Research Article Received: 9 July 2013
Revised: 29 September 2013
Accepted: 30 October 2013
Published online in Wiley Online Library: 22 January 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2593
Insight into the structures of the second and fifth transmembrane domains of Slc11a1 in membrane mimics Li Wang, Dan Wang and Fei Li* Slc11a1 is an integral membrane protein with 12 putative transmembrane domains and functions as a pH-coupled divalent metal cation transporter. In the present study, the structures of the peptides corresponding to the second and fifth transmembrane domains of Slc11a1 (from 88 to 109 for TMD2 and from 190 to 215 for TMD5) were determined in membrane-mimic environments by CD and NMR techniques. It was demonstrated that TMD2 and TMD5 form an α-helical structure in 30% 2,2,2-trifluoroethanol (TFE) and 40% hexafluoro-2-propanol (HFIP) aqueous solution, respectively. The α-helix of TMD5 displays a less space-occupied face consisting of the residues Ala194, Gly197, Thr201, Ala204 and Gly208. The α-helix is partially unfolded in the N-terminal region when Gly197 is substituted by Val. The unfolding of the helix in the N-terminal part and/or increase in volume at the less space-occupied face of the helix may exert an effect on the arrangement of TMD5 in membrane. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: Slc11a1; transmembrane domain; structure; NMR
Introduction
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* Correspondence to: Fei Li, State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Avenue, Changchun 130012, China. E-mail: [email protected] State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130012, China
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
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Slc11 (solute carrier family 11), formally named as natural resistance-associated macrophage protein, is the protein family of proton-coupled transporters with 10–12 transmembrane domains. The homologues of Slc11 have been found in mammals, birds, insects, plants, fish and bacteria [1–5]. The structure and function of the protein family have been remarkably conserved throughout evolution. Slc11a1 and Slc11a2, the two members of the protein family with 12 transmembrane domains, share 63% amino acid sequence identity and an overall homology of 78% in humans and rodents [6,7]. Both proteins can transport divalent metal cations in a pH-dependent fashion [7,8]. Slc11a2 [or divalent metal-ion transporter 1 (DMT1)] is expressed at the apical membrane of duodenal enterocytes [9] and in recycling endosomes of peripheral tissues and mediates transfer of iron internalized by transferrin from the endosomes to the cytoplasm [10]. Deficiency of Slc11a2 is related to a hypochromic microcytic anemia [11–13]. The expression of Slc11a1 is restricted to the membrane of mature phagocytes in steady-state conditions and further stimulated by inflammatory signals [14–16]. Slc11a1 regulates divalent cation concentration within macrophage phagosomes [17–20]. Deficiency of Slc11a1 causes sensitivity to several intracellular pathogens. Some naturally occurred mutations in Slc11a2/DMT1 of murine and human, such as V114 deletion in TMD2 [13], G185R in TMD4 [11,12], G212V in TMD5 [13] and R416C in TMD9 [21], have been identified to be responsible for severe hypochromic microcytic anemia. The single G169D substitution in TMD4 of Slc11a1 has been found to be associated with susceptibility of murine to infections with intracellular parasites such as Leishmania, Mycobacterium and Salmonella [14,22]. Some residues in TMD1, TMD3 and TMD6 of both DMT1 and MntH (Slc11 homologue from Escherichia coli) were also detected by mutation assays to be crucial for the divalent metal-ion transport
of the Slc11 proteins [23–25]. The mutations of the specific residues in these domains may influence the transport activity of the proteins by changing binding affinity of certain residue(s) with metal ions or by changing the interactions between transmembrane domains and thus vary the size or property of the pore formed by the transmembrane domains. A number of model systems have been used to mimic features of membranes in the study of structures of membrane proteins. TFE and HFIP are two of these systems because of their effects on stabilizing the secondary structures of polypeptides. The clusterization property of the fluorinated solvents in aqueous solution was pointed as primary reason for the effect. The clusters of TFE or HFIP may cooperatively associate with the hydrophobic surface of an α-helix (or a β-strand), thus mimicking the environment of a membrane or the interior of a protein. Possible mechanisms by which alcohol-based cosolvents affect polypeptide structure include the enhancement of polypeptide internal hydrogen bonding, the disruption of water structure and lessening of the hydrophobic effect, the penetration of cosolvent molecules into the protein core and preferential solvation of certain groups of the polypeptide chain [26,27]. Many studies have demonstrated that TFE is a suitable model system of membrane [28–32]. In some circumstances, the structures of polypeptides obtained in TFE are even similar to those adopted in their active conformations and could be correlated with their biological activity [33–35].
WANG, WANG, AND LI In our previous work, we have studied the structures and topologies of the peptides corresponding to the TMD1 and TMD6 of Slc11a2/DMT1 [36–39], TMD3 and TMD4 of Slc11a1 [40–45] and some peptides from specific site mutations of these transmembrane domains in both organic solvents and detergent micelles using CD and NMR methods. We also studied the secondary structures and topologies of TMD1-TMD5 of Slc11a1 in lipid membranes by CD and attenuated total reflectance (ATR) FTIR techniques. We found some changes in structure, location relative to membrane surface and assembly of these transmembrane domains because of specific site mutations. In the present paper, we studied the three dimensional structures of the peptides corresponding to TMD2 and TMD5 of mouse Slc11a1 (mSlc11a1) and the mutant G197V of TMD5 (corresponding to G212V of TMD5 in mSlc11a2) in fluoroalcohol aqueous solutions by CD and NMR. Because both sequences of TMD2 and TMD5 are highly conserved in Slc11a1 and Slc11a2 (Scheme 1), the structure of TMDs in Slc11a1 obtained in this study could be considerably suggestive of the structure of corresponding TMDs in Slc11a2.
Experimental Materials The peptides were synthesized by solid phase FMOC chemistry (Shanghai Apeptide Co., Ltd., Shanghai, China) and purified by reverse phase HPLC on a C18 column using a solvent system consisting of 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B). Elution was achieved by applying a linear gradient from 100% to 20% A in 30 min. Three Lys residues were added at each terminal end of TMD2, and two Lys residues were added at the N-terminal ends of both TMD5 and TMD5-G197V mutants to facilitate purification. The molecular weights of the peptides (3306.27, 3259.98 and 3302.06 Da for TMD2, TMD5 and TMD5-G197V, respectively) were confirmed by mass spectroscopy, and their purities of >95% were estimated by HPLC. Deuterated 2, 2, 2-trifluoroethanol (TFE-d2; 98%) and 1,1,1,3,3,3-hexafluoro-2propanol (HFIP-d2, 99.5%) were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Sample Preparation The peptide TMD2 was solubilized using 30%TFE/70%H2O (v/v) for the CD and fluorescence experiments or using 30%TFE-d2/ 70%H2O for the NMR experiments. The peptides TMD5 and TMD5-G197V were solubilized using 40%HFIP/60%H2O for the CD and fluorescence experiments or using 40%HFIP-d2/60%H2O for the NMR experiments. The samples of peptides incorporated with SDS micelles were prepared using the method as described previously [46]. Deionized water was used in preparation of the mSlc11a1 TMD2
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mSlc11a2 TMD2
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mSlc11a1 TMD5
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mSlc11a2 TMD5
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SDS samples for fluorescence experiments, and 10 mM Citric acid/Na2HPO4 buffers at pH 5.5 and 7 were used in preparation of the SDS samples for CD experiments.
