FORMAS COMU

PutP belongs to the solute:sodium symporter (SSS) family (TC 2.A.21), which contains several hundred members of prokaryotic and eukaryotic origin (Jung, 2002; Wright and Turk, 2004; Reizer et al., 1994). Proteins of this family utilize a smf to drive uphill transport of substrates such as sugars, amino acids, vitamins, iodide, myo-inositol, phenyl acetate, and urea (Dohán et al. 2006; Jung 2001; Wright et al. 2007). Most of the functionally characterized transporters feed catabolic pathways (e.g. PutP, SGLT, and Ppa) or are involved in cell adaptation to osmotic stress (e.g. OpuE) (Jung, 2001; Spiegelhalter and Bremer, 1998). Among the eukaryotic members of the SSS family the Na+/glucose transporter (SGLT1) and Na+/iodide symporter (NIS) are implicated in the human diseases glucose-galactose malabsorption and iodide transport defect (Wright et al., 2007; Reed-Tsur

et al., 2008). Both proteins also play an important role in medical therapy (Wright et al., 2007;

Dohán et al., 2006). Furthermore, bacterial transporters such as PutP of Helicobacter pylori and Staphylococcus aureus contribute to bacterial virulence and thus represent putative targets for the development of new drugs against pathogens (Kavermann et al., 2003; Schwan et al., 2006).

Topology and functional properties of PutP - PutP of E. coli is one of the best

characterized prokaryotic members of the SSS family. It is composed of 502 amino acid residues and has a molecular mass of 54.3 kDa. Gene fusion analyses, Cys accessibility studies, site-specific proteolysis, and SDSL EPR measurements revealed a secondary structure model according to which PutP contains 13 TMs with the N terminus located on the periplasmic side of the membrane and the C terminus facing the cytoplasm (Jung et al., 1998; Wegener et al., 2000) (Fig. 1.5.). The transporter catalyzes the coupled translocation

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of Na+ and proline with a stoichiometry of 1:1 (Yamato and Anraku, 1993). The apparent

Km

values for Na+ and proline uptake were determined with 30 and 2 µM, respectively (Yamato and Anraku, 1993). Li+ can also be used as coupling ion for proline transport, although the affinity of the transporter for Li+ (

Km

∼125 µM) is significantly lower than for Na+ (Chen et al., 1985). Kinetic analyses of Na+/proline transport catalyzed by PutP suggested that transport occurs according to an ordered binding mechanism (Yamato and Anraku, 1990; Yamato, 1992). Thereby, the initial binding of Na+ is proposed to induce a conformational alteration that increases the affinity of the transporter for proline. The ternary complex assumedly reorientates in the membrane, leading to release of Na+ and proline on the other side of the lipid bilayer. At high substrate concentrations, proline can also bind to the protein in the absence of Na+, as it was shown by electrophysiological measurements using the rapid solution exchange technique combined with a solid-supported membrane (SSM) (Zhou et al., 2004).

Fig. 1.5.: Secondary structure model of PutP of E. coli highlighting important residues. The model is based on a gene

fusion approach, Cys accessibility analyses, SDSL EPR, and site-specific proteolysis (Jung et al., 1998; Wegener et al., 2000). Putative TMs are represented as rounded rectangles and numbered with Roman numerals; loops are numbered with Arabic numerals starting from the N terminus. The single-letter amino acid code is used and the location in the amino acid sequence is indicated by a number. Residues proposed to be directly involved in ligand binding are shown in red. Amino acids proposed to be involved in ligand-induced conformational alterations based on Cys accessibility analyses, fluorescence, and EPR measurements are highlighted or underlined in blue. Other residues of structural and/or functional importance are represented in green.

