DEBP
Created by Nirav Patel
The Glutamate/Aspartate Binding Protein is a two-chain transport protein that specifically binds the amino acid, L-glutatmate and L-aspartate. DEBP is found in many microbes, particularly Escherichia Coli. DEPB has a molecular weight of 64851.95 Da and an isoelectric point of 8.10.1Specifically, DEBP is a periplasmic binding protein that is part of an ATP binding cassette(ABC) transport system. These systems couple ATP hydrolysis to the transport of solutes across the membranes. They consist of a periplasmic binding protein, an integral membrane protein, and two cytoplasmic nucleotide-binding domains that hydrolyze ATP. Periplasmic proteins are found in the periplasmic space in between the outer and inner membranes of gram-negative bacteria such as E. Coli. The periplasmic protein, such as DEBP, captures the ligand that diffuses through the outer membrane. The periplasmic protein then passes off the ligand to the transmembrane protein for transport into the cell. This is achieved by conformational changes that occur upon ligand binding.2
The transport of glutamate is significant in microbes such as E. Coli because it is useable as a carbon and energy source via alpha ketoglutarate. This process also produces ammonia, which is used by the microbe to assimilate amino acids. Finally, glutamate is important for cell wall synthesis and is therefore a major part of the osmoprotection of the cell.3
The DEBP protein is an asymmetric unit consisting of two similarly folded DEBP molecules. The ligand-binding region exists at the intersection of these two domains. At the heart of each domain exists a five-stranded beta sheet consisting primarily of hydrophobic residues. This central beta sheet is surrounded by 12 alpha helices that shield the beta sheet from water molecules. These two domains are linked together by two antiparallel beta strands that make up the ligand binding region. 4
In fact, the DEBP protein is highly specific to binding only glutamate and aspartate. This high specificity is achieved by hydrogen bonding between the side chains and main chain of the DEBP protein and the glutamate/aspartate molecules. For example, the side chains of the Arg24, Ser72, Arg75, Ser90, and His 164 hydrogen bond to the glutamate and anchor the deprotenated gamma-carboxylate group of glutamate/aspartate with six hydrogen bonds.The side chains of Arg75 and Arg90 also form salt bridges with the deprotenated alpha-carboxylate groups of glutamate/aspartate. In addition, the main chain amide groups of Thr92 and Thr140 form hydrogen bonds with the alpha-carboxylate groups of glutamate. The positively charged alpha amino group of the glutamate is also responsible for forming salt bridges with the side chain carboxylate group of Asp 182 and hydrogen bonds with the main chain carbonyl oxygen of Ser90.1
Hydrogen bonds and salt bridges are not the only interactions that play an important role in the binding of glutamate/aspartate by DEBP. For example, the methylene groups of the glutamate from Van der Waals interactions with the hydrophobic side chains of the DEBP protein.1
The DEBP protein, as mentioned earlier, is highly specific for the ligand it binds and can even discriminate glutamate/aspartate from glutamine and asparagine. Glutamine and asparagine are very similar molecules to glutamate and aspartate respectively. This is because the presence of Arg 24 and Arg75 create a preference for a strongly negatively charged group (the carboxylate groups of glutamate/aspartate) as opposed to a neutral group (amide groups of glutmaine/asparagine). There are also severe steric clashes between the amide hydrogen atoms of DEBP and the alpha carboxylate groups of glutamine /asparagine. It is also interesting to note that glutamate and aspartate are very different in terms of molecular size. Despite these differences, DEBP is able to discriminate molecules because of the very specific binding pocket around the ligand side chain. The reason for this is that the gamma carboxylate group of the ligand only interacts with side chain groups from DEBP and therefore is more flexible. Therefore when glutamate is replaced with asparatate, the alpha carboxylate and alpha amino groups of aspartate occupy the same position that the alpha carboxylate and amino group of glutamate do. This change in the side chain carboxylate group is allowed by conformational rearrangements of the DEBP side chains.1
In summary, the DEBP protein is 36% helical and 23% beta sheet. There are 12 helices consisting of 105 residues and 15 beta strands consisting of 67 residues. There are also 16 beta turns, and 3 beta bridges. It is also interesting to note that the DEBP protein has a disulfide bond between cystine 88 and cystine 213. 5
When a BLAST search was performed on the amino acid sequence of DEBP, one of the homologs found was the arginine-, lysine-, histidine-binding protein Artj from Geobacillus Stearothermophilus (PSB Id:2q2c). The two proteins had overlap for 79% of the amino acid sequence and an e value of 2e-11.6 There are no particular segments of the protein that are conserved, however, the two share overlapping sequences throughout the peptide. Both proteins have a substrate-binding pocket that forms at the intersection of the two domains.7 Both proteins are very specific for the ligands they bind and transport across the cell membrane and therefore there are differences in their sequences. Glutamate has a neutral charge at physiological pH, however, arginine, lysine, and histidine are basic amino acids and have a positive charge at physiological pH. Therefore it is expected that the binding domain would be different between the two proteins, which have very different ligands. However, both are part of an ABC transport system and therefore they both contain highly conserved sequences. Another major difference between the two proteins is that the DEBP protein has a disulfide bond whereas the arginine-, lysine, histidine binding protein ARTJ lacks this tertiary structure.1