DasR

DasR (PDB ID: 4ZS8), a protein found in Streptomyces coelicolor, is a member of the large family of prokaryotic transcription elements, GntR (1, 2). Transcription factors are valuable to bacteria because they allow alteration of gene expression in response to changes in the environment, such as availability of certain food sources or other environmental stresses (2). DasR has been shown to bind phosphorylated sugars and to repress genes containing DasR-responsive elements (dre-sites) in the bacterial genome by inhibiting transcription at that site (2). These sites are often found in genes related to N-acetylglucosamine (GlcNAc) related catabolism, a major source of energy for S. coelicolor (2). DasR also regulates other general processes like cell development and secondary metabolite production in addition to being involved in antibiotic biosynthesis, making it a global regulator of transcription (2). The molecular weight of DasR is 57.23325 kDa and its isoelectric point is 9.36 (3). DasR is a homodimer and has a varied secondary structure, containing beta sheets, alpha helices, and random coils with varied levels of polarity throughout (2). As a transcription factor, DasR must sense changes in the cell to properly alter transcription (2). This is done through the binding of either glucosamine-6-phosphate (GlcN-6-P) or N-acetylglucosamine-6-phosphate (GlcNAc-6-P), which alters the conformation of the protein and inhibits DNA binding (2).

Two databases that can be used to find proteins of similar structure are PSI-BLAST and the Dali Server (4, 5). PSI-BLAST identifies similar proteins based on primary structure (4). It does this by comparing amino acid sequences and assigning scores, E values, based on the number of gaps in the alignment between the queried and the comparison protein (4). Proteins are considered significantly similar if the E value is less than 0.05 (4). Dali Server finds similar proteins based on tertiary structure (5). It does this by assigning a Z-score using a sum of pairs method by comparing intramolecular distances in the protein (5). A Z-score greater than 2 indicates the proteins are significantly similar (5).

One protein identified using these two methods was NagR (PDB ID: 4U0V) from Bacillus subtilis, sharing the GntR family and HutC subfamily with DasR (1, 4, 5). The E value and Z-score of NagR in comparison with DasR were 4e-50 and 21.4, respectively, indicating significant similarity in both primary and tertiary structure (4, 5). Most members of the GntR family are homodimers with each subunit consisting of a DNA binding domain (DBD) near the N-terminus and a globular region that is involved in effector binding, referred to as the effector binding domain (EBD) (6). The effector binding domains vary greatly in the GntR family, allowing the recognition of a large number of molecules and a response to a wide variety of environmental conditions (6, 7). In the HutC subfamily to which DasR and NagR belong, the EBD in each monomer consists of three alpha helices surrounding a six stranded anti-parallel beta sheet (6). Each monomer is able to bind one ligand each (6).

Like DasR, NagR binds the sugar phosphates GlcN-6-P and GlcNAc-6-P, which are coordinated by the N-termini of helices αE1 and αE5 in the EBD of both proteins (2, 7). The EBD of DasR has been crystallized free of ligands (PDB ID: 4ZSB), in complex with GlcN-6-P (PDB ID: 4ZSI), and in complex with GlcNAc-6-P (PDB ID: 4ZSK) (1). In DasR, the motif common among HutC subfamily members that is vital in coordinating the sugar ligand’s phosphate group (2). In DasR, these residues are Arg-142, Leu-143, and Arg-144 in beta strand βE2 (2). DasR also contains residues that act to stabilize other parts of the effector (2). This includes Tyr-98, Glu-154, Glu-193, and Tyr-238, which hydrogen bond with hydroxyl groups of the sugar and Ser-97, Arg-221, Glu-232, Val-234, and Tyr-177, which have hydrophobic segments that interact with the ligand (2). A notable difference between the two proteins is that while NagR binds its ligands exclusively in an alpha-anomeric configuration, DasR preferentially binds the alpha anomer in one binding site and the beta anomer in the other (2).

NagR and DasR share DNA-binding mechanisms (2). The DBD of NagR and DasR are highly similar, and consists of a winged-helix-turn-helix structure (6). This structure contains a tri-helical core, including two helices that interact with the major groove of specific sequences of DNA (6). Surrounding this core are two beta strands that insert into the minor groove of the DNA when bound (6). Both proteins contain an arginine residue at the tip of the helix that interacts with the major groove and a glycine residue at the tip of the wing that anchors it into the minor groove (2, 6). The consensus sequence for DNA binding in the HutC subfamily is (GT-N(1)-TA-N(1)-AC) (8, 9). As a result of primary structure variability, not all GntR family members recognize the same operator sequences, but the target sequences of NagR and DasR are similar (2, 6). The DNA binding activity of DasR, though unclear, can consequently be inferred from information available on NagR (2). For example, NagR recognizes two guanines in its target sequences through interaction with disordered and only loosely associates the two domains (2, 7). In this state, the DBDs are able to adopt a wide variety of conformations, including a DNA binding competent conformation where they point downward in the same direction as the C-terminal beta strand of the protein (2, 7). Upon binding their effectors, both proteins undergo conformational changes that cause a stabilization of the region between the EBD and DBD (2, 7). This is the result of both the formation of a new beta sheet, termed β*, that interacts with the C-terminal beta sheet βE6 in both proteins, and a change in position of helices αE1 and αE5 that are responsible for coordination of the ligand (2, 7). Helix αE1, which is attached to the segment linking the DBD and EBD, is displaced by about 3-4 Å while αE5 is displaced by about 1 Å in DasR (2).

