GLUT1

Human glucose transporter GLUT1 (PDB ID: 4PYP) is a biomolecule found in Homo sapiens. GLUT1 is a member of the sugar porter family, which is a subgroup of the major facilitator superfamily (MFS) (1). Proteins belonging to the MFS function as membrane transporters and exist in all known organisms (2). GLUT1 is also responsible for maintaining a base level of glucose uptake in human tissues and organs, including the brain, through facilitated diffusion (1, 3). GLUT1 transports glucose across biological membranes through an alternating access pump mechanism, whereby the substrate-binding site is accessible from either side of the membrane at different times depending on changes in the conformation of GLUT1 (4). GLUT1 can undergo mutations that inhibit its ability to transport glucose across the membrane, which can lead to diseases of the brain (1). One such disease is GLUT1 deficiency syndrome, which includes symptoms such as epilepsy, impaired cognitive development, and reduced motor capabilities (5). GLUT1 also functions as an indicator for tumorigenesis. Cancerous cells require more glucose than noncancerous cells due to their use of anaerobic glycolysis, meaning that elevated glucose levels can indicate the presence of various cancers (1). The study of GLUT1 transport allows scientists to understand the mechanisms by which human cells maintain their glucose levels and how mutations of GLUT1 negatively affect human health.

All members of the major facilitator superfamily of glucose transporters work to control the glucose uptake of erythrocytes through either active or passive transport. GLUT1 is a uniporter that facilitates diffusion of the glucose molecule B-nonylglucoside (B-NG) into human cells through a passive mechanism. GLUT1 also maintains the average concentration of glucose in human blood cells at approximately 5 mM (1). GLUT1 uses the concentration gradient of B-NG across the cell membrane of erythrocytes to transport sugar molecules into cells via the alternating access mechanism. B-NG interacts with the ligand-binding site inside GLUT1 when it is in the outward-open conformation, which causes the protein to shift to the inward-open conformation. B-NG then dissociates from the binding site and GLUT1 returns to the outward conformation (1, 4). If the concentration of glucose inside of the cell becomes too high, GLUT1 cannot continue glucose uptake because there is no longer a concentration gradient able to power the transport.

GLUT1 from Homo sapiens was expressed for analytical observation and testing through X-ray diffraction at a resolution of 3.166 Å (6). GLUT1 is involved in constant uptake of glucose, so the N45T point mutation was introduced to avoid the heterogeneity caused by glycosylation. Additionally, because GLUT1 has multiple potential conformations, the E329Q mutation was used to arrest GLUT1 in the inward-open conformation so that its structure could be studied effectively (1).

The primary structure of GLUT1 consists of a single subunit containing 492 amino acid residues. The secondary structure of GLUT1 is made of approximately 69% alpha helices and 31% random coils (6). No beta pleated sheets are found in the secondary structure. The dominance of helices in GLUT1 structure allows for hydrophobic residues to be buried inside the structure and its hydrophilic residues to face outward on the surface. The secondary structure provides stabilization to GLUT1 through hydrogen bonding between the amide hydrogens and carboxyl oxygens of the residues in the alpha helices.

Like all members of the sugar porter family, the tertiary structure of GLUT1 consists of a core fold that organizes 12 transmembrane segments into two folded domains called the amino-terminal (N) and carboxy-terminal (C) domains. Each domain contains six transmembrane segments folded into inverted repeats (7). The intracellular helical bundle (ICH) that connects the N and C domains is comprised of four short alpha helices (1). GLUT1 contains only one subunit, so it does not have quaternary structure.

The only ligand associated with GLUT1 is the B-nonylglucoside sugar. B-NG binds to the ligand-binding site on the outward-open GLUT1, which increases interactions between the N and C domains on the extracellular side of the membrane. When the binding affinity of the extracellular groups becomes greater than the binding affinity of the intracellular groups, GLUT1 changes conformation from outward-open to inward-open (1). B-NG is exposed to the low concentration intracellular environment, which causes equilibrium to shift from substrate binding to substrate dissociation (1). B-NG is released from the active site, which allows GLUT1 to return to its outward-open conformation.

