Structural evidence for functional lipid interactions in the betaine transporter BetP
2013; Springer Nature; Volume: 32; Issue: 23 Linguagem: Inglês
10.1038/emboj.2013.226
ISSN1460-2075
AutoresCaroline Koshy, Eva S. Schweikhard, Rebecca M. Gärtner, Camilo Pérez, Özkan Yıldız, Christine Ziegler,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoArticle18 October 2013free access Structural evidence for functional lipid interactions in the betaine transporter BetP Caroline Koshy Caroline Koshy Department of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt am Main, Germany Search for more papers by this author Eva S Schweikhard Eva S Schweikhard Department of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt am Main, Germany Search for more papers by this author Rebecca M Gärtner Rebecca M Gärtner Department of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt am Main, Germany Search for more papers by this author Camilo Perez Camilo Perez Department of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt am Main, Germany Search for more papers by this author Özkan Yildiz Özkan Yildiz Department of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt am Main, Germany Search for more papers by this author Christine Ziegler Corresponding Author Christine Ziegler Department of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt am Main, Germany Department of Protein Crystallography, Institute of Biophysics and Biophysical Chemistry, University of Regensburg, Regensburg, Germany Search for more papers by this author Caroline Koshy Caroline Koshy Department of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt am Main, Germany Search for more papers by this author Eva S Schweikhard Eva S Schweikhard Department of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt am Main, Germany Search for more papers by this author Rebecca M Gärtner Rebecca M Gärtner Department of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt am Main, Germany Search for more papers by this author Camilo Perez Camilo Perez Department of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt am Main, Germany Search for more papers by this author Özkan Yildiz Özkan Yildiz Department of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt am Main, Germany Search for more papers by this author Christine Ziegler Corresponding Author Christine Ziegler Department of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt am Main, Germany Department of Protein Crystallography, Institute of Biophysics and Biophysical Chemistry, University of Regensburg, Regensburg, Germany Search for more papers by this author Author Information Caroline Koshy1, Eva S Schweikhard1, Rebecca M Gärtner1, Camilo Perez1, Özkan Yildiz1 and Christine Ziegler 1,2 1Department of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt am Main, Germany 2Department of Protein Crystallography, Institute of Biophysics and Biophysical Chemistry, University of Regensburg, Regensburg, Germany *Corresponding author. Department of Structural Biology, Max-Planck Institute of Biophysics, Max-von-Laue Strasse 3, 60438 Frankfurt, Germany. Tel.:+49 69 6303 3054; Fax:+49 69 6303 2209; E-mail: [email protected] The EMBO Journal (2013)32:3096-3105https://doi.org/10.1038/emboj.2013.226 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Bilayer lipids contribute to the stability of membrane transporters and are crucially involved in their proper functioning. However, the molecular knowledge of how surrounding lipids affect membrane transport is surprisingly limited and despite its general importance is rarely considered in the molecular description of a transport mechanism. One reason is that only few atomic resolution structures of channels or transporters reveal a functional interaction with lipids, which are difficult to detect in X-ray structures per se. Overcoming these difficulties, we report here on a new structure of the osmotic stress-regulated betaine transporter BetP in complex with anionic lipids. This lipid-associated BetP structure is important in the molecular understanding of osmoregulation due to the strong dependence of activity regulation in BetP on the presence of negatively charged lipids. We detected eight resolved palmitoyl-oleoyl phosphatidyl glycerol (PG) lipids mimicking parts of the membrane leaflets and interacting with key residues in transport and regulation. The lipid–protein interactions observed here in structural detail in BetP provide molecular insights into the role of lipids in osmoregulated secondary transport. Introduction Transmembrane proteins need to function in a fluid lipid bilayer environment. Over the years, many observations have indicated that the bilayer often plays an essential part in the organization and function of membrane proteins (Lee, 2004; Phillips et al, 2009). Specific bilayer lipids can be essential cofactors for proteins in folding and function, as seen in the