 Residues involved in the formation of the helix mostly retained their intensities in the first h, except Thr11 and Asp14 A NOESY spectrum recorded after 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 conformations 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 Fig 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 showed 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 after addition of D2O to a Fig Comparison of NMR structures of DMT-TM4 at 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 12-DoxylPtdCho Information from 1H line-broadening caused by the lipid-soluble spin-labeled compound 12doxylPtdCho 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/12doxylPtdCho 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- 1946 H Li et al (Eur J Biochem 271) Ó FEBS 2004 Fig Effects of paramagnetic agents on the TOCSY spectra (HN-Ha region) of mM DMT1-TM4 in 300 mM SDS-d25 at 298 K and pH 5.5 (A) In the absence of paramagnetic agents (B) In the presence of mM 12-doxylPtdCho (12-doxylPC) (C) In the presence of 0.2 mM Mn2+ (D) In the presence of 0.1 mM Gd3+ peaks 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 previously 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 H 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 mM DMT1-TM4 in 300 mM SDS-d25, and some of the results are shown in Figs and Addition of 0.1 mM Mn2+ 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 Fig pH-regulated location of the C-terminus in SDS micelles Residual relative intensities of Ha-HN 2D cross-peaks of DMT1-TM4 in SDS micelles in the presence of 0.2 mM Mn2+ at pH 5.5 (j) and 4.0 (d) Those calculated from Ha-Hb cross-peaks are represented as s 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 Ó FEBS 2004 Structure and topology of TM4 of DMT1 (Eur J Biochem 271) 1947 the presence of 0.2 mM Mn2+ (Figs 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 embedded in the SDS micelles and was solvent inaccessible When Mn2+ concentration was increased to mM, only the N-terminal residues were observable in the TOCSY spectrum 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 significantly 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 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 mM Gd3+ at pH 5.5 This is in agreement with the Mn2+ titrations, but the paramagnetic 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 mM Mn2+ 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 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) Similarly, 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 ÔblockedÕ 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 slow amide 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 low pH 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 paramagnetic 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 draw a 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 compared 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 significant 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 1948 H Li et al (Eur J Biochem 271) estimate the exact position of peptides relative to micelle surface because doxyl spin-labels broaden the proton resonances from a large distance Paramagnetic broadening via addition of paramagnetic metal ions was therefore applied as an additional probe to address the topology of the peptide Our results showed that few broadening effects have been observed for the N-terminal residues e.g Val2– Leu9 (Figs and 7) at both pH 5.5 and 4.0, implying that the N-terminus was located inside the micelles This is unexpected considering the high energetic cost of partitioning the peptide bonds into the hydrocarbon core [53] The Ha-HN resonances of the C-terminal residues Phe18–Tyr24 were unobservable in the presence of paramagnetic metal ions at pH 5.5, suggesting that these residues are situated at the micelle surface or outside micelles at this pH value Surprisingly, a periodic residue broadening was observed for the residues in the a-helical region at pH 5.5 (Fig 7) Asp14 completely disappeared, while the intensities of Thr11, Ile12 and Thr15 significantly decreased and this effect was propagated in the presence of Gd3+ This phenomenon seems at odds with the results obtained from spin-labeling experiments as the paramagnetic metal ions not enter the micelle and should only broaden the residues located in aqueous phase and/or at the surface of SDS micelles [49] Previously, Mn2+ has also been used as a paramagnetic probe to explore solvent-exposed residues for membrane peptides Relative high concentrations ( 1– mM) were used to broaden the residues located outside the micelles, and membrane-spanning regions were found to be unaffected even at such high concentrations, while the intensities for solvent-exposed segments either completely disappeared or were drastically reduced [54–56] However, in the present study, mM Mn2+ caused complete disappearance of the cross-peaks of almost all residues at pH 5.5, except for the N-terminal residues A possible explanation is that there is solvent accessibility in the peptide-micelle aggregate, and this possibility is also supported by the fast amide proton exchange for those residues situated in the helical-core region in SDS micelles (Fig 2) The amide protons of Thr11, Asp14 and Thr15, which were buried inside SDS micelles, were