Far-UV CD Spectra Far-UV CD spectra were recorded on a Jasco J-810 spectropolarimeter (Jasco Co., Tokyo, Japan) using a 0.5-mm path length cuvette. The concentrations of 20 μM peptide and 10 mM SDS detergent were used in the experiments. The spectra were scanned over the wavelength range of 190–260 nm with data pitch 0.1 nm and bandwidth 1.0 nm at a sweep speed of 50 nm min 1. Three scans were acquired and averaged, and the CD signal of solvent was subtracted.
Fluorescence Measurements All fluorescence spectra were recorded on RF-5301PC fluorescence spectrophotometer (Shimadzu, Kyoto, Japan) at room temperature. In the fluorescence experiments, the concentration of SDS detergent was fixed at 8 mM, while the concentration of peptide was changed in a range of peptide : detergent from 1 : 25 to 1 : 200. The spectra were scanned in a range of 290–420 nm with excitation of 280 nm and slit size of 3 nm (excitation) × 5 nm (emission). Three spectra were recorded in an interval of ca. 5 min and averaged, and the reference spectrum of the respective medium was subtracted.
NMR Experiments All NMR spectra were recorded on a Bruker Avance 500 spectrometer (Bruker, Fällanden, Switzerland) with a z-gradient coil and a 5-mm triple resonance broadband inverse (TBI) probe at 298 K. The samples of 2 mM TMD2 peptide in 240 mM SDS detergent and 2 mM TMD5 peptides in 180 mM SDS detergent were used in all NMR experiments. The TOCSY and NOESY experiments were performed using a mixing time of 100 and 200 ms, respectively. The transients of 48 for TMD2 and 64 for TMD5 and TMD5G197V were acquired for each increment in t1. Water signal was suppressed using WATERGATE technique. Sodium salt of 2,2-dimethyl-2-sila-pentane-5-sulfonic acid (DSS) were used as an internal standard for proton chemical shifts. The 2D NMR spectra were measured with 2048 data points in F2 dimension and 512 data points in F1 dimension and processed using standard Bruker software (Bruker, Fällanden, Switzerland). NMR assignments were achieved using software Sparky [47]. The structures of the peptides were calculated, evaluated and displayed using the programs CYANA (1.0.6) [48], PROCHECK-NMR [49] and MOLMOL [50], respectively.
KLLWVLLWATVLGLLCQRLAA109R KLLWVLL LAT IVGLL LQRLAA124R
RKLEAFFGLLITIMALTFGYEYVVA215H RKLEAFFGFLITIMALTFGYEY ITV230K
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Scheme 1. Sequences of the peptides studied (mSlc11a1 TMD2 and TMD5) and their homologies in mSlc11a2. The residues with gray shade are non-conserved in mSlc11a1 and mSlc11a2, and the tilted residues in mSlc11a2 TMD2 and TMD5 (V114 and G212) are mutation sensitive for microcytic anemia.
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STRUCTURES OF THE SECOND AND FIFTH TRANSMEMBRANE DOMAINS OF Slc11a1 in the same conditions. This suggests that the mutation may make part of helix unfolded. A common difference between the CD spectra of the peptides in organic solvents and in SDS micelles is found. The ratios of θ222/θ208 for the peptides in SDS micelles are closer to 1 than those for the peptides in organic solvents. More negative absorbance at 222 nm for the peptides in SDS micelles is associated to the aggregation of the peptides via coiled-coil interactions in the micelles [51,52]. As mentioned above, three Lys residues were added at each terminal end of TMD2, and two Lys residues were added at the N-terminal ends of both TMD5 and TMD5-G197V mutants in order to increase the hydrophilicity of the peptides and facilitate purification. The addition of the Lys residues at both ends of the peptides could decrease the propensity of peptide aggregation because of repulsive interaction among these positively charged residues and facilitate the incorporation of the peptides in SDS micelles because of increasing interaction between the positively charged Lys residues and the negatively charged headgroups of the micelles. The addition of the Lys residues may also have effect on the secondary structures of the peptides in certain situation [53]. However, the effect could be rather small in TFE or HFIP aqueous solution because the stabilization of helical conformations in the cosolvents is mainly correlated with the conformational biases of the primary sequence [54]. Aggregation of the Peptides
Figure 1. The CD spectra of the peptides TMD2 (A), TMD5 (B) and TMD5G197V (C) in different environments.
Results and Discussion Structural Evaluation of the Peptides by CD
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The CD spectra of the peptides TMD2, TMD5 and TMD5-G197V in organic solvents and SDS micelles at pH 5.5 and 7 were recorded to analyze their secondary structures. As shown in Figure 1, the CD spectra of all the peptides are characterized by double negative minima at ca. 208 and 222 nm and a positive maximum at ca. 194 nm. This suggests that the α-helix structures are induced by these environments. Moreover, the CD spectra of the peptides in SDS micelles are basically independent of pH conditions. Compared with the wild type, the mutant peptide TMD5-G197V displays lower helicity both in 40%HFIP aqueous solution and in SDS micelles, as indicated by less negative molar ellipticity either at 208 nm or at 222 nm in the CD spectra of the mutant peptide
We measured the NMR spectra of the three peptides in SDS micelles. The spectral lines were severely broad and overlapped. When we solubilized TMD2 peptide in 30%TFE-d2 aqueous solution or solubilized TMD5-related peptides in 40%HFIP-d2 aqueous solution, we observed a dramatic improvement of the 1H NMR spectra (Figure 2). This indicates that the peptides may be aggregated in SDS micelles. The aggregate is disaggregated in TFE or HFIP aqueous solution because of strong ability of TFE and HFIP in dissociating peptide aggregate. Accordingly, the structures of the peptides at molecular level were studied by NMR in fluoroalcohol aqueous solutions but not in SDS micelles (see Structural Determination of the Peptides by NMR section). The aggregate of TMD2 in SDS micelles was further probed by self-quenching experiment of Trp fluorescence at various peptide concentrations. It was predicted that the fluorescence intensity should increase linearly with increasing peptide concentration if no peptide aggregation occurs, while the dependence should be nonlinear if peptide aggregates. Our result demonstrates that the fluorescence emission increases nonlinearly with increasing [TMD2]/[SDS] ratio and the intensities are lower than those predicted by a linear increase in the case of no aggregation (Figure 3). The quenching of fluorescence with increasing peptide concentration suggests a strong aggregate of the peptide in SDS micelles. The intermolecular interactions among tryptophan residues in an aggregate result in the decrease in fluorescence intensity. The fluorescence experiments of TMD5 peptides were not performed because of the absence of Trp in the peptides. It is noted that TMD2 includes two Trp residues located at positions 91 and 95, but only one fluorescence emission at 334 nm is observed in the fluorescence spectra of TMD2 incorporated with SDS micelles. This implies that the two Trp residues are involved in the similar environment in SDS micelles. Previous studies have shown that the fluorescence emission at ~330 nm is related to a hydrophobic ambient of Trp, while the fluorescence emission at ~350 nm is related to the exposure of
WANG, WANG, AND LI
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Figure 2. The 1D- H NMR spectra of TMD2 (A) and TMD5 (B) in different environments.