crystal structure of the potassium channel KcsA (Zhou et al, 2001; Valiyaveetil et al, 2002). Membrane lipids can control function by deforming locally to compensate for the hydrophobic mismatch between protein and bilayer thickness, as in the gating of bacterial mechanosensitive channels, which respond to membrane mechanical stress (Perozo et al, 2002; Phillips et al, 2009). Lipid head-group compositions have also been shown to influence the activity of proteins like the diacylglycerol kinases and cation-pumping ATPases (Lee, 1998, 2004; Quick et al, 2012). Recently, experimental and computational studies on the secondary transporter LeuT indicated that the surrounding environment (detergent micelle or lipid bicelle) could play an important role in its function (Zhao et al, 2011; Quick et al, 2012; Wang et al, 2012). The membrane environment of LeuT was further shown to respond dynamically to the structural features of conformational changes in this transporter, by computational studies (Mondal et al, 2013). LeuT is an important prototype for secondary transport in that many functionally unrelated transporters share its fold (Forrest et al, 2011). The betaine symporter BetP is one such secondary transporter, which shares the conserved LeuT-like fold of two structurally related, topologically inverted domains (Supplementary Figure S1a and b). It is a key player in the hyperosmotic stress response of Corynebacterium glutamicum (Peter et al, 1996; Morbach and Krämer, 2003; Krämer and Morbach, 2004). This soil bacterium exhibits a very uncommon plasma membrane composition comprising lipids with only negatively charged head groups namely phosphotidyl glycerol (PG), phosphotidyl inositol (PI) and cardiolipin (CL), with exclusively 16:0/18:1 fatty acyl chains (Hoischen and Krämer, 1990; Schiller et al, 2006; Özcan et al, 2007). Regulation of transport activity in BetP is triggered by an increase in the internal concentrations of K+ in hyperosmotic stress situations (Rübenhagen et al, 2001). The response of betaine uptake rate to increasing external osmolality is severely compromised in membranes lacking negatively charged lipids (Rübenhagen et al, 2000; Schiller et al, 2006). Its activation optimum also crucially depends on the fraction of 16:0/18:1 fatty acyl chains (Özcan et al, 2007). It has been suggested that BetP is able to sense stress and achieve full activity at low temperatures via a signal that stems directly from the membrane (Özcan et al, 2005). Structural data obtained from BetP reconstituted in 2D crystals implied that these might include changes in membrane curvature or thickness (Tsai et al, 2011). This as yet undefined lipid–protein interaction is an intriguing proposition because other osmotic stress-regulated transporters (van der Heide et al, 2001; Poolman et al, 2004) also depend on the presence of specific lipids for their optimal activation. In fact, lipid–protein interactions are often found in stress-triggered activation, for example, during salt stress to affect water transport via aquaporins (Walz et al, 1997) or during hypotonic stress to activate mechanosensitive channels (Yoshimura and Sokabe, 2010). The stringent causality between transport regulation of BetP and the unique composition of the C. glutamicum membrane (Özcan et al, 2007) coupled with the wealth of structural data makes BetP a prime example to structurally investigate regulatory lipid–protein interactions. Atomic structures of BetP in different conformational states allowed recently for the molecular description of the alternating access cycle of BetP that involves a hinge-like gating movement along the midsection of mainly two transmembrane helices from each inverted repeat, TM1′ (The numbering of the BetP TM helices was adapted to the LeuT numbering for better comparison. Therefore, TM1′–TM10′ correspond to TM3–TM12, while TM1 and TM2 are now assigned as TM(−2) and TM(−1), respectively; Supplementary Figure S1c.) and TM5′ to open the cytoplasmic pathway and TM6′ and TM10′ to open the periplasmic pathway (Perez et al, 2012; Figure 1B). These structures unambiguously identified key players in the co-transport of sodium and betaine. Figure 1.(A) Inward-open state of BetP at 2.7 Å: surface cut of the inward-open state (Ci) with citrate bound in the pathway, adopted by all three chains (A–C). The inset panel shows the functionally important and evolutionarily conserved tryptophans in TM6′—Trp373, Trp374 and Trp377, stacked like the rungs of a ladder in this conformation. The citrate buffer molecule is shown in black stick representation with the 2Fo-Fc map for citrate contoured at 1σ. This site is occupied by choline and betaine in previously reported structures PDB: 3P03 at 3.3 Å and PDB: 4AIN at 3.1 Å resolution, respectively. (B) Resolved lipids are shown in trimeric context with helices of each chain involved in conformational changes highlighted. Lipids are closely associated