The outward-facing conformation of GLUT1 is maintained primarily by hydrogen bonds between Glu-329 on the ICH and the amide groups of Gly-154 and Ala-155 on the peptide backbone. The intracellular residue Arg-153 forms two hydrogen bonds with the carbonyl oxygen on Lys-458 to contribute to the stabilization of the outward-open conformation as well. The outward-to-inward conformation shift is triggered by Arg-126 on transmembrane segment 4 (TM4) forming a cation-? interaction with Tyr-292 on TM7 in the C domain. This is a strong interaction that increases both extracellular binding affinity and the strength of the extracellular gate that occludes the binding site. Once in the inward-facing conformation, Asn-34 on transmembrane segment 1 (TM1) forms hydrogen bonds with Ser-294 and Thr-295 on TM7 and Thr-310 on TM8. This network of bonds blocks the ligand-binding site when GLUT1 is in the inward-open conformation (1).

In addition to the outward-facing and inward-facing conformations, there exists an occluded conformation, wherein the binding site cannot be accessed from either the extracellular or intracellular side of the membrane. This conformation appears between the outward-facing and inward-facing structures in the alternating access mechanism of GLUT1. B-NG can either be bound to the site or not present at the site when in this conformation (1). The ligand-bound, occluded conformation occurs during the transition from outward-open to inward-open, and the ligand-free, occluded conformation occurs moving from inward-open to outward-open.

According to the ExPASy database, GLUT1 has a molecular weight of 55683.48 Da and an isoelectric point of 8.93 (8). The Basic Local Alignment Search Tool (BLAST), in particular PSI-BLAST for position-specific iterated queries, database finds proteins with a primary structure similar to that of a protein input. BLAST works through comparison of E values, which are assigned to various proteins based on the gaps in the homology of their sequences compared to the sequence of the protein query. High E values indicate a larger number of gaps, while low E values indicate fewer gaps and greater sequence homology. Only E values less than 0.05 are considered significant when analyzing similarities between proteins. D-xylose proton symporter XylE (PDB ID: 4JA3) from Escherichia coli has an E value of 2*10-152, meaning that it shares a significantly similar primary structure with GLUT1 (6, 9). The Dali server uses the sum-of-pairs method to evaluate the similarities between the tertiary structures of proteins. Dali uses a Z score to signify structural similarities, where a Z score greater than 2 means that the similarities are significant for protein analysis. XylE has a Z score of 43.5, which indicates that XylE and GLUT1 have a high degree of tertiary structure similarity (6, 10).

GLUT1 and XylE are both transport proteins that move glucose across cell membranes. They both belong to the sugar porter family of the MFS because of their similar function but are found in different organisms; GLUT1 is found in humans while XylE is present in E. coli (11). Both GLUT1 and XylE have outward-facing and inward-facing conformations that are dictated by the interaction of a glucose molecule with the binding site. Nonetheless, their method of transport is fundamentally different from each other. GLUT1 is a uniporter and XylE is a proton-driven symporter (1). GLUT1 acts as a facilitator that moves B-NG down its concentration gradient into the cell. XylE undergoes active transport and relies on proton motive force generated by the proton gradient across the membrane in order to transport D-xylose.

Specific residue changes also have an effect on the functions of XylE and GLUT1. For example, Arg-133 on XylE forms a network of bonds with Asp-27 that is essential for proton coupling to power the active transport of D-xylose. In GLUT1, Asp-27 and Arg-133 become Asn-29 and Arg-126, respectively, and Arg-126 does not associate with Asn-29 (1). This small shift in amino acid sequence dictates whether or not the protein can perform proton driven active transport.

Further similarities and differences between GLUT1 and XylE can be found within their structure. XylE has 970 residues, which is nearly double the 504 residues of GLUT1. However, both structures are comprised of only one subunit and one unique protein chain. In regards to secondary structure, neither XylE and GLUT1 contain beta pleated sheets and are dominated by alpha helices, though GLUT1 (69%) has slightly fewer alpha helices than XylE (70%) (6). Since both proteins are members of the sugar porter family, they have the same domains in C, N, and the ICH (1).

In essence, the overall biological significance of GLUT1 rests in its role as a glucose uptake monitor, as well as its functional ubiquity across various species. The interactions of residues on different domains of the protein, both extracellularly and intracellularly, cause GLUT1 to change conformation and allow B-NG molecules into human erythrocyte cells in order to maintain the concentration of glucose necessary for the cells to function properly. Inactivating GLUT1 mutations have been found to cause a variety of diseases resulting from an insufficient energy supply in the brain, such as GLUT1 deficiency syndrome. A large influx of GLUT1 in humans has also been shown to be an indicator of cancerous cell growth and tumorigenesis (1). Ultimately, the physiological significance of the systems of glucose transport undertaken by GLUT1 and the pathophysiological impact of its role as a marker for serious diseases make it of utmost importance for scientists to study.