Figure 3. The fluorescence spectra (A) and the emission maxima at 334 nm (B) of TMD2 in SDS micelles at various peptide/SDS ratios.
Trp to a polar ambient [55]. Although the Trp at position 91 is close to the N-terminal end of the peptide, it is enclosed by the hydrophobic residues such as two Leu residues before it and one Val and two Leu residues after it. Therefore, both tryptophan residues are embedded in the hydrophobic region of the micelles, and the maximum emission at 334 nm is observed for TMD2 aggregated in SDS micelles. Structural Determination of the Peptides by NMR
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All 1H NMR resonances of TMD2 in 30%TFE-d2 or TMD5 and TMD5-G197V in 40%HFIP-d2 aqueous solution were assigned by combined analyses of their 2D TOCSY and NOESY spectra (see Table S1-S3 in Supporting information). The fingerprint regions of 2D NOESY spectra of the peptides in fluoroalcohol aqueous solutions with assignments of Hα-HN cross-peaks and NOE connectivities obtained from the NOESY spectra are shown in Figure 4. In the assignments of NMR cross-peaks, the residues in the sequences belonging to the putative transmembrane domains are numbered sequentially, while the extra added Lys residues at the N-terminal and C-terminal ends are defined as N1, N2 and so on and C1, C2 and so on, respectively. Some NOE connectivities of Hα(i)-HN(i + 3) and Hα(i)-HN(i + 4) for TMD5 and TMD5-G197V indicated in Figure 4D and 4F are not observed in Figure 4C and 4E because the intensities of these cross-peaks are lower than the intensity levels that are set for clear presentation of the spectra.
The NOE connectivities and chemical shift index [56] suggest a predominant α-helix structure for TMD2 peptide in 30%TFE-d2 aqueous solution. On the basis of the NOE connectivities, the three dimensional structure of the peptide was calculated. The NMR restraints used in calculations and structural statistics extracted from calculation results are listed in Table 1. Figure 5A shows ensemble of backbone atoms of the 20 structures with the lowest target functions for TMD2 in 30%TFE-d2 aqueous solution. The structures are well converged in the region of Leu3-Leu19. Ramachandran analyses for the 20 structures indicate that the dihedral angles of all residues involved in this region fall within the allowed regions of an α-helix. In the helix, the conservative residues Thr10, Gly13 and Gln17 face the same side, making this side more polar and less space occupied as compared with other sides of the helix. The similar structure could be predicted for the TMD2 of Slc11a2 protein based on high conservation of the two proteins in TMD2. This specific structure of TMD2 may be associated with the specific contact of the transmembrane domain with others. The deletion of V114 in TMD2 of Slc11a2 changes the arrangement of these residues in helix, e.g., the position of polar Thr is replaced by apolar Ala, by which the function of the protein may be influenced. The NOE connectivities of TMD5 in 40%HFIP-d2 aqueous solution predict an α-helical structure for nearly entire peptide, although some medium-range NOE connectivities of Hα(i)-HN (i + 3) and Hα(i)-HN(i + 4) are relatively weak. Compared with the
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STRUCTURES OF THE SECOND AND FIFTH TRANSMEMBRANE DOMAINS OF Slc11a1
Figure 4. The Hα-HN region of the NMR spectra and NOE connectivities with chemical shift index for TMD2 in 30%TFE-d2 solution (A and B), TMD5 in 40%HFIP-d2 solution (C and D) and TMD5-G197V in 40%HFIP-d2 solution (E and F).
of specific steric structure, which is associated with the function of the protein. The high conservation of TMD5 in Slc11a1 and Slc11a2 implies the structural similarity of this domain in the two proteins, i. e., the TMD5 of Slc11a2 may also fold as an α-helix with a less occupied face. The mutation of G212V in Slc11a2 (corresponding to G197V for Slc11a1) not only introduces a larger hydrophobic residues in the less occupied side but also leads to unfolding of the N-terminal part of the helix, which may have an effect on the interactions of the fifth transmembrane domain with other transmembrane domain(s). Deficiency in the activity of Slc11a2 due to G212V mutation may be associated with the change in the interaction of TMD5 with other(s), which is related to arrangement and topology of the transmembrane domains in membrane. Slc11a2 structural model has been predicted by homology threading based on the crystal structures of Mhp1, LeuT and vSGLT, which may represent discrete steps of a structural fold in cationdriven transport cycle shared by different families (e.g., Slc23, 6 and 5). The structural model shows twofold symmetry in the arrangement of transmembrane helices for the N-domain TMD1-5
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wildtype TMD5, the mutant TMD5-G197V loses the mediumrange NOE connectivities in the N-terminal part. Moreover, the chemical shifts of the α protons in the N-terminal parts of the mutant (such as Arg1-Glu4) are more downfield than those of the wildtype peptide (Figure 4C–F). These results suggest that the N-terminal part of the mutant peptide is less helical than that of the wildtype peptide. The structures of TMD5 and its G197V substitute in 40%HFIP-d2 aqueous solution were calculated based on the restraints from NOE connectivities (Figure 5B and 5C). The NMR restraints used in calculations and structural statistics extracted from calculation results are listed in Table 1. The backbone atoms of the 20 structures with the lowest target functions are basically converged in the region of Leu3-Val24 for TMD5 and Ala5-Val24 for TMD5-G197V. Nearly 100% residues in the converged portion fall in the allowed region of an α-helix according to the Ramachandran statistics for the dihedral angles of the 20 structures. It is noted that there is a less occupied face consisting of the residues Ala5, Gly8, Thr12, Ala15 and Gly19 in the α-helix of TMD5. This alignment of residues may be needed for the formation
WANG, WANG, AND LI Table 1. Statistics of 20 structures with the lowest target functions for TMD2 in 30%TFE-d2/70%H2O, TMD5 and TMD5-G197V in 40%HFIP-d2/60% H2O at 298 K TMD2 2
Average target functions (Å ) Number of nonredundant distance restraints Intraresidual (|i j| = 0) Sequential (|i j| = 1) Medium (|i j| ≤ 4) Long range (|i j| > 4) Average sum of distance restraint violations (Å) Average max. distance restraint violation (Å) Average sum of torsion angle restraint violations (°) Average max. of torsion angle restraint violation (°) RMS deviation from the mean structure (Å) All residues Backbone heavy atoms All heavy atoms Residues Backbone heavy atoms All heavy atoms Ramachandran plot statistics (for each helix span) Residues in most favored region (%) Residues in additionally allowed region (%) Residues in generously allowed region (%) Residues in disallowed region (%)
