Sandbox Reserved 1063

This Sandbox is Reserved from 02/09/2015, through 05/31/2016 for use in the course "CH462: Biochemistry 2" taught by Geoffrey C. Hoops at the Butler University. This reservation includes Sandbox Reserved 1051 through Sandbox Reserved 1080.

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Zinc-Dependent Transcriptional Regulator AdcRZinc-Dependent Transcriptional Regulator AdcR

IntroductionIntroduction

AdcR is a zinc-dependent transcriptional regulator that controls the activation of over seventy genes within the bacteria Streptococcus pneumoniae. Zinc plays a vital role in organism homeostasis acting as a co-factor and a regulator of enzymatic activity, however zinc can lead to cell toxicity and deficiency of other vital metals that are also necessary for protein function. Given the importance of zinc in general homeostasis the vital role of AdcR in Streptococcus pneumoniae can be understood given its ability to regulate zinc transfer proteins within the bacteria. Consistent with AdcR's identity as a member of the MarR protein family, AdcR exhibits a triangular shape consisting of a two fold pseudosymetric homo dimer with its own unique winged helix-turn-helix (wHTH) binding domain. This structure calls for multiple zinc binding sites that facilitate protein conformational change allowing for DNA binding and regulation through the wHTH domain.

Figure Legend

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Zn(II) BindingZn(II) Binding

Zinc-Dependent Transcriptional Regulator AdcR has on each of its two protomers (important amino acids for Zn(II) binding shown in white) and can bind a total of four Zn(II) per dimer. 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 hydrogen bonding of the Nd1 atom of H108 and then liganding Oe1 atom of E41.

Binding Site 1Binding 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.

Binding Site 2Binding 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 LigandsOther 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 BindingDNA Binding

Zinc's Allosteric ActivationZinc's Allosteric Activation

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. Most importantly, the hydrogen bonding network connects the metal binding pockets to the alpha 4 helix, which is known as the recognition helix. Alpha 4 helix plays a crucial role in binding DNA. The specific sequence of DNA that is recognized by alpha helix 4 is unknown at the moment; however, scientists believe 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 same is seen across the MarR family as a whole. Now the protein is ready to bind DNA.

Helix-Turn-Helix DomainHelix-Turn-Helix Domain

The AdcR MarR transcriptional regulator's structure resembles the other proteins in the same family; however, the most notable differences are found in the winged helix-turn-helix (wHTH) motif that assists in binding DNA. This wHTH motif is consistent among all marR family proteins that bind DNA. Although AdcR is a highly alpha helical protein, the "wings" of the DNA binding domain consist of two anti parallel beta strands. There is one on each domain of the protein.

The HTH domain is made up of the alpha 2, alpha 3, alpha 4 helices. 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 Arginine residues and other positive amino acids that stabilize this interaction between DNA.

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