with loops connecting helices involved in functional movement. Inset shows distinct elements in the conformational changes in BetP during the alternating access mechanism for substrate translocation. Bundle helices are in red, scaffold helices in blue and arm helices in green. The cytoplasmic halves of TM1′ and 5′ are the gating elements for the inward opening of the transporter while their symmetry equivalents TM6′ and TM10′ gate the periplasmic opening. Download figure Download PowerPoint Here, we describe a new X-ray structure of a BetP trimer, in which all three protomers adopt a similar inward-facing state resolved to 2.7 Å with specifically bound PG lipids. The structure reveals in atomic detail how the gating helix TM1′ (Forrest et al, 2011), which also plays an important role in conformational changes in LeuT-like fold transporters, interacts with lipids. The observed lipid–protein interaction sites involving residues important in both transport and regulation of BetP highlight the functional impact of lipids in the structure-based transport mechanism of a LeuT-like fold transporter. Results Structure of BetP trimer with all three protomers in inward-facing state We determined the crystal structure of a BetP trimer with each chain adopting an inward-open state (Ci) at a resolution of 2.7 Å (Figure 1A). This structure was obtained after removing unspecifically bound lipids by thorough washing steps during detergent exchange on the affinity column (Supplementary methods). Washing would also remove any endogeneous betaine that could bind in the structure and no external betaine was added during purification and crystallization. The main-chain conformations of the Ci state at 2.7 Å are identical to previously reported inward-facing substrate bound (CiS) structures at lower resolution (Ressl et al, 2009; Perez et al, 2011b, 2012), which crystallized as asymmetric trimers with respect to their conformational state. A citrate buffer molecule is wedged into the cytoplasmic funnel in all three chains, and is located below the central S1 substrate-binding site that was described previously in betaine-bound closed-state structures (Perez et al, 2012). It is coordinated at a similar site between TM1′ and TM6′ as betaine (Perez et al, 2012) or choline (Perez et al, 2011b, 2012) in previously reported structures of inward-open substrate bound conformations, although it has no functional impact on BetP, that is, as an inhibitor or a competitor for betaine (data not shown). Although the transporter state is identical in all there protomers, each chain differs in the folding and orientation of its C-terminal domain that forms an α-helix in chains A and C (Supplementary Figure S3). The C-terminal helix contains a zipper-like arrangement of several arginines that are crucial in stress sensing and regulation (Ott et al, 2008). In chain B only a few residues are resolved, reflecting its flexibility. In its helical arrangement, the C-terminal domain interacts with loop 2 of the adjacent chain establishing the cytoplasmic network (Ressl et al, 2009) for which the C-terminal domain of chain A interacts with loop 2 of chain C, while the C-terminal domain of chain C interacts with loop 2 in chain B (Supplementary Figure S3b and c). The structurally observed interaction sites are in good agreement with mutagenesis studies (Ott et al, 2008) and especially restrict the gating helices TM1′ and TM6′ on the cytoplasmic side of chains B and C. Lipid densities resolved in the BetP trimer The improved resolution crystals of BetP revealed strong elongated densities in the Fo-Fc map, which could be assigned to lipid moieties (Supplementary Figure S4). From the observed densities, the most easily identified were used to build lipid chains into the model during refinement, after having systematically excluded other possibilities like detergents from the crystallization conditions. Specifically, they correspond to eight ordered lipids bound to the BetP trimer. The head groups of the lipids could be identified and the acyl chains are resolved almost completely along their entire length, even at a modest resolution of 2.7 Å, albeit with different levels of flexibility, suggesting that these lipids are reasonably well ordered. Head groups were assigned to negatively charged phosphatidyl glycerol (PG) based on the 2Fo-Fc and Fo-Fc difference maps as well as the mainly positively charged residues coordinating them (Figures 2, 3, 4; Supplementary Figure S4). The extent of the densities in the 2Fo-Fc maps additionally argued for the placement of the longer PG heads as opposed to the shorter phosphatidyl ethanolamine (PE) head group and model building with the latter led to unsatisfied head-group electron densities. Cardiolipin, which is the other predominant negatively charged lipid in the C. glutamicum lipidome and also a component in the E. coli membrane, was also tested. However, model building