TMD5
TMD5-G197V
0.21 ± 0.06 252 130 60 62 0 1.8 ± 0.4 0.19 ± 0.05 0.0 ± 0.01 0.04 ± 0.14
0.03 ± 0.01 262 124 76 62 0 0.5 ± 0.1 0.09 ± 0.02 0.0 ± 0.0 0.0 ± 0.0
0.24 ± 0.05 275 132 72 71 0 1.7 ± 0.4 0.2 ± 0.04 0.5 ± 0.8 0.41 ± 0.69
3.75 ± 0.87 5.47 ± 1.18 3–19 0.70 ± 0.24 1.64 ± 0.32
2.16 ± 0.85 3.12 ± 0.89 3–24 1.20 ± 0.50 2.08 ± 0.55
2.85 ± 0.83 4.09 ± 0.89 5–24 1.26 ± 0.51 2.30 ± 0.73
81.6 17.8 0.6 0
82.6 16.8 0.6 0
73.4 21.9 4.4 0.3
Figure 5. Ensemble of backbone atoms of the 20 structures with the lowest target functions fitted over helix region for TMD2 in 30%TFE-d2/70%H2O (A), TMD5 (B) and TMD5-G197V (C) in 40%HFIP-d2/60%H2O.
and the C-domain TMD6-10 (conserved hydrophobic core of Slc11). Comparison of the TMDs arrangements in LeuT (open to outside/ occluded) with those in vSGLT (open to inside) shows the movements of the N-domain TMD1, 2 and 5 and the C-domain 6, 7 and 10 between the two models [57]. This indicates that the TMD2 and 5 may play a role in substrate uptake of Slc11 transporters by structural/topological modulations, although they may not directly participate in the binding with substrates.
in Slc11a1) with a more polar and a relatively less space-occupied face. The structural study of the peptide from the fifth transmembrane domain of Slc11a1 in 40%HFIP aqueous solution reveals that the peptide adopts an α-helical structure from Leu3 to Val24 (Leu192-Val213 in Slc11a1) with a full less spaceoccupied face. The specific structural characteristic of the two TMDs may determine their arrangement in membrane and function of the protein. The substitution of Gly197 by Val in TMD5 of Scl11a1 results in partial unfolding of α-helix in the Nterminal region, which may be function associated.
Conclusion
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The structural study of the peptide from the second transmembrane domain of Slc11a1 in 30%TFE aqueous solution reveals that the peptide folds as an α-helix from Leu3 to Leu19 (Leu90-Leu106
Acknowledgement This work was financially supported by the National Natural Science Foundation of China (20973083 and 20934002).
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STRUCTURES OF THE SECOND AND FIFTH TRANSMEMBRANE DOMAINS OF Slc11a1
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23 Lam-Yuk-Tseung S, Govoni G, Forbes J, Gros P. Iron transport by Nramp2/DMT1: pH regulation of transport by 2 histidines in transmembrane domain 6. Blood 2003; 101: 3699–3707. 24 Cohen A, Nevo Y, Nelson N. The first external loop of the metal ion transporter DCT1 is involved in metal ion binding and specificity. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10694–10699. 25 Haemig H, Brooker R. Importance of conserved acidic residues in mntH, the Nramp homolog of Escherichia coli. J. Membr. Biol. 2004; 201: 97–107. 26 Buck M Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins. Q. Rev. Biophys. 1998; 31: 297–355. 27 Guo H, Karplus M. Solvent influence on the stability of the peptide hydrogen bond: a supramolecular cooperative effect. J. Phys. Chem. 1994; 98: 7104–7105. 28 Mao D, Wachter E, Wallace B. Folding of the mitochondrial proton adenosine triphosphatase proteolipid channel in phospholipid vesicles. Biochemistry 1982; 21: 4960–4968. 29 Rizo J, Blanco FJ, Kobe B, Bruch MD, Gierasch LM. Conformational behavior of Escherichia coli OmpA signal peptides in membrane mimetic environments. Biochemistry 1993; 32: 4881–4894. 30 Li S-C, Deber C. Peptide environment specifies conformation. Helicity of hydrophobic segments compared in aqueous, organic, and membrane environments. J. Biol. Chem. 1993; 268: 22975–22978. 31 Chaloin L, Vidal P, Heitz A, Van Mau N, Méry J, Divita G, Heitz F. Conformations of primary amphipathic carrier peptides in membrane mimicking environments. Biochemistry 1997; 36: 11179–11187. 32 Fregeau Gallagher NL, Sailer M, Niemczura WP, Nakashima TT, Stiles ME, Vederas JC. Three-dimensional structure of leucocin A in trifluoroethanol and dodecylphosphocholine micelles: spatial location of residues critical for biological activity in type IIa bacteriocins from lactic acid bacteria. Biochemistry 1997; 36: 15062–15072. 33 Greff D, Fermandjian S, Fromageot P, Khosla MC, Smeby RR, Bumpus FM. Circular-dichroism spectra of truncated and other analogs of angiotensin II. Eur. J. Biochem. 1976; 61: 297–305. 34 Wray V, Federau T, Gronwald W, Mayer H, Schomburg D, Tegge W, Wingender E. The structure of human parathyroid hormone from a study of fragments in solution using 1H NMR spectroscopy and its biological implications. Biochemistry 1994; 33: 1684–1693. 35 Mabrouk K, Van Rietschoten J, Rochat H, Loret EP. Correlation of antiviral activity with beta-turn types for V3 synthetic multibranched peptides from HIV-1 gp120. Biochemistry 1995; 34: 8294–8298. 36 Wang D, Song Y, Li J, Wang C, Li F. Structure and metal ion binding of the first transmembrane domain of DMT1. Biochem. Biophys. Acta Biomembranes 2011; 1808: 1639–1644. 37 Xiao S, Li J, Wang Y, Wang C, Xue R, Wang S, Li F. Identification of an “α-helix-extended segment-α-helix” conformation of the sixth transmembrane domain in DMT1. Biochem. Biophys. Acta - Biomembr. 2010a; 1798: 1556–1564. 38 Song Y, Wang D, Qi H, Xiao S, Xue R, Li F. Metal ion binding of the first external loop of DCT1 in aqueous solution. Metallomics 2009; 1: 392–394. 39 Xiao S, Wang C, Li J, Li F. Folding and assembly of TMD 6-related segments of DMT 1 in trifluoroethanol aqueous solution. J. Pept. Sci. 2011; 17: 505–511. 40 Xue, R, S Wang, C Wang, T Zhu, F Li, H Sun. HFIP-induced structures and assemblies of the peptides from the transmembrane domain 4 of membrane protein Nramp1. Pept. Sci. 2006; 84: 329–339. 41 Xue R, Wang S, Qi H, Song Y, Wang C, Li F. Structure analysis of the fourth transmembrane domain of Nramp1 in model membranes. Biochem. Biophys. Acta - Biomembr. 2008; 1778: 1444–1452. 42 Qi H, Yang L, Xue R, Song Y, Wang S, Li F. The third and fourth transmembrane domains of Slc11a1: comparison of their structures and positioning in phospholipid model membranes. Pept. Sci. 2009; 92: 52–64. 43 Xue R, Wang S, Qi H, Song Y, Xiao S, Wang C, Li F. Structure and topology of Slc11a1 (164–191) with G169D mutation in membranemimetic environments. J. Struct. Biol. 2009; 165: 27–36. 44 Xiao S, Wang Y, Yang L, Qi H, Wang C, Li F. Study on structure and assembly of the third transmembrane domain of Slc11a1. J. Pept. Sci. 2010b; 16: 249–255. 45 Xiao S, Yang L, Li F. The structure and assembly model of the third transmembrane domain of Slc11a1 in SDS micelles revealed by NMR study of the Leu-substituted peptide. J. Pept. Sci. 2012; 18: 45–51.
