Zn(II) Binding
Zinc-Dependent Transcriptional Regulator AdcR has on each of its two protomers and can bind a total of four Zn(II). is made up of the alpha helix 1, alpha helix 5, and the alpha helix 6. This domain is connected to the HTH winged domain with the long alpha helix 5. This dimerization domain connects to the DNA binding domain and together with the alpha 1 alpha 2 loop make up the [4]. Each protomer has one high affinity site (KZn1 = 10^12 M; pH 8) and one low affinity binding site (KZn2 = 10^7 M; pH 8). The metal binding pockets of the AdcR MarR transcriptional regulator are made up of the DNA binding domain with the extended alpha 1 and alpha 2 loop. The two different Zn(II) binding sites are connected via of the Nd1 atom of H108 and then liganding Oe1 atom of E41.
Binding Site 1
consists of a distorted tetrahedral geometry around Zn(II). The four amino acids involved in zinc binding are E24 Oe1, H42 Nd1, H108 Ne2, and H112 Ne2. Binding site 1 is the only binding site that plays a significant role in the protein's regulatory function. The ability of binding site 1 to coordinate to the Zn(II) ion is pH dependent. At pH 6 the binding affinity for the Zn(II) ion is 10^9 - 10^10 M^-1, but at pH 8 the binding affinity increases to 10^12 M^-1. This is due to the charges on the histidines of the binding site. At pH 8, the histidines are positively charged and can interact with the negatively charged Zn(II) ion. However, at pH 6 the histidines are neutrally charged and will not coordinate as well with Zn(II).
Binding Site 2
consists of a highly distorted tetrahedral geometry around the zinc ion. There are three amino acids involved in the binding of the zinc ion (C30, E41, and E107) as well as a water molecule. If a C30A AdcR missense is present in binding site 2, it will have no effect on the ability of the protein to bind DNA. Therefore, binding site 2 has no significant role in DNA binding.
Other Ligands
The AdcR MarR transcriptional regulator is able to bind Co(II) in binding site 1 in a way that induces similar conformational changes to Zn(II) binding. Co(II) coordination in binding site 1 is able to allosterically activate DNA binding similarly to Zn(II) binding.
DNA Binding
Hydrogen Bond Network
The Hydrogen Bonding Network is shown with dotted green lines approximately 2.8 angstroms between residues. The network consists of 4 major residues as follows from right to left: E24, N38, Q40, S74.
The binding of Zinc allows for the conformational change that induces the binding of DNA in order to activate genes. The binding of Zinc metals creates a hydrogen bond network within the protein that connects the metal binding sites and the DNA binding domain. More importantly, the hydrogen bonding network connects the metal binding pockets to the alpha 4 helix. Alpha 4 helix on each monomer plays a crucial role in binding DNA because it acts as the . Specific residues in the recognition helix recognize a sequence of DNA that is unknown at the moment; however, scientists do know that the hydrogen bond network acts as an allosteric activator for the protein to bind DNA. The hydrogen bond network connects the alpha 2 and alpha 4 helix via hydrogen bonding between specific residues. After zinc is bound, a glutamate (E24) residue from a random coil accepts a hydrogen bond from the carboxamide end of an asparagine (N38) residue from the alpha 2 helix. Then, a glutamine (Q40) residue from alpha 2 helix accepts a hydrogen bond from a serine (S74) residue from the alpha 4 helix. The color coding in the previous sentence represents the (), which is seen across the MarR family as a whole.
A charge map of AdcR shows the general triangular shape and the positive charged (blue) area on HTH domains
Helix-Turn-Helix Domain
The AdcR MarR transcriptional regulator's structure resembles the other proteins in the same family as mentioned before; however, the most notable differences are found in the winged helix-turn-helix (wHTH) motif that assists in binding DNA. Although AdcR is a highly alpha helical protein, the "wings" of the DNA binding domain consist of two anti parallel beta strands that are made up of several positively charged residues such as Arg. The major groove of DNA is bound to the recognition helix while the wings grip onto the minor grooves. The charge map on the right highlights the positively charged areas, which stabilize the negatively charged backbone of the major and minor grooves of DNA. There is a wHTH domain on each monomer of the protein.
The domain is made up of the alpha 2 and alpha 4 helices along with anti-parallel beta sheets on each side. Only one monomer is shown. The recognition helix, or the alpha 4 helix, binds the major groove of DNA through hydrogen bonding and Van der Waals interactions between exposed bases. The wings of the helix bind the minor groove of DNA while the other helices stabilize the DNA and Protein upon binding. The two anti parallel beta sheets contain several that stabilize this interaction between DNA.