In DasR, a large interaction region consisting of a number of hydrogen bonds and van der Waals forces forms between the EBD and DBD upon ligand binding (2). These interactions involve the EBD residues in beta strands βE4, βE5, and the region between βE5 and βE6 interacting with DBD residues in helix αD3 (2). Furthermore, Val-70 and Arg-242 from the C-terminal region of the EBD form hydrogen bonds upon ligand binding and help separate the DBDs, as do Lys-86, Pro-87, and Lys-88 from the linker region between the EBD and DBD (2).These conformational changes effectively separate and lock the DBDs of both monomers into an upward conformation that is DNA binding incompetent (2, 7). Indeed, assays done to assess the binding of DasR in response to the addition of GlcNAc in vivo show a significantly reduced level of binding of DasR to its target dre-sequences (10). This contrasts with NagR’s apparent mechanism of DNA binding (11). When DNA-bound NagR is exposed to its sugar ligands, it is not significantly displaced from the DNA strand (11). NagR tends to bind to specific target sequences and polymerize, continuing to recruit dimers to the site in vitro (11). This extends the protein coverage beyond just the target region recognized by NagR (11). The binding of the sugar ligand disrupts the interactions that allow polymerization of NagR, but another unknown signal is required to remove it from the DNA (11).

Bacteria face the challenge of altering their metabolic machinery to utilize the various carbon sources available to them at different times (12). This is particularly relevant to NagR and DasR, both of which target and repress operons that encode proteins that allow uptake and utilization of GlcNAc (2, 7). Carbon catabolite repression (CCR) is the process by which the expression or activity of proteins related to secondary carbon source utilization are reduced in response to the availability of a more preferable carbon source (10, 12). The mechanism for the process in S. coelicolor, where DasR is used, has not yet been elucidated, but it is known to differ from that in B. subtilis, where NagR is used (10, 12). In B. subtilis, CCR is controlled by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in which glucose molecules are transported into the cell and phosphorylated simultaneously (10, 12). The phosphorylation state of the transporter subunit that acts as a phosphate donor and the accumulation of certain glycolysis products are general indicators of which nutrients are available (12, 13). NagR is involved when GlcNAc in particular accumulates outside the cell and upregulates the transcription of genes specific to GlcNAc utilization, including a subunit of the PTS system specific for GlcNAc (7).

The exact method of CCR has not been discovered in S. coelicolor, but it is known that as GlcNAc accumulates, PTS switches the cell from a growth state to one of developmental arrest and halts antibiotic production (10). It is also likely that DasR is a crucial component of PTS in S. coelicolor (13). Though DasR plays a similar role to NagR in controlling GlcNAc utilization, DasR is not restricted to regulating sugar metabolism (2). For Streptomyces bacteria, the chitin monomer GlcNAc is the primary source of carbon and nitrogen, making GlcNAc a general marker for nutrient availability in the soil-dwelling S. coelicolor (10, 13). DasR’s regulative capacity has, therefore, been expanded to include a vast number of other genes, including those that are involved in glycolysis, development, production of antibiotics, and the metabolism of lipids and nitrogen (10, 13). This larger role is evidenced by the larger number of dre-sites in S. coelicolor than NagR binding sites in B. subtilis (10). In addition, knocking out DasR results in a significantly altered transcription level in around 1,200 genes, which comprises about 15% of the genome in S. coelicolor (10). Furthermore, knocking out DasR prevents sporulation in S. coelicolor, a process that protects the bacteria during nutrient deprivation, indicating a role for DasR in regulating the growth cycles of the organism (13).

This behavior has a clear function in the survival of the bacteria (13). When GlcNAc is readily available, proteins required for GlcNAc utilization are created as a result of the inhibition of DasR, antibiotics are not produced, and the bacteria enter developmental arrest (10, 13). In this scenario, there is no pressure to outcompete neighboring bacteria, making this is an advantageous behavior (10, 13). When GlcNAc is scarce, microbes must compete for resources, causing S. coelicolor to enter a growth phase and produce antibiotics to eliminate its competitors (13). These antibiotics are of great interest to pharmaceutical companies, as the Streptomyces genus produces 80% of the world’s antibiotics, 10,000 tons of which are produced each year (14). In addition, growing antibiotic resistance necessitates the discovery of ever more antibiotics to fight disease (14). Understanding the conditions under which the bacteria produce these compounds and the roles of the various transcription factors involved is highly important (13).