WANG, WANG, AND LI 46 Li F, Li H, Hu L, Kwan M, Chen G, He Q-Y, Sun H. Structure, assembly, and topology of the G185R mutant of the fourth transmembrane domain of divalent metal transporter. J. Am. Chem. Soc. 2005; 127: 1414–1423. 47 Goddard T, Kneller D. Sparky 3 NMR software. University of California: San Francisco, 2001. 48 GuČntert P, Mumenthaler C, WuČthrich K. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 1997; 273: 283–298. 49 Laskowski RA, Rullmann JAC, MacArthur MW, Kaptein R, Thornton JM. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 1996; 8: 477–486. 50 Koradi R, Billeter M, Wüthrich K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 1996; 14: 51–55. 51 Choy N, Raussens V, Narayanaswami V. Inter-molecular coiled-coil formation in human apolipoprotein E C-terminal domain. J. Mol. Biol. 2003; 334: 527–539. 52 Dutta K, Alexandrov A, Huang H, Pascal SM. pH-induced folding of an apoptotic coiled coil. Protein Sci. 2001; 10: 2531–2540. 53 Accardo A, Morisco A, Palladino P, Palumbo R, Tesauro D, Morelli G. Amphiphilic CCK peptides assembled in supramolecular aggregates:
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Supporting Information Additional supporting information may be found in the online version of this article at the publisher’ web site.
172 wileyonlinelibrary.com/journal/jpepsci Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 165–172
Revised: 29 September 2013
Accepted: 30 October 2013
Published online in Wiley Online Library: 22 January 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2593
Insight into the structures of the second and fifth transmembrane domains of Slc11a1 in membrane mimics Li Wang, Dan Wang and Fei Li* Slc11a1 is an integral membrane protein with 12 putative transmembrane domains and functions as a pH-coupled divalent metal cation transporter. In the present study, the structures of the peptides corresponding to the second and fifth transmembrane domains of Slc11a1 (from 88 to 109 for TMD2 and from 190 to 215 for TMD5) were determined in membrane-mimic environments by CD and NMR techniques. It was demonstrated that TMD2 and TMD5 form an α-helical structure in 30% 2,2,2-trifluoroethanol (TFE) and 40% hexafluoro-2-propanol (HFIP) aqueous solution, respectively. The α-helix of TMD5 displays a less space-occupied face consisting of the residues Ala194, Gly197, Thr201, Ala204 and Gly208. The α-helix is partially unfolded in the N-terminal region when Gly197 is substituted by Val. The unfolding of the helix in the N-terminal part and/or increase in volume at the less space-occupied face of the helix may exert an effect on the arrangement of TMD5 in membrane. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: Slc11a1; transmembrane domain; structure; NMR
Introduction
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* Correspondence to: Fei Li, State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Avenue, Changchun 130012, China. E-mail: [email protected] State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130012, China
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
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Slc11 (solute carrier family 11), formally named as natural resistance-associated macrophage protein, is the protein family of proton-coupled transporters with 10–12 transmembrane domains. The homologues of Slc11 have been found in mammals, birds, insects, plants, fish and bacteria [1–5]. The structure and function of the protein family have been remarkably conserved throughout evolution. Slc11a1 and Slc11a2, the two members of the protein family with 12 transmembrane domains, share 63% amino acid sequence identity and an overall homology of 78% in humans and rodents [6,7]. Both proteins can transport divalent metal cations in a pH-dependent fashion [7,8]. Slc11a2 [or divalent metal-ion transporter 1 (DMT1)] is expressed at the apical membrane of duodenal enterocytes [9] and in recycling endosomes of peripheral tissues and mediates transfer of iron internalized by transferrin from the endosomes to the cytoplasm [10]. Deficiency of Slc11a2 is related to a hypochromic microcytic anemia [11–13]. The expression of Slc11a1 is restricted to the membrane of mature phagocytes in steady-state conditions and further stimulated by inflammatory signals [14–16]. Slc11a1 regulates divalent cation concentration within macrophage phagosomes [17–20]. Deficiency of Slc11a1 causes sensitivity to several intracellular pathogens. Some naturally occurred mutations in Slc11a2/DMT1 of murine and human, such as V114 deletion in TMD2 [13], G185R in TMD4 [11,12], G212V in TMD5 [13] and R416C in TMD9 [21], have been identified to be responsible for severe hypochromic microcytic anemia. The single G169D substitution in TMD4 of Slc11a1 has been found to be associated with susceptibility of murine to infections with intracellular parasites such as Leishmania, Mycobacterium and Salmonella [14,22]. Some residues in TMD1, TMD3 and TMD6 of both DMT1 and MntH (Slc11 homologue from Escherichia coli) were also detected by mutation assays to be crucial for the divalent metal-ion transport
of the Slc11 proteins [23–25]. The mutations of the specific residues in these domains may influence the transport activity of the proteins by changing binding affinity of certain residue(s) with metal ions or by changing the interactions between transmembrane domains and thus vary the size or property of the pore formed by the transmembrane domains. A number of model systems have been used to mimic features of membranes in the study of structures of membrane proteins. TFE and HFIP are two of these systems because of their effects on stabilizing the secondary structures of polypeptides. The clusterization property of the fluorinated solvents in aqueous solution was pointed as primary reason for the effect. The clusters of TFE or HFIP may cooperatively associate with the hydrophobic surface of an α-helix (or a β-strand), thus mimicking the environment of a membrane or the interior of a protein. Possible mechanisms by which alcohol-based cosolvents affect polypeptide structure include the enhancement of polypeptide internal hydrogen bonding, the disruption of water structure and lessening of the hydrophobic effect, the penetration of cosolvent molecules into the protein core and preferential solvation of certain groups of the polypeptide chain [26,27]. Many studies have demonstrated that TFE is a suitable model system of membrane [28–32]. In some circumstances, the structures of polypeptides obtained in TFE are even similar to those adopted in their active conformations and could be correlated with their biological activity [33–35].
