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Introduction

Adhesin Competence Regulator () is a transcriptional regulator that controls the activation of over seventy genes within the bacteria Streptococcus pneumoniae and is a member of the multiple antibiotic resistance regulator (MarR) protein family ^[1]. Members of the MarR protein family conserve a number of features including a general triangular shape, a two fold pseudosymmetric homodimer, and a winged helix-turn-helix pattern (wHTH) which can be seen in Figure 1. AdcR exhibits these conserved features as well. While conserving these important characteristics, AdcR exhibits several distinct features.

Figure 1. Proteins MarR (3BPX), HucR (2FBK), TcaR (3KP5), and OhrR (2PFB) are pictured above with conserved features of the MarR protein family highlighted

In contrast with other members of the MarR family, AdcR is metal dependent. 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 ^[2]. Binding of Zinc allows AdcR to bind DNA and activate the transcription of high-affinity Zinc specific uptake transporters. The importance of AdcR in Streptococcus pneumoniae can be understood provided its ability to regulate zinc transfer proteins within the bacteria.

Structural Overview

One of the two functional domains of AdcR is . This domain connects and stabilizes the two pseudosymmetric dimers and is composed of the , the C-terminus of the , and the . This domain is connected to the DNA binding domain by the long α5 helix. The DNA binding domain stabilizes the major and minor groove of DNA via the motif. The binding of Zinc to the induces a conformational change that allows for a between helices of the binding domain. It is believed that this hydrogen bond network is the allosteric activator needed to expose residues that bind the bases along the major groove of the DNA ^[3]. The binding sites are found adjacent to the DNA binding domain.

DNA Binding

Helix-Turn-Helix Motif

Figure 2. A generic protein representing the wHTH motif binding the major and minor groove of DNA similar to AdcR.

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 ^[3]. The motif is made up of the alpha 3 and alpha 4 helices along with on each side. There is one wHTH motif per monomer. 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 ^[3]. 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 (The residues are only shown on one side because this same section of amino acids on the other protein monomer were not crystallized).. The charge map down below highlights the dark blue tips consisting of lysine and arginine residues, which stabilize the negatively charged backbone of DNA.

Zn(II) Binding

Zinc-Dependent Transcriptional Regulator AdcR has on each of its two protomers and can bind a total of four Zn(II). The combined with the and make up the . Each protomer has one high affinity site (Binding site 1; KZn₁ = 10¹² M; pH 8) and one low affinity binding site (Binding Site 2; KZn₂ = 10⁷ M; pH 8) ^[4]. The metal binding pockets of the AdcR MarR transcriptional regulator are made up of the DNA binding domain with the extended α1-α2 loop. The two different Zn(II) binding sites are connected via of H108 and E41.

Binding Site 1

consists of a distorted tetrahedral geometry around Zn(II). The four amino acids involved in zinc binding are E24, H42, H108, and H112. 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 10⁹ - 10¹⁰ M^-1, but at pH 8 the binding affinity increases to 10¹² M^{-1 [4]}. This is due to the charges on the histidines of the binding site. At pH 6, the histidines are positively charged and are not able to interact with the positively charged Zn(II) ion. However, at pH 8 the histidines are neutrally charged and are able to coordinate with Zn(II), which increases the overall binding affinity. 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 ^[3].

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 (shown as a red sphere). If Cys30 in binding site 2 is mutated to an alanine, it will have no effect on the ability of the protein to bind DNA ^[3]. Therefore, binding site 2 has no significant role in the ability of AdcR to bind to DNA and AdcR is still able to function with no zinc bound present in binding site 2.

Hydrogen Bond Network

Figure 3. A charge map of AdcR shows the general triangular shape and the positively charged area on the tips of the wHTH domains

The binding of zinc metals creates a hydrogen bond network within the protein that connects the metal binding sites and the DNA binding domain. The () is represented by each atom type in the 3D model. The hydrogen bond network is characteristic of the MarR family as a whole. More importantly, the hydrogen bonding network connects the metal binding pockets to the α4 helix also known as the recognition helix. 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 α2 and α4 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 α2 helix. Then, a glutamine (Q40) residue from α2 helix accepts a hydrogen bond from a serine (S74) residue from the α4 helix ^[3]. The binding of zinc allows for these conformational changes that induces the binding of DNA in order to activate genes.

Figure 4. The Hydrogen Bonding Network is shown with dotted green lines approximately 2.8 angstroms between residues.

Medical Relevancy

Streptococcus pneumoniae, the host to AdcR, is a significant pathogenic bacterium. Although asymptomatic in healthy individuals S. pneumoniae can lead to Bronchitis, meningitis conjunctivitis, or brain abscesses in those with weaker immune systems. Host regulation of zinc is often used to combat pathogens such as S. pneumoniae. A better understanding of AdcR, the regulator that controls the transcription of zinc specific uptake transporters, could potentially help illuminate better ways to combat not only S. pneumoniae, but other comparable bacteria .