Glucose_H_p__symporter

The glucose/H+ symporter, also referenced in short as GlcPSe, (PDB ID: 4LDS) is a glucose transporter protein found in the Staphylococcus epidermidis (S. epidermidis) (1). The capacity for cells to take in and metabolize glucose is a characteristic that is shared among a vast majority of organisms (2). Glucose transporters allow the movement of glucose into cells in which glucose acts as a vital source of energy and precursor for protein and lipid synthesis. The specific glucose transporter from S. epidermidis was chosen for investigation due its high sequence identity and homology to human glucose transport (GLUT) family and inhibition by human glucose transport inhibitors cytochalasin B, phloretin and forskolin. An interference in the activity of GLUTs has been connected to an array of diseases including GLUT deficiency syndrome, Fanconi-Bickel syndrome, cancer and diabetes. The examination and analysis of the crystal structure of GlcPSe with site-directed mutagenesis and transport-activity studies can provide understanding into the mechanism of glucose transport (1).

For the crystallization process, the glucose/H+ transporter from S. epidermidis was expressed in E. coli C41 cells using bacterial transformation and purification techniques. GlcPSe was crystalized in an inward-facing conformation by hanging-drop vapor diffusion through the combination of 1 µL of 10 mg/mL purified GlcPSe with 1 µL of precipitating solution. Under the conditions of 22-26% PEG 400m 0.1 M calcium acetate, 0.1 M NaCL, 0.1 M MOPS (pH 7.0) at 18 °C, rod-like crystals of 0.05 × 0.05 × 0.2 mm appeared within 2 to 3 days. The crystals were then dehydrated to improve their diffraction and increase resolution from 10 Å to 3-4 Å. The inward-facing structure was obtained through the use of many programs including ShelxC/D/E to obtain initial phases from Hg-derivative crystal by single anomalous diffraction, COOT to manually trace the 12 transmembrane helices and XtalView, Phenix and Refmac to build and refine the model (1).

According to Expasy database, GlcPSe has a molecular weight of 96731.65 Da and an isoelectric point of 6.08 (3). Comprised of two identical subunits, A and B, the unliganded wildtype inward-facing GlcPSe consists of a total of 892 residues with 446 residues in each subunit. The GlcPSe was crystalized from residues 22 to 446 for each full-length, physiological subunit of GlcPSe, missing 22 amino acids on the N-terminus. Each subunit includes a repeating helix motif (residues 27-54, 61-83, 95-114, 120-141, 153-175, 181-203, 260-283, 295-320, 327-349, 355-379. 391-411 and 423-443), major facilitator superfamily (MFS) domain (residues 29- 446) and the sugar transporter domain (residues 32-446) (4).

The primary structure of GlcPSe is composed of two identical chains, each consisting of 446 residues for a total of 892 hydrophobic, hydrophilic, acidic and basic amino acid residues. Each subunit of GlcPSe has a secondary structure that is approximately 72% helical (22 helices, 323 residues) with the majority being α-helix (20 out of 22 helices), but 2 out of the 22 helices are 3/10 helices, and the remaining secondary structure is made up of random coils (4). The α-helices consists of a mixture of hydrophobic and hydrophilic amino acids which interact to stabilize the structure through the hydrogen bonds formed between carbonyl oxygens and amide hydrogens.

Relating to the tertiary structure of GlcPSe, the random coils are located towards the exterior of the structure and link between helices. The outer ends of the helices and the random coils linking the helices consist of a notable amount of acidic and basic amino acids which contribute to the strong interaction between helices within a structure through the formation of hydrogen bonds and salt bridges (1, 5). The mixed hydrophobic and hydrophilic residues of helices 1, 4, 7 and 10 form a central amphipathic cavity of GlcPSe. The quaternary structure shows the interaction of the subunits by cytoplasmic loops made up by three helical segments. As a result, the structure demonstrates twelve transmembrane helices arranged as two twofold pseudosymmetrical domains connected by a cytoplasmic loop made up of three helical segments (1).

The GlcPSe protein takes part in transport of glucose into the cell through the mechanism of glucose/H+ symport, which is demonstrated by the optimum glucose uptake activity of the protein at acidic pH. The protein shows high substrate specificity for glucose substrate and high kinetic affinity. The mechanism of glucose and H+ symport exhibits a stoichiometry ratio of 1:1. Through site-specific mutagenesis of residues, Gln-137, Gln-250, Gln- 251 and Asn-256 are identified critical residues of glucose binding sites. Asp-22 is identified as a critical residue involved in H+ binding by catalyzing uniport. Closely related to Asp-22, the Arg-102 in helix 4 is also important in glucose transport and H+ binding. The salt bridge between these two residues of distant helices pushes out surrounding helices and opens the cavity housing the glucose binding site when H+ is absent. At low pH, protonated Asp-22 releases Arg-102 and lowers energetic barrier between inward and outward conformation transitions, closing the glucose substrate cavity. Ser-105 is also a notable residue in glucose transport due to its ability to create a hydrogen bond with the Asp-22 side chain, blocking formation of salt bridge between Asp-22 and Arg-102. As GlcPSe was chosen for research due to its homology to human GLUTs, a number of GLUT inhibitors, including phloretin, cytochalasin B, forskolin and phlorizin, were introduced to test their effects on the GlcPSe. Phloretin fully inhibits and cytochalasin B and forskolin partially inhibits glucose transport activity while phlorizin did not significantly inhibit glucose transport activity (1). Research on the side effects of antipsychotic drug olanzapine on glucose metabolism has found that olanzapine inhibits glucose transport activity by binding to polar region of the cytosolic part of the transporter such as residues Asn-136 and Arg-129 and disrupting the key salt bridge formed between Arg-129 and Glu-362, disrupting the opening and closing of the substrate cavity for glucose transport (6).