Functional important residues of PutP - Site-directed mutagenesis studies identified

several amino acids in PutP that are crucial for transport activity (Fig. 1.5.). Most of the found residues are located in TM II of PutP, suggesting that this segment is of particular functional importance. Thus, the carboxylate of Asp55 in TM II was proved to be essential for transport, and significant, albeit highly reduced activity was detected only with Glu at this position (Quick and Jung, 1997). The latter substitution caused an about 50-fold decrease of the

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apparent affinity of the protein for Na+ compared to the wild-type transporter. On the other hand, only a relatively small alteration of the apparent affinity for proline was observed, suggesting that Asp55 is at or close to the cation-binding site (Quick and Jung, 1997). Met56 was also identified as a residue which is crucial for high-affinity ion binding (Pirch et al., 2002). However, in contrast to Asp55 it is not essential for transport and is assumed to be not involved in direct binding of the coupling ion (Pirch et al., 2002). In addition to Asp55 and Met56, individual replacements of Ser57 and Gly58 in TM II led to significantly altered transport kinetics. Substitutions of Ser57 by Ala, Cys, Gly, or Thr caused a reduction of the apparent Na+ and proline affinities by up to two orders of magnitude with little influence on

Vmax

values (Quick et al., 1996). Similarly, Cys substitution of Gly58 decreased the apparent affinity of PutP for Na+ and proline although the effect was less dramatic as in case of Ser57 (Pirch et al., 2002). The influence of these replacements on Na+ and proline affinities supports the idea of a close cooperativity between the cation- and substrate-binding sites in PutP. Furthermore, Ala48 and Gly63 of TM II were found to be crucial for transport function, since replacement of these conserved residues by Cys significantly impaired proline uptake (Pirch et al., 2003). Analysis of the accessibility of single Cys individually placed at different positions in TM II to N-ethylmaleimide (NEM) and fluorescein-5-maleimide indicated a participation of the TM in the formation of a hydrophilic cleft open to the cytoplasmic side of the membrane (Pirch et al., 2003). Together with the transport analyses, the data support the conclusion that residues of TM II of PutP contribute to the formation of an ion- and/or substrate-translocation pathway.

Besides amino acids of TM II, residues of TM IX were shown to be of functional importance. In particular, experiments on Ser340 and Thr341 of TM IX demonstrated that these residues are crucial for proline transport (Böhm and Jung, unpublished information). This is supported by the fact that Ser340 as well as Thr341 are conserved within the SSS family. Replacement of the corresponding amino acids in NIS (Ser353 and Thr354) revealed that the hydroxyl groups are involved in Na+ binding and/or translocation (De la Vieja et al., 2007). Furthermore, a similar amino acid arrangement was found in the X-ray structure of the leucine transporter LeuTAa of the NSS family in which two adjacent polar residues (Thr354,

Ser355) were shown to participate in Na+ binding (Yamashita et al., 2005). Combined with the observed accessibility of the native Cys residue at position 344 in TM IX of PutP to NEM (Yamato and Anraku, 1988; Hanada et al., 1992), this suggests that TM IX forms part of a ligand-translocation pathway similar to TM II of PutP.

In addition to the functionally important residues identified in transmembrane domains of PutP, four conserved charged residues (Arg40, Asp187, Arg257, and Glu311) in loop regions of PutP were identified as being relevant for the transport process (Quick et al., 1999; Quick and Jung, 1998; Ohsawa et al., 1988; Böhm and Jung unpublished information)

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(Fig. 1.5.). Removal of the charged side chain at the individual positions led to a reduced Na+ dependence of proline binding. In the case of Arg40 this was accompanied by decreased apparent affinities of the transporter for Na+ while the apparent affinity for proline was only slightly altered. Therefore, it is proposed that Arg40 is located close to the site of ion binding and is important for the coupling of ion and proline transport (Quick et al., 1999). Replacement of Asp187 with Cys led to high-affinity proline binding even at very low Na+ concentrations, which was attributed to an enhanced Na+ affinity of the transporter (Quick and Jung, 1998). The same was expected for Arg257. However, the effect of the Cys substitution of this residue on proline uptake was significantly lower than that for the alteration of Asp187 (Ohsawa et al., 1988). These results indicate that Asp187 as well as Arg257 are located close to the pathway of the coupling ion through the membrane and are probably involved in the release of Na+ to the cytoplasmic side of the membrane. Finally, the PutP derivative PutP-E311C showed biphasic kinetics with one component manifesting the kinetic parameters of active transport (i.e. high apparent affinity for proline comparable to the wild-type) and the other exhibiting the characteristics of facilitated diffusion (i.e. low apparent affinity for proline) as it is typically for an uncoupled transport process (Böhm and Jung, unpublished information). Thereby, the binding of the proline was shown to be independent of the presence of Na+, suggesting that this residue is important for Na+ binding and/or coupling of ion and proline transport.