with cardiolipin resulted in unconvincing fitting of densities at head-group position and was subsequently excluded. Although given the resolution there is ambiguity in assigning the correct acyl chains, the extended densities in the structure best fitted a 16/18 acyl chain length during refinement. The major 16/18 acyl chain composition of PG lipids in the phospholipidome of the heterologous expression system E. coli has been identified by mass spectrometric analysis as 16:0/18:1 (Oursel et al, 2007; Matyash et al, 2008). Consequently, we assigned the acyl chains of the identified lipids to 16:0 palmitoleyl–18:1 oleoyl. Figure 2.Non-annular lipids: (A) Membrane view of lipids bound in the centre of the trimer with helices providing co-ordination residues shown as grey oval helices, while the rest of the trimer is shown as cylindrical helices. The inset figure shows five completely resolved POPG lipids L1–L5 in stick representations. An acyl chain for which no head-group density was resolved is shown in dark salmon. The final 2Fo-Fc maps for the lipids (which were modelled after multiple iterative rounds of refinement prior to placement indicated their positions) are contoured at 0.8σ. (B) Detailed residue coordination for each non-annular lipid: coordinating helices that are also involved in conformational changes are coloured according to Figure 1B, in order to recognize regions that are both membrane associated and part of the dynamics of the transport cycle. The head group of lipid L1 is coordinated by K121 (loop 2) and R395 (IL3) from chain A and main chain carbonyl of K121 from chain B. Head group of L5 is coordinated by K542 and S545 of chain B. Coordination for the head group of L2 is via K121 (loop 2-chain C), K121 (loop 2-chain B) and R395 (IL3-chain B). L3 is similarly coordinated by K121 (loop 2-chain A), K121 (loop 2-chain C) and R395 (IL3-chain C). POPG head group for L4 is coordinated between R554, R558, Q557 (C-terminal helix-chain A), R126 (loop 2-chain C) and R395 (IL3-chain C). The acyl chains of these lipids are coordinated by hydrophobic residues from helices lining the trimer core and are shown in stick representations. Download figure Download PowerPoint Figure 3.Annular lipids: Membrane view of lipid L6 resolved at the trimer periphery. The final 2Fo-Fc map density is contoured at 0.8σ. A Cymal5 molecule shown in grey provides additional head-group coordination for lipid L6 (inset). Download figure Download PowerPoint Figure 4.Annular lipids: (A) Membrane view of lipids U1and L7 resolved at the trimer periphery. Final 2Fo-Fc map densities are contoured at 0.8σ. (B) Detailed residue coordination for upper leaflet lipid U1 and lower leaflet lipid L7 is shown. Helices and residues involved in lipid coordination are highlighted and coloured according to Figure 1B if also involved in conformational changes. (C) Normalized activity regulation in BetP TM1′ unwound helix mutants in E. coli MKH13 cells: regulation of betaine uptake is shown for BetP WT (black circle/116.5 nmol/min × mg dw), BetP M150I (red triangles/75.6 nom/min × mg dw), BetP M150F/I152A (orange diamonds/146.9 nmol × mg dw) and stretch mutants BetP G149A (green triangles/38.1 nmol/min × mg dw) and BetP G153A (blue squares/45.2 nmol/min × mg dw). Normalization was carried out for each mutant related to its highest activity, indicated in parenthesis. Download figure Download PowerPoint Lipid analysis of purified BetP by thin layer chromatography identifies PG In order to resolve any ambiguity in lipid assignment in the structure given the modest resolution, we performed TLC on BetP protein prepared for crystallization. Chromatography using different solvent systems enabled the detection of phospholipids in BetP. An initial one-dimensional separation clearly excluded the presence of positively charged PE from the protein sample (Supplementary Figure S5a) and showed the presence of negatively charged CL/PG species. However, interpretation was confounded since Cymal-5, the detergent in BetP, also runs at the same height (Supplementary Figure S5a and b). The negatively charged species was further separated using an acetone:acetic acid:water:methanol system (Supplementary methods). In this 1D separation run, a phospholipid spot in BetP co-migrating with the PG standard was observed (Supplementary Figure S5b), indicating the presence of anionic PG lipids in purified BetP. No distinct CL species could be identified, but the possibility of some amounts of the cardiolipin being present and migrating at the same height as the Cymal-5 spot could not be eliminated. Although cardiolipin could not be identified from the better-resolved lipid densities within the structure, it may well be a component of the annular lipids present along the periphery of the trimer. Eight POPG binding sites within trimeric BetP The lipids are resolved along the membrane limits (Supplementary Figure S6) with seven