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Adhesin Competence Regulator (AdcR)

spinqualitylabelsall models Adhesin Competence Regulator (3TGN) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Introduction Adhesin Competence Regulator (AdcR) is a transcriptional regulator that controls the activation of over seventy genes within the bacterium Streptococcus pneumoniae and is a member of the multiple antibiotic resistance regulator (MarR) protein family . Members of the Mar R 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 also exhibiting its own distinct features. Figure 1: MarR protein family features. 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 . 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 Figure 2. A generic protein representing the wHTH motif binding the major and minor groove of DNA similar to AdcR. One of the two functional domains of AdcR is the dimerization domain. This domain connects and stabilizes the two pseudosymmetric protomers and is composed of the α1 helix, the α6 helix . and the C-terminus of the α5 helix . This domain is connected to the DNA binding domain by the long α5 helix. The DNA binding domain interacts with the major and minor grooves of DNA via the winged helix-turn-helix (wHTH) motif (Figure 2). The binding of Zinc to the Zinc binding pocket induces a conformational change that allows for a hydrogen bond network between 4 specific residues. This network connects multiple helices from the metal binding pockets and DNA binding domain, and is believed play a critical role in the allosteric activation of AdcR, allowing the protein to bind exposed bases along the major and minor grooves of the DNA ligand . Thus, the protein is able to perform its biological function by activating transcription after binding DNA. DNA Binding Helix-Turn-Helix Motif Figure 3. A charge map of AdcR shows the general triangular shape and the positively charged area on the tips of the wHTH motif The AdcR MarR transcriptional regulator's structure resembles that of other proteins in the MarR family; however, the most notable differences are found in the winged helix-turn-helix (wHTH) motif that assists in binding DNA . The winged helix turn helix motif is made up of the α3 and α4 helices along with anti-parallel β sheets on each side. There is one wHTH motif per monomer. The recognition helix, or the α4 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 β sheets contain several Arginine, Asparagine, and Lysine residues that stabilize this interaction between DNA. The charge map (Figure 3) highlights the dark blue tips of the wHTH motif consisting of lysine and arginine residues, which stabilize the negatively charged backbone of DNA. The residues are only shown on the random loop of one monomer because the random loop on the other protein monomer was not crystallized. Zn(II) Binding Zinc-Dependent Transcriptional Regulator AdcR has two binding sites for zinc on each of its two protomers and can bind a total of four Zn(II) ions. The α1-α2 loop combined with the α5 helix and α2 helix contribute residues to the metal binding sites. 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) . The two different Zn(II) binding sites are connected via hydrogen bonding of H108 and E41. Binding Site 1 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 . 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 . Binding Site 2 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). When Cys30 in binding site 2 is mutated to an alanine, it has no effect on the ability of the protein to bind DNA . 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. In fact, the presence of binding site 2 may simply be due to an excess of zinc during the crystallization process. Hydrogen Bond Network Figure 4. The Hydrogen Bonding Network is shown with dotted green lines approximately 2.8 angstroms between residues. The binding of zinc metals creates a hydrogen bond network (Figure 4) within the protein that connects the metal binding sites and the DNA binding domain. The hydrogen bond network (with measurements) (residues in stick structures, colored by atom type) is characteristic of the MarR family as a whole and connects the metal binding pockets to the α4 helix also known as the DNA recognition helix. Several residues in this helix recognize the DNA ligand. The hydrogen bond network connects the α2 and α4 helices 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. A glutamine (Q40) residue from α2 helix accepts a hydrogen bond from a serine (S74) residue from the α4 helix . Clinical Relevance Streptococcus pneumoniae 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 help to illuminate better mechanism for combating not only S. pneumoniae, but other comparable bacteria.