57 D55C/T341C were cross-linked by all three cross-linkersalbeit the cross-linking efficiencies were lower (20-40% intramolecular cross-linking) than that of S57C/T341C. Finally, M62C/A327C showed significant cross-linking by CuPh (30% intramolecular cross-linking) only at 25 °C, whereas reaction at 4 °C was ineffective, proposing that a conformational alteration preceded disulfide bridge formation. In contrast to CuPh, M62C/A327Ccould be cross-linked with BMH or p-PDM (40 to 45% intramolecular cross-linking) already at 4 °C, suggesting that under thiscondition the distance between both positions lied in the rangeof 9.2 Å and 12.3 Å (distance spanned by rigid p-PDM).
To test for possible ligand-induced changes of distances betweenTMs II and IX, the effect of Na+ and proline on Cys cross-linkingwas analyzed (suppl. Fig. 2.4.). The results did not revealany significant effect of the ligands on cross-linking.
Taken together, these results demonstrated an at least temporalproximity of TMs II and IX. The distances between selected residuesof these domains were not well defined probably due to the structuralflexibility of this protein region.
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58 surroundings of the ion-binding siteof PutP. Alternatively, it may participate in ion binding via its main-chain carbonyl group.
Substitution of the neighboring Thr341 results in a somewhat different phenotype, indicating a specific role for the hydroxylgroup of the side chain in ion binding. Although the most conservativereplacement with Ser has only little effect on ion binding, Na+ does not significantly stimulate proline uptake into intactcells containing PutP-T341C or -T341V. Using the more definedproteoliposomes system, Na+ stimulation of transport containingeither one of these mutants is observed only at elevated ion concentrations, suggesting a highly reduced (minimum two ordersof magnitude) ion affinity. Why is this stimulation not seenwith intact cells? Clearly, it is easier to establish defined energetic conditions with proteoliposomes than with intact cells in which not only PutP but a number of other membrane proteins (e.g. Na+/H+ antiporter) interfere with electrochemical ion gradients.
Furthermore, high concentrations of Na+ have an inhibitoryeffect on various proteins and cellular processes (Padan and Krulwich, 2000). In fact, proline uptake into PutP wild-type cells is inhibited already at Na+ concentrations of 250 mM and higher. This is exactly the concentration range by which proline uptake into PutP-T341C or -T341V proteoliposomes becomes stimulated. Therefore, becauseof the complex scenario in intact cells, stimulatory and inhibitoryeffects of Na+ on proline transport may partially compensateeach other. In any event, the observed distinctive effects ofthe Thr341 substitutions on the apparent ion affinity implicatethe residue in binding of the coupling ion.
Besides the effects of the amino acid replacements on ion binding, proline binding and transport is also affected. The substitutionsinhibit also counterflow activity, which does not require anelectrochemical ion gradient. In addition, the proline affinityis affected, albeit to a lesser extent than the ion affinities(Fig. 2.3.). These results provide additional evidence for the particular functional significance of Ser340 and Thr341 and indicate that both residues are important for ion and prolinebinding. Similar observations have already been made upon replacement of other residues (Asp55, Ser57, Gly58 of TM II) supposed to be involved in ligand binding (Pirch et al., 2002; Quick et al., 1996; Quick and Jung, 1997). Obviously, there is an interdependence of ion and proline binding as shown bythis and other investigations (Jung et al., 1998a; Zhou et al., 2004; Yamato and Anraku, 1993; Yamato and Anraku, 1990). A simple explanationfor the observed phenomena is a close proximity or even an overlapping of ion- and proline-binding sites. Such an arrangement would ensure tight coupling of ion and substrate transport. But whatwould be the primary function of Ser340 and Thr341? The followingresults argue for a primary role of the residues in ion binding:(i) the amino acid replacements have a stronger impact on ion than on proline binding; ii) apparent ion affinities remainreduced even in presence of a saturating proline concentration;
(iii) Cys at position 340 or 341 is not protected by prolineas, e.g. observed for positions 57
59 and 58, which are suggestedto participate in proline binding (Pirch et al., 2002; Quick et al., 1996).
A location of Ser340 and Thr341 in or close to a ligand-translocation pathway is further supported by the result that Cys placed ateither position in the middle of TM IX reacts with methanethiosulfonate compounds of different polarities. This result indicates an accessibility of both positions from the water phase. The lackof efficient Cys protection by Na+ may be attributed to thehighly reduced Na+ affinities of the mutants. In addition, coupling ions are small and, therefore, a priori less efficient in protectingCys at or close to a ligand-binding site than a larger co-transportedsubstrate (e.g. amino acid, sugar).
If Ser340 and Thr341 participate in ligand binding, are theresidues located close to other amino acids implicated in Na+ (Asp55) and proline binding (Ser57, Gly58)? Here, it is shown that pairs of Cys residues placed at these positions 55/340, 55/341, 57/340, and 57/341 form disulfide bridges. Albeit thecross-linking efficiency varies between the pairs, the resultssupport the idea of a functional interaction of TMs II and IX.However, the necessary amino acid replacements in (or close to) the proposed ligand-binding sites of PutP impair transportactivity almost completely. Therefore, pairs of Cys residuesare also introduced at either end of TMs II and IX. The resulting functional PutP variants are also cross-linked, indicating anat least temporal proximity of TMs II and IX. Furthermore, theobservation that cross-linkers of varying lengths cross-linkpositions in both TMs suggests a conformational flexibility of these parts of the transporter. The latter idea is further supported by the temperature dependence of cross-linking ofM62C/A327C. The proposed proximity between TMs II and IX isin general agreement with earlier analyses reporting a clusteringof mutations causing an altered substrate specificity withinthe N- and C-terminal domains of PutP (Dila and Maloy, 1986).