lipids aligned with the lower (L1–7) cytoplasmic leaflet and one lipid resolved from the upper (U1) periplasmic membrane leaflet. Five of these lipids are non-annular and appear to mediate intra-trimeric contacts in the BetP core (Figure 2A and B). Three other lipids are annular, present on the outer surface of the protein (Figures 3, 4A and B). These lipids are less well ordered reflecting the faster exchange of lipids on the surface with those in the membrane bulk (Lee, 2011) while electron densities for the non-annular lipids are better resolved, presumably due to their restriction within the trimer. Consequently, the assignment of annular lipids to PG was based mainly on the extent of densities for the head groups. Three completely resolved lipid molecules (L1–3) are present in the trimer centre, each coordinated by residues from adjacent chains (Figure 2A). Positively charged residues from loop 2 (Lys121) and internal loop IL3 (Arg395) from adjacent chains coordinate these lipid moieties, which are resolved at symmetric sites within the trimer (Figure 5A). L1 extends between chains A and B, whereas L2 and L3 are coordinated by chains B-C and A-C, respectively (Figure 2B). Figure 5.(A) L1, L2 and L3 marked in a black circle are resolved at symmetrical sites in the central hydrophobic cavity formed by the three chains. (B) L4, L5, L6, L7 and U1 (red circles) on the other hand do not have equivalently resolved lipid molecules in the trimer. (C) Asymmetrically occupied lipid-binding sites: membrane view of each monomer from the crystallized trimer showing lipids individually associated with it in spheres. The lipids are coloured according to Figures 2 and 3. Bulk lipids are shown in grey. In general, monomers in the membrane may have different lipid sites occupied. These bound lipids can serve as cofactors providing additional electrostatically favourable interaction sites for trimeric assembly (lower panel). Download figure Download PowerPoint Lipid molecule L4 mediates a cytoplasmic interaction network, involving the longest resolved C-terminal helix of chain A and loops from adjacent chains. Positively charged residues from loops 2 (Arg126) and IL3 (Arg395) in chain C and the C-terminal domain of chain A (Arg554, Arg558 and side chain -NH of Gln557) coordinate the negatively charged head group of L4 (Figure 2B). The corresponding lipid position between loops 2 and IL3 is not occupied between chains A and B, since the C-terminal domain of chain B is flexible and not resolved in the structure. Some density consistent with a lipid head group is present between chains B and C at this position, but was not modelled in the structure due to its limited resolution. The acyl chains of L1, L2, L3 and L4 (Figure 2A and B) extend into the central core of the trimer and are coordinated by hydrophobic side chains from TM(−1), TM10′, TM7′, TM1′ and h7 the amphipathic helix unique to the Betaine-Choline-Carnitine Transporter (BCCT) family (Ziegler et al, 2010), lying parallel to the membrane (Figure 2B; Supplementary Figures S2 and S9). A single acyl chain for which head group is not resolved is also observed in the centre of the trimer coordinated by h7, forming a lipid plug between the chains (Figure 2A and inset). Lipid L5 is resolved close to the trimer core between chains A and B and is coordinated by residues in the loop preceding the C-terminal domain of chain B, namely Lys542 and Ser545. Some density is observed at the same site between chains A and C, which might correspond to a lipid head group, but was not modelled in due to the ambiguity. Peripheral lipid molecule L6 is resolved close to this site between chains C and B, coordinated by residues from chain C's TM(−1) and TM10′—a key element in the conformational changes during transport (Perez et al, 2012) (Figure 3). Another annular lipid L7 is resolved on the trimer periphery associated with residues on the cytoplasmic half of TM1′, which navigates the maximum movement in the inward opening of BetP (Perez et al, 2012) (Figure 4A and B). Some density which best fitted a lipid head group was resolved between residues from the periplasmic end of TM(−1) in chain A and amphipathic h7 from chain C (Figure 4A). The head group and acyl chains of this lipid (U1) from the upper membrane leaflet are also coordinated by residues from TM5′ of chain C. Density for acyl chains in this lipid is visible but flexible reflecting the lack of a distinct coordination. Both annular lipids, U1 and L7, are resolved close to symmetry molecules in the structure, which could influence their packing though they are not directly coordinated by crystal contacts (Supplementary Figure S10). Consequently, these lipid sites are asymmetrical and no corresponding lipid moieties are distinctly identifiable in other chains (Figure 5B). One of the acyl chains of the annular lipid L7 accesses a hydrophobic cleft halfway through chain C lined by residues