Aspartate Receptor Tar (PDB ID: 4Z9I) from Escherichia coli 

Created by: James Chung 

            The homodimer, consisting of two chains, A and B and occurs due to reduced exposure to solvent and better subunit association (3, 5).  While the tertiary structure contains no disulfide bonds, scientists have engineered some of these chemical bonds between subunits in order to crystallize the structure (6). 

            The secondary structure of Asp-Tar consists of eight alpha helices, three beta turns, and five random coils with no beta sheets (1).  The periplasmic domain contains the binding site of this protein and consists of a dimer of four alpha helix bundles.  It is symmetrical when the ligand, aspartate, is unbound but is asymmetrical upon aspartate binding (3).  These four helices continue throughout the periplasmic domain to the transmembrane region and connect with the cytoplasmic portion as well.  Alpha helices are well suited to span membranes and normally contain non-polar amino acid residues throughout the membrane portion.  Hence, the alpha helical portion helps situate the Asp-Tar throughout the Escherichia coli’s cell membrane (3).

            Since the two subunits are identical, they both contain binding pocket in the periplasmic domain of the protein.  Important residues in the binding pocket include Thr-154 of chain A, Gln-158 of the first subunit, chain A, and Trp-57 of the second subunit, chain B. In these residues, the carbonyls of Thr-154 and Gln-158 and the nitrogen of Trp-57 hydrogen bond to the ligand, aspartate (3).  Additionally, Tyr-149 of chain A has polar interactions occurring between its aromatic group and the methylene group of the aspartate ligand (3).  In addition to the residues mentioned previously, in the binding pocket, Arg-69 and Arg-73 are specialized for seeking out aspartate and do so through hydrogen bonding with the aspartate ligand  (3).  The chemical nature of these residues in the binding pocket creates a positively charged area that will serve as an attractor for the negatively charged aspartate ligand.  In addition to electrostatic interactions, four water molecules serve as a bridge between the aspartate ligand and the residues in the binding pocket.  These are of great importance, because they only appear in the presence of the ligand.  Research indicates that the water molecules help neutralize the positive charge of the pocket through polar interactions (3).  To summarize, it is the combination of positive electrostatic potential with water bridging that contributes to the specific binding of the Asp-Tar to aspartate.  One example of water bridging, one of these water molecules hydrogen bonds to the carboxyl of the aspartate ligand, the hydroxyl of Tyr-149, and the gamma hydroxyl of Ser-68 (3).

             While both of these binding sites could technically bind two aspartates, binding is negatively cooperative.  This means that upon the binding of one aspartate to one binding site, the other binding site does not bind another aspartate (3).  When this ligand binds, the protein undergoes a conformational change, which scientists believe is the trigger for this signaling transduction pathway (1).  Furthermore, while either of the two binding sites can bind the first aspartate, it is this conformational change that prevents the binding of a second aspartate to the other binding site.  This binding creates a destabilization among the subunits, but allows for flexibility of movement of the receptor subunit (1).  In this change, the fourth alpha helix of the periplasmic domain displaces in a piston-like fashion.  The result is an asymmetric shape between the two subunits.  Electrostatic surface potentials of the binding site show that it is initially positively charged due to its residues but upon aspartate binding, the potential loses its net charge due to the neutralization of charge (1). While the Asp-Tar displays great specificity for the aspartate, it can also bind to glutamate albeit weakly (3). 

            PSI-BLAST and Dali server results provided possible comparison proteins to the Asp-Tar protein.  PSI-BLAST is a program that can compare amino acid sequences of a protein and match these to other similar proteins.  It does so by evaluating the gaps in the comparison protein’s sequence and from this, assigns an E value to the comparison structure.  In comparing sequences, PSI-BLAST only evaluates primary structure.  The smaller the E value, the closer the two proteins are similar in sequence with an E value less than 0.05 indicating a significant similarity (7).  Dali server by contrast, uses tertiary structure to compare two proteins.  In comparing proteins, Dali server calculates the intramolecular distances using a sum-of-pairs method and assigns a Z-score to the comparison protein of interest.  Unlike E values, a higher Z-score indicates a closer similarity with a score of 2 indicating a good match (8). 

            Using the results from the Psi-Blast and Dali servers, the Aspartate Receptor from Salmonella enterica (PDB ID: 1WAT) served as a good comparison protein, with an E Value of 4x10^-66 and Z-score of 17.8 (7, 8).  Like the Asp-Tar, the Aspartate Receptor (Asp-Tar II) binds to aspartate, triggering a signal transduction pathway that leads to chemotaxis (6).  Important functional residues of Asp-Tar II include Arg-64, Arg-73, and Tyr-149, and are conserved among the Asp-Tar protein (6).  Like Asp-Tar, these residues of the Asp-Tar II protein stabilize the aspartate through hydrogen bonding (6).  This protein is of biological significance because it highlights another protein that organisms may use to detect vital nutrients (6).  Furthermore, this protein can elucidate the roles of other chemotaxis proteins by mapping out the similarities between it and other similar proteins (6). 

            Asp-Tar II is similar in weight and length to Asp-Tar with 326 residues and a molecular weight of 32865 Daltons (6).  Unlike Asp-Tar, Asp-Tar II is not completely helical in structure and has beta strands in addition to alpha helices, beta turns, and random coils.  For quaternary structure, however, Asp Tar II is also a homodimer with a binding site on each of its chains (6).   Furthermore, the binding of this protein also exhibits negative cooperative binding, allowing for only one of the binding sites to be bound to a ligand in a single time (6).                

            To conclude, the Asp-Tar protein of Eshirichia coli is a type of chemotaxis protein used for detecting molecules.  This protein is a homodimer and its ligand is aspartate. Although it has two binding pockets, upon the binding of one aspartate, this protein will undergo a conformational change prohibiting it from binding a second aspartate.  By comparison, the Asp-Tar II from Salmonella enterica is also a homodimer and contains aspartate as its ligand.  However, its secondary structure has beta strands unlike the secondary structure of Asp-Tar.  Hence, Asp-Tar II is a good comparison protein to Asp-Tar and further portrays the chemotaxis role that vital to unicellular organisms.