Despite comprehensive trials, a significant effect of Na+ andproline binding on cross-linking efficiencies (e.g. via alteringintramolecular distances) was not observed, although a participation of TM II in ligand-induced conformational alterations was previouslyreported (Pirch et al., 2003). Also, in view of the in-part dramatic effectsof the Cys substitutions on apparent ligand affinities, it isassumed that the PutP variants are even in the presence of ligands still dynamic enough to allow efficient cross-linking of Cys pairs within the given labeling period. In addition, Cys cross-linking is known to underestimate distances (Abramson et al., 2003), thereby potentiallyobscuring ligand-induced changes.
Taken together, the data lead to a working hypothesis accordingto which amino acids of TM II (Asp55) and TM IX (Ser340, Thr341) form part of the ion- and/or substrate- translocation pathway of PutP, thereby Asp55 and Thr341 may directly participate in Na+ binding (Fig. 2.6.). Furthermore, since binding of Na+ andproline is interdependent and side- chain alterations affectnot only binding of Na+ but also of proline, it is proposedthat binding
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60 of both ligands occurs in close proximity. Thereby, the carboxylate of proline may even directly interact with thecoupling ion.
Such an arrangement resembles in part the situation found in thethree-dimensional structure of the leucine transporter LeuTAa (NSS family) (Yamashita et al., 2005). Here, binding of two Na+ ions occurs in cavitieslocated also in about the middle of the membrane with Na1+ being in direct contact with the substrateleucine. Furthermore, ligand binding also involves residuesin N-terminal and C-terminal domains (e.g. Na1+, TMs I and 6; Na2+, TMs I and VIII). Na2+, for example, makes direct contact to five oxygen atoms out of which three originate from main-chain carbonyl groups and two come fromside-chain hydroxyl groups of two adjacent polar amino acids,Thr354 and Ser355 (TM VIII). Although the neighborhood of Thr and Ser resembles the situation in PutP, the results presented here suggest a direct involvement in Na+ binding only for theside chain of Thr341 of PutP.
Figure 2.6.: Model showing the participation amino acids of TM II and IX in ligand binding and transport. The model is based on the current analysis as well as on previous investigations (Pirch et al., 2002; Quick et al., 1996; Quick and Jung, 1997). It represents a working hypothesis according to which TMs II and IX participate in the formation of a ligand-translocation pathway; Asp55, Ser57, and Thr341 directly participate in Na+ or proline binding. Further structural information is necessary to prove or disprove the model.
Can the model presented in Fig. 2.6. be generalized for all members of the SSS family? The conclusions drawn here are in good agreementwith previous analyses of NIS, which like PutP is a member ofthe SSS family. In fact, the corresponding amino acids in NIS (Ser353 and Thr354) are demonstrated to be particularly crucial for Na+ binding and/or translocation (De la Vieja et al., 2007). The general importanceof Asp55 and Ser57 is less clear as these residues are notas conserved within the SSS family as Ser340 and Thr341.
So, it is possible that Na+ binding to the N-terminal domains differs significantly between members of the SSS family. These differences may be attributed to the different Na+:substrate stoichiometries(e.g. 1:1 in PutP; 2:1 in NIS, SGLT1). In addition, becauseion- and substrate-binding sites are supposed to closely interactwith each other, the properties of the substrates (e.g. chargedproline versus polar sugar) may also have influenced the precise mechanism of Na+ binding. In human SGLT1, the N-terminal halfof the protein is thought to contain two Na+-binding sites,whereas the C-terminal part is made responsible for sugar binding and translocation (Meinild et al., 2001; Panayotova-Heiermann et al., 1996;
Panayotova-Heiermann et al., 1997; Kumar et al., 2007). Thereby, sugar binding is supposed
61 to occur in a hydrophilic cavity formed by TMs X-XIII (Diez-Sampedro et al., 2001; Wright et al., 2007). Residues directly participating in Na+ binding are not yetidentified.
Finally, it must be considered that the complete sets of residuesinvolved in ion and/or substrate binding are neither known forPutP, SGLT1, or other members of the SSS family.
Furthermore, although the results presented here support an involvement of Ser340 and Thr341 of PutP in ligand binding, it must be statedthat the observed binding defects can also be explained by indirecteffects, e.g. distortion of the structural arrangement of neighboring residues participating in the formation of a binding cavity.Along this line, Ser340 and Thr341 could simply be requiredfor stabilizing intramolecular contacts between N- and C-terminal domains, which in turn may be necessary for high affinity ligand binding. Clearly, more information on transporter structureis required to fully understand the phenomena described here.
In summary, the results demonstrate that Ser340 and Thr341of PutP are particularly important for high affinity Na+ andproline binding. Being placed in the middle of TM IX, the residuesare accessible from the water phase and located close to residuesof TM II, which are also implicated in ligand binding. For these reasons it is suggested that Ser340 and Thr341 are locatedin a ligand-translocation pathway. Furthermore, it is proposedthat Thr341 directly participates in Na+ binding.