Conformational dynamics of PutP - A series of protein chemical and spectroscopic

studies was employed to explore Na+- and/or proline-induced conformational alterations at specific sites in PutP (Wegener et al., 2000; Pirch et al., 2002; Pirch et al., 2003; Pirch and Jung, unpublished information). SDSL of Cys introduced at position of Leu37 or Phe45 in loop 2 and TM II of the transporter and analysis of the corresponding EPR line shapes revealed ligand-induced mobility changes of the attached nitroxide (Wegener et al., 2000) (Fig. 1.5.). In the case of PutP-L37R1 (R1 designates the modified nitroxide side chain, see above), binding of Na+ and/or proline to the transporter led to an immobilization of the spin label side chain while the nitroxide at position 45 becomes more mobile upon addition of proline, and Na+ alone had no effect. From these findings it was concluded that proline binding induces a conformational alteration of PutP that involves at least parts of TM II and the preceding cytoplasmic loop. Na+ could only be shown to affect the structure of loop 2 (Wegener et al., 2000). These results are further supported by DEER distance measurements between spin labels attached to positions 37 (loop 2) and 187 (loop 6). The measurements revealed an increase of the mean interspin distance between the nitroxides at these positions upon Na+ binding, suggesting that one or both positions are involved in Na+- induced structural alterations (Jeschke et al., 2004a). The EPR studies were confirmed and extended by Cys accessibility analyses in the presence and absence of ligands. Thus, Na+

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was shown to increase the accessibility of Cys placed at the position of Ser57, Gly58 in TM II and Ser71 or Glu75 in loop 3 (Pirch et al., 2003; Pirch and Jung, unpublished information) (Fig. 1.5.). In contrast to Ser57 and Gly58, proline did not affect the accessibility of Cys at positions 71 and 75. Furthermore, proline inhibited the reaction of sulfhydryl-specific reagents with Cys at positions in TM II (Phe45, Val46, Thr47, Leu49, Ser50, Ala51, Ser54, Gly63, and Leu64), TM VII (Pro252), Loop 8 (Arg257), and TM IX (Cys344) (Pirch et al., 2003; Pirch and Jung, unpublished information). Since substitution of the native amino acids at most of these positions has no or only very little effect on PutP transport kinetics, the altered Cys accessibility can be attributed to conformational alteration of the protein upon ligand binding and not to direct steric hindering as it is discussed for Ser57 and Gly58 (Pirch

et al., 2002; Pirch et al., 2003). In addition to these results, fluorescence of PutP site-

specifically labeled with a fluorescence group at position 223 (loop7) was altered by addition of proline if Na+ was present (Pirch and Jung, unpublished information). In conclusion, the studies suggest that at least parts of TM II, TM VII, and TM IX and loops 2, 3, 7, and 8 are involved in structural alterations induced by Na+ and/or proline, which correlate well with the ordered binding model of Na+/proline transport.

Tertiary structural information - To understand the transport mechanism of PutP at the

molecular level, knowledge of the structure is indispensable. However, the only information on the tertiary structure of PutP available so far was gained by DEER distance measurements between spin labels attached to positions in loops 2, 4, 6, and 7 (Jeschke et

al., 2004a). The results revealed that the proposed cytoplasmic loops 2 and 6 and the loops

2 and 4 are in close proximity (∼2 nm) to each other, whereas the cytoplasmic loop 4 and the periplasmic loop 7 are separated by a distance of 4.8 nm. This DEER data strongly supports the idea of loops 2, 4, and 6 being located at the same side and loops 4 and 7 at opposite sides of the membrane, which is in good agreement with the proposed secondary structure model of PutP (Jung et al., 1998; Wegener et al., 2000) (Fig. 1.5.).

In document Representaciones sociales de la convivencia escolar, como estrategia para la creación teatral (página 148-156)