Val408 and Trp412 from TM7′ and Ala313, Ala314, Ala317 and Ile318 from TM5′ (Supplementary Figure S7). The tip of this acyl chain is involved in direct van der Waal's interactions with Met150 in the glycine-rich unwound region of TM1′ (Figure 4B). This stretch makes a 6-Å movement during conformational changes from outward to inward via the closed state (Supplementary Figure S11). Being an annular lipid, this moiety was recognized by patches of head-group density and acyl chain density close to the interaction site with the transporter. Discussion Structure of state-symmetric trimer reveals specific lipid binding Compared to other LeuT-like fold transporters the cytoplasmic funnel in the inward-facing state of BetP is narrower (Perez et al, 2012). The Ci state was only observed in chain C in previously reported structures (Ressl et al, 2009; Perez et al, 2011b, 2012). This chain exhibits the closest contact with a C-terminal domain within the trimer, also the major crystal contact, and we could not exclude a restrictive effect on the cytoplasmic opening of BetP (Perez et al, 2012). Here, we observe a structurally identical inward-facing state also in chains B and A, the latter being free from any C-terminal interactions or crystal contacts, and thus demonstrate that the functional substrate-free open inward-facing (Ci) state of BetP is in fact narrower than in other LeuT-like fold transporters. Therefore, with respect to the transporter states this new structure is the most symmetric of all BetP structures obtained thus far. It is also the one with the best resolution so far, which allowed us to identify eight PG lipid-binding sites within the trimer. Strikingly, negatively charged PG lipids constitute only 15% of the membrane fraction in the E. coli expression system whose major membrane component is PE (Rübenhagen et al, 2000), whereas PG is the major membrane component in C. glutamicum (Özcan et al, 2007). This implies that native lipids are selected for and retained during the heterologous expression, purification and crystallization of BetP emphasizing their structural necessity. The assignments to PG lipids are in perfect agreement with multiple biochemical and functional studies (Schiller et al, 2006; Özcan et al, 2007), all of which show that this lipid species is crucially involved in transport regulation in BetP. Lipids associate with helices involved in conformational changes Some of the resolved lipids are coordinated by residues from helices or loops involved in conformational changes. The head groups of all three of the lipids resolved at symmetric sites in the trimer centre L1–L3 (Figure 5A) are coordinated by residues from loops 2 and IL3. Both these loops link together functionally important helices. Loop 2 connects peripheral helix TM(−1) to TM1′, which is one of the gating elements opening the cytoplasmic half of BetP during transport (Perez et al, 2012). IL3 connects TM6′ and TM7′, which like TM1′, are also part of the 4TM bundle domain, containing within them the substrate translocation pathway (Perez et al, 2012). Previously, we showed that disruption of a cation-π interaction between residues from loops 2 and IL3 led to a dramatic decrease in activity and upregulation by osmotic stress was no longer possible (Gärtner et al, 2011). Therefore, between them, these two loops form an important lipid head-group coordination site. Lipid L4 in addition to being coordinated by loops 2 and IL3 also involves the C-terminal helix of chain A (Figure 2B). The negatively charged lipid molecule here provides an interaction site to effectively restrain the positively charged stretch in the C-terminal helix and consequently restrict movement of the loops connecting the bundle helices in a regulatory manner (Gärtner et al, 2011). To date, it is not known whether this regulatory restriction from the membrane results in upregulation or downregulation of the transport activity, which is attributed to the fact that mutations in the ionic network of loop 2 are not tolerated. However, having key components in the functional conformational changes directly accessible by the membrane may prove to be beneficial to communicate changes in the state of the membrane due to osmotic stress, an important factor in osmoregulation (Wood, 1999), through the core of the protein. Structural interaction of annular lipid with the catalytic core Lipid L7 is nested between TM5′ and TM1′, with one of its acyl chains interacting with the functionally important glycine-rich unwound stretch in TM1′ (Figure 4A and B). Along with being crucial in conformational changes, mutation of a glycine in this unfolded region to an aspartate was sufficient to alter substrate specificity in BetP and allow for transport of choline as well (Perez et al, 2011b). This lipid interaction is only resolved in the one chain which has a symmetry molecu
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