The structure of an alternate outward-facing conformation of GlcPSe was modeled based on Xy1E (PDB ID:4GC0) to compare the inward-facing and outward-facing conformations as the both conformations seem to have a role in the glucose transport mechanism. The transition between conformations occurs through a relative rotation of the N and C domains around an axis parallel to the membrane that passes through the middle of the molecule and close to the glucose-binding site. A number of structural changes between conformations could be observed through the comparison. The helices on the cytoplasmic side are moved closer towards the center of molecule and the helices on the periplasmic side are moved further away from the center in the outward-facing conformation than in the inward-facing conformation. Furthermore, a salt bridge between Arg-71 and Arg-369 occurs in the outward-facing conformation not found in inward-facing conformation, exhibiting the changing interactions between residues (1).

Two different servers, BLAST and Dali server, were used to search for proteins with homologous sequence and structure, respectively. BLAST stands for Basic Local Alignment Search Tool. PSI-BLAST, which stands for position-specific iterated BLAST, was specifically used to compare the protein of interest with proteins of similar primary structure. The degree of similarity is represented by an assigned E value per sequence comparison calculated by determining gaps between total sequence homology. An E value of less than 0.05 is counted as significant for protein analysis. The Dali server compares the tertiary structure of proteins by calculating the differences in intramolecular distances through a sum-of-pairs method. A Z-score is assigned to indicate degree of tertiary structure similarity between the protein of interest and the comparison protein. A Z-score greater than 2 is indicated to be structures with significant similarities. The Arabidopsis thaliana (A. thaliana) Sugar Transport protein 10 (PDB ID: 6H7D), also referred to as STP10, has a significant E-value of 8*10-67 and Z-score of 28.9, showing significant similarity in both primary and tertiary structure (7, 8).

Both STP10 and GlcPSe are sugar transport proteins directly involved in the cellular uptake of glucose. While STP10 is expressed in the plant A. thaliana, the GlcPSe is expressed in gram-positive bacterium S. epidermis. Both proteins exhibit a high affinity sugar recognition and proton-coupled symport of glucose. However, the sugar transport mechanism of STP10 is highly dependent on the Lid domain that contains a cluster of aromatic residues Phe-55, Phe-59, Phe-60, Phe-79, Phe-87 and Trp-202 and disulfide bridge between Cys-77 and Cys-449 while the salt bridge between Arg-129 and Glu-362 and between Asp-22 and Arg-102 is critical in the GlcPSe glucose transport mechanism (1, 9). Although both proteins carry out similar functions, major structural differences can be found between the two proteins. GlcPSe is a dimer made up of two identical subunits, A and B, while STP10 is a monomer of a single subunit A. The primary structure of the two proteins differs in that GlcPSe has a total of 892 residues while the STP10 protein has a total of 521 residues, exhibiting that the STP10 protein is smaller that GlcPSe by 371 residues. GlcPSe does not have any ligands while the STP10 has four ligands, including 1-Oleoyl-R-glycerol, phosphate ion, Heptaethylene glycol and Beta-D-Glucose, associated. The secondary structures are similar in that they are predominantly helical (~70%) for both proteins. However, the secondary structure of STP10 has a higher proportion of 3/10 helices (6 out of 32 helices) than that of GlcPSe (2 out of 22 helices) (4, 10).

In conclusion, the vital role of glucose as an energy source and precursor in lipid and protein synthesis in a vast majority of organisms can portray the biological importance of glucose transporters in general. In particular, the GlcPSe protein is examined due to its high sequence identity and homology with human GLUTs which have been found to be associated with a variety of diseases such as GLUT deficiency syndrome, Fanconi-Bickel syndrome, cancer and diabetes (1). The closer investigation and analysis of the structure of GlcPSe and its mechanism of glucose transport allows scientists to gain insight into the mechanism of human GLUTs that are involved in common diseases. Since there is a variety of glucose transport mechanisms for different types of GLUTs such as uniport or myo-inositol/H+ symport, further examination and comparison of other homologous proteins that have corresponding mechanisms of specific GLUTs would be helpful in attaining deeper understanding of the role of GLUTs in biological processes and diseases.