WANG, WANG, AND LI In our previous work, we have studied the structures and topologies of the peptides corresponding to the TMD1 and TMD6 of Slc11a2/DMT1 [36–39], TMD3 and TMD4 of Slc11a1 [40–45] and some peptides from specific site mutations of these transmembrane domains in both organic solvents and detergent micelles using CD and NMR methods. We also studied the secondary structures and topologies of TMD1-TMD5 of Slc11a1 in lipid membranes by CD and attenuated total reflectance (ATR) FTIR techniques. We found some changes in structure, location relative to membrane surface and assembly of these transmembrane domains because of specific site mutations. In the present paper, we studied the three dimensional structures of the peptides corresponding to TMD2 and TMD5 of mouse Slc11a1 (mSlc11a1) and the mutant G197V of TMD5 (corresponding to G212V of TMD5 in mSlc11a2) in fluoroalcohol aqueous solutions by CD and NMR. Because both sequences of TMD2 and TMD5 are highly conserved in Slc11a1 and Slc11a2 (Scheme 1), the structure of TMDs in Slc11a1 obtained in this study could be considerably suggestive of the structure of corresponding TMDs in Slc11a2.
Experimental Materials The peptides were synthesized by solid phase FMOC chemistry (Shanghai Apeptide Co., Ltd., Shanghai, China) and purified by reverse phase HPLC on a C18 column using a solvent system consisting of 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B). Elution was achieved by applying a linear gradient from 100% to 20% A in 30 min. Three Lys residues were added at each terminal end of TMD2, and two Lys residues were added at the N-terminal ends of both TMD5 and TMD5-G197V mutants to facilitate purification. The molecular weights of the peptides (3306.27, 3259.98 and 3302.06 Da for TMD2, TMD5 and TMD5-G197V, respectively) were confirmed by mass spectroscopy, and their purities of >95% were estimated by HPLC. Deuterated 2, 2, 2-trifluoroethanol (TFE-d2; 98%) and 1,1,1,3,3,3-hexafluoro-2propanol (HFIP-d2, 99.5%) were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Sample Preparation The peptide TMD2 was solubilized using 30%TFE/70%H2O (v/v) for the CD and fluorescence experiments or using 30%TFE-d2/ 70%H2O for the NMR experiments. The peptides TMD5 and TMD5-G197V were solubilized using 40%HFIP/60%H2O for the CD and fluorescence experiments or using 40%HFIP-d2/60%H2O for the NMR experiments. The samples of peptides incorporated with SDS micelles were prepared using the method as described previously [46]. Deionized water was used in preparation of the mSlc11a1 TMD2
88
mSlc11a2 TMD2
103
mSlc11a1 TMD5
190
mSlc11a2 TMD5
205
SDS samples for fluorescence experiments, and 10 mM Citric acid/Na2HPO4 buffers at pH 5.5 and 7 were used in preparation of the SDS samples for CD experiments.
Far-UV CD Spectra Far-UV CD spectra were recorded on a Jasco J-810 spectropolarimeter (Jasco Co., Tokyo, Japan) using a 0.5-mm path length cuvette. The concentrations of 20 μM peptide and 10 mM SDS detergent were used in the experiments. The spectra were scanned over the wavelength range of 190–260 nm with data pitch 0.1 nm and bandwidth 1.0 nm at a sweep speed of 50 nm min 1. Three scans were acquired and averaged, and the CD signal of solvent was subtracted.
Fluorescence Measurements All fluorescence spectra were recorded on RF-5301PC fluorescence spectrophotometer (Shimadzu, Kyoto, Japan) at room temperature. In the fluorescence experiments, the concentration of SDS detergent was fixed at 8 mM, while the concentration of peptide was changed in a range of peptide : detergent from 1 : 25 to 1 : 200. The spectra were scanned in a range of 290–420 nm with excitation of 280 nm and slit size of 3 nm (excitation) × 5 nm (emission). Three spectra were recorded in an interval of ca. 5 min and averaged, and the reference spectrum of the respective medium was subtracted.
NMR Experiments All NMR spectra were recorded on a Bruker Avance 500 spectrometer (Bruker, Fällanden, Switzerland) with a z-gradient coil and a 5-mm triple resonance broadband inverse (TBI) probe at 298 K. The samples of 2 mM TMD2 peptide in 240 mM SDS detergent and 2 mM TMD5 peptides in 180 mM SDS detergent were used in all NMR experiments. The TOCSY and NOESY experiments were performed using a mixing time of 100 and 200 ms, respectively. The transients of 48 for TMD2 and 64 for TMD5 and TMD5G197V were acquired for each increment in t1. Water signal was suppressed using WATERGATE technique. Sodium salt of 2,2-dimethyl-2-sila-pentane-5-sulfonic acid (DSS) were used as an internal standard for proton chemical shifts. The 2D NMR spectra were measured with 2048 data points in F2 dimension and 512 data points in F1 dimension and processed using standard Bruker software (Bruker, Fällanden, Switzerland). NMR assignments were achieved using software Sparky [47]. The structures of the peptides were calculated, evaluated and displayed using the programs CYANA (1.0.6) [48], PROCHECK-NMR [49] and MOLMOL [50], respectively.
KLLWVLLWATVLGLLCQRLAA109R KLLWVLL LAT IVGLL LQRLAA124R
RKLEAFFGLLITIMALTFGYEYVVA215H RKLEAFFGFLITIMALTFGYEY ITV230K
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Scheme 1. Sequences of the peptides studied (mSlc11a1 TMD2 and TMD5) and their homologies in mSlc11a2. The residues with gray shade are non-conserved in mSlc11a1 and mSlc11a2, and the tilted residues in mSlc11a2 TMD2 and TMD5 (V114 and G212) are mutation sensitive for microcytic anemia.