found to exchange more rapidly compared with other residues in the helical region in SDS This was particularly the case for Asp14, which completely exchanged with deuterium (D2O) within 30 in SDS micelles Interestingly, the paramagnetic effect of Mn2+ (0.2 mM) on NMR resonances of the peptide was barely observable at pH 4.0, except for the residues of Leu21– Tyr24, which lost  60% of their intensities This suggests that the C-terminus of the peptide is partially embedded into SDS micelles at pH 4.0, which may therefore block the entrance of either solvent molecules or metal ions The movement of the C-terminus relative to SDS micelles at different pH values is also supported by the calculated structures, in which the C-terminus was folded into helix only at low pH values (e.g pH 4.0) This phenomenon, that the residues situated near micellar aqueous boundary can move Ôup and downÕ at different pH values, has been noticed previously [57] Alternatively, at such a pH ( 4), a relatively high concentration of protons may compete with Mn2+ and thus retard the entry of this metal ion at pH 4.0 For the best explanation of our experimental data, we hypothesize that a channel comprised of several peptide Ó FEBS 2004 Fig Self association of DMT1-TM4 in TFE CD spectra of DMT1TM4 at different concentrations ranging from lM to 750 lM (top) and dependence of molar residue ellipiticity at 222 nm on peptide concentration, indicative of intermolecular interactions (bottom) monomers might be formed, which allows the permeation of metal ions Our CD study has shown the change of the molar residue ellipiticity of the peptide at 222 nm on peptide concentration (Fig 8), indicating the presence of intermolecular interactions Neither the oligomerization by SDSTricine-PAGE [24], nor intermolecular NOE peaks in NOESY spectra were observed An attempt to test aggregation behavior in SDS micelles by means of a similar approach was not made, as the aggregation probably depends on both SDS concentrations and the ratio of SDS to the peptide It is reasonable to assume that peptide may have a similar aggregation behavior in SDS micelles The peptide helical monomers are probably orientated with the polar face (e.g Thr11, Asp14 and Thr15) pointing to each other in the inner lumen of the oligomer, to reduce the unfavorable free energy caused by immersing these residues in the hydrophobic milieu Interactions between the polar residues may be mediated by water molecules The oligomeric assembly in the membrane thus may be a loose association between several monomers, which is probably highly symmetric judging from unobservable intermolecule NOEs Twenty one out of 33 known structures possess a helical bundle topology (http://blanco.biomol.uci.edu/ Membrane_Proteins_xtal.html), suggesting that pore or channel-forming assembly may be the common features for the function of transmembrane proteins It has been Ó FEBS 2004 Structure and topology of TM4 of DMT1 (Eur J Biochem 271) 1949 reported previously that model peptides, corresponding to a segment of integral proteins, have been found to be able to resume functional assembly in lipids or even in detergent micelles, such as formation of channels or pores, as their integral proteins [15,58,59] The significant broadening effects of paramagnetic metal ions, e.g Mn2+ and Gd3+ on the peptide NMR signals in the present study are probably either due to permeation directly or exchange of Mn2+ and Gd3+ bound water with the water and amino protons in the channel In contrast, no observable changes in 2D NOESY were found upon addition of diamagnetic metal ions (Zn2+) under similar conditions (H Li, F Li, Z M Qian & H Sun, unpublished observation) Interestingly, when the pH value was lowed to 4.0, almost all the resonances in NMR spectra recovered their intensities, indicating the movement of the C-terminus towards SDS micellar interior, thus making either metal ions or solvent molecules inaccessible In conclusion, we have determined the solution structures of a synthetic peptide, corresponding to the TM4 of DMT1 in membrane-mimetic TFE and SDS micelles The peptide adopts remarkably similar structures in both environments The structures consist of three regions: a well-defined helical region Leu9–Phe20 in TFE, a highly disordered N-terminus (Arg1–Gly7) and a pH-sensitive C-terminus, which folds into a helix only at 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Copper, M.A., Arkin, I.T & Bowie, J.H (2002) The orientation of the antibiotic peptide maculatin 1.1 in DMPG and DMPC lipid bilayers Support for a pore-forming mechanism FEBS Lett 512, 47–51 Supplementary material The following material is available from http:// blackwellpublishing.com/products/journals/suppmat/EJB/ EJB4104/EJB4104sm.htm Table S1 Chemical shifts for DMT1-TM4 in TFE and SDS  ... 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 Ó FEBS 20 04 Structure and topology of TM4 of. .. Phe16 Ó FEBS 20 04 Structure and topology of TM4 of DMT1 (Eur J Biochem 271) 1 941 Fig Fingerprint region of the 600 MHz NOESY spectra of DMT1-TM4 (A) 200 ms NOE spectrum of mM DMT1-TM4 in 300 mM SDS-d25... residues of Ile10–Phe18 The intensities of the Ha-HN cross-peaks of Thr11, Ile12 and Thr15 were drastically reduced, and Asp 14 completely disappeared in Ó FEBS 20 04 Structure and topology of TM4 of