Projection structure at 8 A resolution of the melibiose permease, an Na-sugar co-transporter from Escherichia coli

Journal of Biological Chemistry

Nurva

Ankeshwarapu

2011

154

In eukaryotic cells, the primary cation responsible for the electrochemical gradient across membranes is Na ϩ , to which most symporters are coupled. In certain Na ϩ symporters, H ϩ and Li ϩ can substitute for Na ϩ ; examples are the human Na ϩ /nucleoside cotransporter (CNT3) (1) and the human Na ϩ /glucose cotransporter (SGLT) (2). In bacterial membranes, the electrochemical gradient is built by H ϩ , and H ϩ / solute cotransporters are most common. Bacterial melibiose permease (MelB) 2 exhibits a unique property that couples sugar transport with Na ϩ , Li ϩ , or H ϩ and selects the cation depending on the transported sugar structure (3). The multiple coupling feature of this group of transporters yields a useful tool to study the mechanism of cation/sugar symport.Bacterial MelB belongs to the glycoside/pentoside/hexuronide:cation family (4), a subgroup of the major facilitator superfamilies of membrane transport proteins, where the lactose permease (LacY) is the best characterized member (5, 6). MelB of Escherichia coli (MelB-EC) is the best characterized member among all MelB orthologues (7-16). MelB-EC catalyzes the coupled stoichiometric symport of a galactoside with a cation (Na ϩ , Li ϩ , or H ϩ ) utilizing the free energy from the downhill translocation of one cosubstrate to catalyze the translocation of the other (3,(17)(18)(19)(20), and all three cations compete for a common binding pocket (21-23).The primary sequence alignment between MelB-EC and LacY is relatively poor with ϳ37% sequence similarity and ϳ15% identity; however, membrane topology studies of MelB (24 -26) suggested a topology similar to LacY, with 12 transmembrane helices and cytoplasmically located N and C termini. A three-dimensional structure model of MelB was recently built by threading analysis (27), using a crystal structure (PDB ID 1PV6) of LacY (28,29) as the template. The model suggested a similar overall fold between these two permeases; i.e. MelB is organized in two-helix bundles connected with a central loop and separated by an internal cavity facing the cytoplasmic side. From bioinformatics data, this overall fold seems conserved among MelB orthologues (27). Moreover, this model is consistent with numerous previous biochemical/biophysical data (14, 30 -37), as well as low-resolution EM structures obtained from MelB-EC (38, 39).Diverse cation selectivity was identified in MelB orthologues (40). Although it shares high sequence identity with MelB-EC, MelB of Klebsiella pneumoniae couples melibiose transport only to H ϩ and Li

supporting

confidence: 90%

Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium

Journal of Biological Chemistry

Nurva

Ankeshwarapu

2011

154

“…2 A and B). The overall shape of the MelB model is consistent with previous structural results based on 2D crystallographic projections and 3D EM maps (15,16). The calculated electrostatic surface potential of the MelB model reveals a hydrophobic transmembrane region and charged/polar extramembrane loops (Fig.…”

supporting

confidence: 88%

A 3D structure model of the melibiose permease of Escherichia coli represents a distinctive fold for Na ⁺ symporters

Yousef

Proc. Natl. Acad. Sci. U.S.A.

2009

The melibiose permease of Escherichia coli (MelB) catalyzes the coupled stoichiometric symport of a galactoside with a cation (either Na ؉ , Li ؉ , or H ؉ ), using free energy from the downhill translocation of one cosubstrate to catalyze the accumulation of the other. Here, we present a 3D structure model of MelB threaded through a crystal structure of the lactose permease of E. coli (LacY), manually adjusted, and energetically minimized. The model contains 442 consecutive residues (Ϸ94% of the polypeptide), including all 12 transmembrane helices and connecting loops, with no steric clashes and superimposes well with the template structure. The electrostatic surface potential calculated from the model is typical for a membrane protein and exhibits a characteristic ring of positive charges around the periphery of the cytoplasmic side. The 3D model indicates that MelB consists of two pseudosymmetrical 6-helix bundles lining an internal hydrophilic cavity, which faces the cytoplasmic side of the membrane. Both sugar and cation binding sites are proposed to lie within the internal cavity. The model is consistent with numerous previous mutational, biochemical/biophysical characterizations as well as low-resolution structural data. Thus, an alternating access mechanism with sequential binding is discussed. The proposed overall fold of MelB is different from the available crystal structures of other Na ؉ -coupled transporters, suggesting a distinctive fold for Na ؉ symporters.bioenergetics ͉ ligand binding ͉ MelB ͉ protein threading ͉ sugar/cation symporter M elibiose permease of Escherichia coli (MelB), encoded by the melB gene in the mel operon (1), is a well-studied representative of the glycoside-pentoside-hexuronide/cation symporter family of membrane transporters (2). MelB utilizes free energy released from the energetically downhill movement of a cation (Na ϩ , Li ϩ , or H ϩ ), in response to an electrochemical cation gradient, to drive the uphill stoichiometric accumulation of a galactopyranoside (3-5). The type of the cotransported cation depends on the stereostructure of the transported sugar (6). ␣-Galactopyranosides (melibiose, raffinose, and p-nitrophenyl-␣-galactoside) are cotransported with Na ϩ , H ϩ , or Li ϩ , whereas ␤-galactopyranosides (lactose, methyl-1-␤-D-galactopyranoside, and p-nitrophenyl-␤-galactoside) are cotransported with Na ϩ or Li ϩ but not H ϩ (6). In the absence of an electrochemical cation gradient, MelB catalyzes the reverse reaction, using free energy from the downhill translocation of the sugar to drive the stoichiometric transport of the coupled cation in either direction across the membrane (5,7,8). Similar features of transport exist in the lactose permease of E. coli (LacY) (9, 10), the best-studied representative of the major facilitator superfamily (MFS) of membrane transporters (2). Both MelB and LacY transport D-galactopyranosides, with similar substrate specificity; however, sugar transport in LacY is coupled solely with H ϩ (9, 10). MelB consists of 473 aa with Ϸ65% apo...

“…1). This model is consistent with numerous (9-11, 14, 15, 18, 21, 28, 29, 34, 36, 46, 47) biochemical and biophysical results, as well as with low-resolution electron microscopy (EM) structures of MelB Ec (20,37).…”

supporting

confidence: 87%

Reduced Na ⁺ Affinity Increases Turnover of Salmonella enterica Serovar Typhimurium MelB

Jakkula

2012

J Bacteriol

؉ , Li؉ , or H ؉ . Bioinformatics and mutational analyses indicate that a conserved Gly117 (helix IV) is a component of the Na ؉ -binding site. In this study, Gly117 was mutated to Ser, Asn, or Cys. All three mutations increase the maximum rate (V max ) for melibiose transport in Escherichia coli DW2 and greatly decrease Na ؉ affinity, indicating that intracellular release of Na ؉ is facilitated. Rapid melibiose transport, particularly by the G117N mutant, triggers osmotic lysis in the lag phase of growth. The findings support the previous conclusion that Gly117 plays an important role in cation binding and translocation. Furthermore, a spontaneous second-site mutation (P148L between loop 4-5 and helix V) in the G117C mutant prevents cell lysis. This mutation significantly decreases V max with little effect on cosubstrate binding in G117C, G117S, and G117N mutants. Thus, the P148L mutation specifically inhibits transport velocity and thereby blocks the lethal effect of elevated melibiose transport in the Gly117 mutants.