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STRUCTURES OF THE SECOND AND FIFTH TRANSMEMBRANE DOMAINS OF Slc11a1 in the same conditions. This suggests that the mutation may make part of helix unfolded. A common difference between the CD spectra of the peptides in organic solvents and in SDS micelles is found. The ratios of θ222/θ208 for the peptides in SDS micelles are closer to 1 than those for the peptides in organic solvents. More negative absorbance at 222 nm for the peptides in SDS micelles is associated to the aggregation of the peptides via coiled-coil interactions in the micelles [51,52]. As mentioned above, three Lys residues were added at each terminal end of TMD2, and two Lys residues were added at the N-terminal ends of both TMD5 and TMD5-G197V mutants in order to increase the hydrophilicity of the peptides and facilitate purification. The addition of the Lys residues at both ends of the peptides could decrease the propensity of peptide aggregation because of repulsive interaction among these positively charged residues and facilitate the incorporation of the peptides in SDS micelles because of increasing interaction between the positively charged Lys residues and the negatively charged headgroups of the micelles. The addition of the Lys residues may also have effect on the secondary structures of the peptides in certain situation [53]. However, the effect could be rather small in TFE or HFIP aqueous solution because the stabilization of helical conformations in the cosolvents is mainly correlated with the conformational biases of the primary sequence [54]. Aggregation of the Peptides
Figure 1. The CD spectra of the peptides TMD2 (A), TMD5 (B) and TMD5G197V (C) in different environments.
Results and Discussion Structural Evaluation of the Peptides by CD
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The CD spectra of the peptides TMD2, TMD5 and TMD5-G197V in organic solvents and SDS micelles at pH 5.5 and 7 were recorded to analyze their secondary structures. As shown in Figure 1, the CD spectra of all the peptides are characterized by double negative minima at ca. 208 and 222 nm and a positive maximum at ca. 194 nm. This suggests that the α-helix structures are induced by these environments. Moreover, the CD spectra of the peptides in SDS micelles are basically independent of pH conditions. Compared with the wild type, the mutant peptide TMD5-G197V displays lower helicity both in 40%HFIP aqueous solution and in SDS micelles, as indicated by less negative molar ellipticity either at 208 nm or at 222 nm in the CD spectra of the mutant peptide
We measured the NMR spectra of the three peptides in SDS micelles. The spectral lines were severely broad and overlapped. When we solubilized TMD2 peptide in 30%TFE-d2 aqueous solution or solubilized TMD5-related peptides in 40%HFIP-d2 aqueous solution, we observed a dramatic improvement of the 1H NMR spectra (Figure 2). This indicates that the peptides may be aggregated in SDS micelles. The aggregate is disaggregated in TFE or HFIP aqueous solution because of strong ability of TFE and HFIP in dissociating peptide aggregate. Accordingly, the structures of the peptides at molecular level were studied by NMR in fluoroalcohol aqueous solutions but not in SDS micelles (see Structural Determination of the Peptides by NMR section). The aggregate of TMD2 in SDS micelles was further probed by self-quenching experiment of Trp fluorescence at various peptide concentrations. It was predicted that the fluorescence intensity should increase linearly with increasing peptide concentration if no peptide aggregation occurs, while the dependence should be nonlinear if peptide aggregates. Our result demonstrates that the fluorescence emission increases nonlinearly with increasing [TMD2]/[SDS] ratio and the intensities are lower than those predicted by a linear increase in the case of no aggregation (Figure 3). The quenching of fluorescence with increasing peptide concentration suggests a strong aggregate of the peptide in SDS micelles. The intermolecular interactions among tryptophan residues in an aggregate result in the decrease in fluorescence intensity. The fluorescence experiments of TMD5 peptides were not performed because of the absence of Trp in the peptides. It is noted that TMD2 includes two Trp residues located at positions 91 and 95, but only one fluorescence emission at 334 nm is observed in the fluorescence spectra of TMD2 incorporated with SDS micelles. This implies that the two Trp residues are involved in the similar environment in SDS micelles. Previous studies have shown that the fluorescence emission at ~330 nm is related to a hydrophobic ambient of Trp, while the fluorescence emission at ~350 nm is related to the exposure of
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Figure 2. The 1D- H NMR spectra of TMD2 (A) and TMD5 (B) in different environments.
Figure 3. The fluorescence spectra (A) and the emission maxima at 334 nm (B) of TMD2 in SDS micelles at various peptide/SDS ratios.
Trp to a polar ambient [55]. Although the Trp at position 91 is close to the N-terminal end of the peptide, it is enclosed by the hydrophobic residues such as two Leu residues before it and one Val and two Leu residues after it. Therefore, both tryptophan residues are embedded in the hydrophobic region of the micelles, and the maximum emission at 334 nm is observed for TMD2 aggregated in SDS micelles. Structural Determination of the Peptides by NMR
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All 1H NMR resonances of TMD2 in 30%TFE-d2 or TMD5 and TMD5-G197V in 40%HFIP-d2 aqueous solution were assigned by combined analyses of their 2D TOCSY and NOESY spectra (see Table S1-S3 in Supporting information). The fingerprint regions of 2D NOESY spectra of the peptides in fluoroalcohol aqueous solutions with assignments of Hα-HN cross-peaks and NOE connectivities obtained from the NOESY spectra are shown in Figure 4. In the assignments of NMR cross-peaks, the residues in the sequences belonging to the putative transmembrane domains are numbered sequentially, while the extra added Lys residues at the N-terminal and C-terminal ends are defined as N1, N2 and so on and C1, C2 and so on, respectively. Some NOE connectivities of Hα(i)-HN(i + 3) and Hα(i)-HN(i + 4) for TMD5 and TMD5-G197V indicated in Figure 4D and 4F are not observed in Figure 4C and 4E because the intensities of these cross-peaks are lower than the intensity levels that are set for clear presentation of the spectra.
The NOE connectivities and chemical shift index [56] suggest a predominant α-helix structure for TMD2 peptide in 30%TFE-d2 aqueous solution. On the basis of the NOE connectivities, the three dimensional structure of the peptide was calculated. The NMR restraints used in calculations and structural statistics extracted from calculation results are listed in Table 1. Figure 5A shows ensemble of backbone atoms of the 20 structures with the lowest target functions for TMD2 in 30%TFE-d2 aqueous solution. The structures are well converged in the region of Leu3-Leu19. Ramachandran analyses for the 20 structures indicate that the dihedral angles of all residues involved in this region fall within the allowed regions of an α-helix. In the helix, the conservative residues Thr10, Gly13 and Gln17 face the same side, making this side more polar and less space occupied as compared with other sides of the helix. The similar structure could be predicted for the TMD2 of Slc11a2 protein based on high conservation of the two proteins in TMD2. This specific structure of TMD2 may be associated with the specific contact of the transmembrane domain with others. The deletion of V114 in TMD2 of Slc11a2 changes the arrangement of these residues in helix, e.g., the position of polar Thr is replaced by apolar Ala, by which the function of the protein may be influenced. The NOE connectivities of TMD5 in 40%HFIP-d2 aqueous solution predict an α-helical structure for nearly entire peptide, although some medium-range NOE connectivities of Hα(i)-HN (i + 3) and Hα(i)-HN(i + 4) are relatively weak. Compared with the
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STRUCTURES OF THE SECOND AND FIFTH TRANSMEMBRANE DOMAINS OF Slc11a1
Figure 4. The Hα-HN region of the NMR spectra and NOE connectivities with chemical shift index for TMD2 in 30%TFE-d2 solution (A and B), TMD5 in 40%HFIP-d2 solution (C and D) and TMD5-G197V in 40%HFIP-d2 solution (E and F).
of specific steric structure, which is associated with the function of the protein. The high conservation of TMD5 in Slc11a1 and Slc11a2 implies the structural similarity of this domain in the two proteins, i. e., the TMD5 of Slc11a2 may also fold as an α-helix with a less occupied face. The mutation of G212V in Slc11a2 (corresponding to G197V for Slc11a1) not only introduces a larger hydrophobic residues in the less occupied side but also leads to unfolding of the N-terminal part of the helix, which may have an effect on the interactions of the fifth transmembrane domain with other transmembrane domain(s). Deficiency in the activity of Slc11a2 due to G212V mutation may be associated with the change in the interaction of TMD5 with other(s), which is related to arrangement and topology of the transmembrane domains in membrane. Slc11a2 structural model has been predicted by homology threading based on the crystal structures of Mhp1, LeuT and vSGLT, which may represent discrete steps of a structural fold in cationdriven transport cycle shared by different families (e.g., Slc23, 6 and 5). The structural model shows twofold symmetry in the arrangement of transmembrane helices for the N-domain TMD1-5
J. Pept. Sci. 2014; 20: 165–172 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
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wildtype TMD5, the mutant TMD5-G197V loses the mediumrange NOE connectivities in the N-terminal part. Moreover, the chemical shifts of the α protons in the N-terminal parts of the mutant (such as Arg1-Glu4) are more downfield than those of the wildtype peptide (Figure 4C–F). These results suggest that the N-terminal part of the mutant peptide is less helical than that of the wildtype peptide. The structures of TMD5 and its G197V substitute in 40%HFIP-d2 aqueous solution were calculated based on the restraints from NOE connectivities (Figure 5B and 5C). The NMR restraints used in calculations and structural statistics extracted from calculation results are listed in Table 1. The backbone atoms of the 20 structures with the lowest target functions are basically converged in the region of Leu3-Val24 for TMD5 and Ala5-Val24 for TMD5-G197V. Nearly 100% residues in the converged portion fall in the allowed region of an α-helix according to the Ramachandran statistics for the dihedral angles of the 20 structures. It is noted that there is a less occupied face consisting of the residues Ala5, Gly8, Thr12, Ala15 and Gly19 in the α-helix of TMD5. This alignment of residues may be needed for the formation
WANG, WANG, AND LI Table 1. Statistics of 20 structures with the lowest target functions for TMD2 in 30%TFE-d2/70%H2O, TMD5 and TMD5-G197V in 40%HFIP-d2/60% H2O at 298 K TMD2 2
Average target functions (Å ) Number of nonredundant distance restraints Intraresidual (|i j| = 0) Sequential (|i j| = 1) Medium (|i j| ≤ 4) Long range (|i j| > 4) Average sum of distance restraint violations (Å) Average max. distance restraint violation (Å) Average sum of torsion angle restraint violations (°) Average max. of torsion angle restraint violation (°) RMS deviation from the mean structure (Å) All residues Backbone heavy atoms All heavy atoms Residues Backbone heavy atoms All heavy atoms Ramachandran plot statistics (for each helix span) Residues in most favored region (%) Residues in additionally allowed region (%) Residues in generously allowed region (%) Residues in disallowed region (%)
TMD5
TMD5-G197V
0.21 ± 0.06 252 130 60 62 0 1.8 ± 0.4 0.19 ± 0.05 0.0 ± 0.01 0.04 ± 0.14
0.03 ± 0.01 262 124 76 62 0 0.5 ± 0.1 0.09 ± 0.02 0.0 ± 0.0 0.0 ± 0.0
0.24 ± 0.05 275 132 72 71 0 1.7 ± 0.4 0.2 ± 0.04 0.5 ± 0.8 0.41 ± 0.69
3.75 ± 0.87 5.47 ± 1.18 3–19 0.70 ± 0.24 1.64 ± 0.32
2.16 ± 0.85 3.12 ± 0.89 3–24 1.20 ± 0.50 2.08 ± 0.55
2.85 ± 0.83 4.09 ± 0.89 5–24 1.26 ± 0.51 2.30 ± 0.73
81.6 17.8 0.6 0
82.6 16.8 0.6 0
73.4 21.9 4.4 0.3
Figure 5. Ensemble of backbone atoms of the 20 structures with the lowest target functions fitted over helix region for TMD2 in 30%TFE-d2/70%H2O (A), TMD5 (B) and TMD5-G197V (C) in 40%HFIP-d2/60%H2O.
and the C-domain TMD6-10 (conserved hydrophobic core of Slc11). Comparison of the TMDs arrangements in LeuT (open to outside/ occluded) with those in vSGLT (open to inside) shows the movements of the N-domain TMD1, 2 and 5 and the C-domain 6, 7 and 10 between the two models [57]. This indicates that the TMD2 and 5 may play a role in substrate uptake of Slc11 transporters by structural/topological modulations, although they may not directly participate in the binding with substrates.
in Slc11a1) with a more polar and a relatively less space-occupied face. The structural study of the peptide from the fifth transmembrane domain of Slc11a1 in 40%HFIP aqueous solution reveals that the peptide adopts an α-helical structure from Leu3 to Val24 (Leu192-Val213 in Slc11a1) with a full less spaceoccupied face. The specific structural characteristic of the two TMDs may determine their arrangement in membrane and function of the protein. The substitution of Gly197 by Val in TMD5 of Scl11a1 results in partial unfolding of α-helix in the Nterminal region, which may be function associated.
Conclusion
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The structural study of the peptide from the second transmembrane domain of Slc11a1 in 30%TFE aqueous solution reveals that the peptide folds as an α-helix from Leu3 to Leu19 (Leu90-Leu106
Acknowledgement This work was financially supported by the National Natural Science Foundation of China (20973083 and 20934002).
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STRUCTURES OF THE SECOND AND FIFTH TRANSMEMBRANE DOMAINS OF Slc11a1
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