Metalloproteins and metal sensing

Cells respond to changes in environment by shifting their gene expression profile to deal with the new conditions. The cellular response to changes in metal homeostasis is an important example of this. Transition metals such as iron, zinc, and copper are essential micronutrients but other metals such as cadmium are simply toxic. The cell must maintain metal concentrations in a window that supports efficient metabolic function but must also protect against the damaging effects of high concentrations of these metals. One way a cell regulates metal homeostasis is to control genes involved in metal mobilization and storage. Much of this regulation occurs at the level of transcription and the protein most responsible for this is the conserved metal responsive transcription factor 1 (MTF-1). Interestingly, the nature of the changes in the gene expression profile depends on the type of exposure. The cell somehow senses the kind of the metal challenge and responds appropriately. We have been using the Drosophila system to try to understand the mechanism of this metal discrimination. Using genome-wide mapping of MTF-1 binding under different metal stresses we find that, surprisingly, MTF-1 chooses different DNA binding sites depending on the specific nature of the metal insult. We also find that the type of binding site chosen is an important component of the capability to induce the metal-specific transcription activation.heavy metal | MED26 D NA recognition by sequence-specific DNA binding transcription factors is the basic mechanism that links the cisregulatory information encoded in the genome with transcriptional control of gene expression. Although it is the DNA binding domain of these factors that dictates the sequence element bound by the protein, it is now clear that, at least for some factors, the DNA sequence of the binding site itself can influence the activation potential of the transcription factor (1, 2). Clearly the process of reading the genome and activating transcription is more intricate than a simple DNA binding event.Some transcription factors respond to signaling events and activate the transcription of different sets of genes in a signalspecific manner. The cellular response to changes in metal homeostasis is one example of this observation. The major transcription factor involved in metal homeostasis is the metal responsive element (MRE) binding transcription factor-1 (MTF-1) (for a recent review, see ref.3). MTF-1 is required for both constitutive and metal inducible transcription of some genes; it is also required for activated transcription of at least one gene in low metal conditions (4, 5). Thus, it responds to both excess and limiting metal conditions by activating different sets of genes.

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confidence: 99%

Single nucleotide in the MTF-1 binding site can determine metal-specific transcription activation

Sims

Chirn

Marr

2012

“…Even the essential metals, for example iron and copper, can become detrimental above a certain concentration inside cells. Many biological processes are thus present to regulate and maintain intracellular metal homeostasis (1)(2)(3)(4)(5)(6)(7)(8)(9). One of them is through metalloregulators, which respond to metal ions and regulate the transcription of genes that protect the bacteria from metal-induced stress (5)(6)(7)10).…”

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confidence: 99%

Direct substitution and assisted dissociation pathways for turning off transcription by a MerR-family metalloregulator

Joshi¹,

Panda²,

Martell³

et al. 2012

107

Metalloregulators regulate transcription in response to metal ions. Many studies have provided insights into how transcription is activated upon metal binding by MerR-family metalloregulators. In contrast, how transcription is turned off after activation is unclear. Turning off transcription promptly is important, however, as the cells would not want to continue expressing metal resistance genes and thus waste energy after metal stress is relieved. Using single-molecule FRET measurements we studied the dynamic interactions of the copper efflux regulator (CueR), a Cu þ -responsive MerR-family metalloregulator, with DNA. Besides quantifying its DNA binding and unbinding kinetics, we discovered that CueR spontaneously flips its binding orientation at the recognition site. CueR also has two different binding modes, corresponding to interactions with specific and nonspecific DNA sequences, which would facilitate recognition localization. Most strikingly, a CueR molecule coming from solution can directly substitute for a DNA-bound CueR or assist the dissociation of the incumbent CueR, both of which are unique examples for any DNA-binding protein. The kinetics of the direct protein substitution and assisted dissociation reactions indicate that these two unique processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely.single-molecule imaging | protein-DNA interaction dynamics B acteria often dwell in environments with high concentrations of metals. Some of these metals are essential, but many are toxic. Even the essential metals, for example iron and copper, can become detrimental above a certain concentration inside cells. Many biological processes are thus present to regulate and maintain intracellular metal homeostasis (1-9). One of them is through metalloregulators, which respond to metal ions and regulate the transcription of genes that protect the bacteria from metal-induced stress (5-7, 10). The MerR-family metalloregulators respond to many metal ions with high selectivity and sensitivity, such as Hg 2þ and Cu 2þ (5,11,12).All MerR-family metalloregulators are homodimeric proteins. They regulate transcription via a DNA distortion mechanism (5, 13-16). They recognize specific dyad-symmetric DNA sequences within a promoter, and both their apo and holo forms bind DNA tightly. In the absence of metal, the metalloregulator bends the DNA; in this configuration RNA polymerase (RNAp) cannot interact with both −10 and −35 sequences properly and transcription is repressed. Upon binding metal, the metalloregulator changes its conformation and further unwinds the DNA slightly to allow proper RNAp interactions with the −10 and −35 sequences; transcription is then activated.Although the mechanisms of transcription activation by MerRfamily metalloregulators are well-studied (5, 13-16), little is yet known about how transcription activation is reversed. Turning off transcription promptly is important, however, as the cells would not want to conti...

“…protein metallation | di-metal carboxylate protein | ferritin superfamily | X-ray crystallography | EPR spectroscopy H alf of all enzymes are estimated to contain metallocofactors (1). An important subset uses transition metal ions to perform key redox reactions such as oxygen activation.…”

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confidence: 99%

Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein

Griese¹,

Roos

Cox

et al. 2013

283

Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving-Williams series (Mn II < Fe II < Ni II < Co II < Cu II > Zn II ). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from Mn II and Fe II in vitro, thus diverging from the Irving-Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds Fe II over Mn II as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a twostep process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine-valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.H alf of all enzymes are estimated to contain metallocofactors (1). An important subset uses transition metal ions to perform key redox reactions such as oxygen activation. The diiron cofactor of the ferritin-like superfamily of proteins is particularly versatile (2). While ferritin itself simply oxidizes and sequesters iron (3), in other family members the diiron center acts as a transient one-or two-electron oxidant. In the R2 subunits of class I ribonucleotide reductases (RNRs) it generates a redoxactive tyrosyl radical (4, 5), whereas in the bacterial multicomponent monooxygenases (BMMs) it catalyzes the hydroxylation of a variety of hydrocarbons (6). For four decades it was assumed that all ferritin superfamily proteins contained diiron cofactors. However, in recent years new subfamilies harboring either a dimanganese or heterodinuclear Mn/Fe cofactor have been documented (7)(8)(9)(10)(11)(12)(13)(14). The Mn/Fe cofactor was discovered in class Ic RNR R2 subunits, where its Mn IV /Fe III state functionally replaces the diiron-tyrosyl radical cofactor of class Ia R2s (9, 10). After a long controversy, class Ib R2 proteins were shown to use a dimanganese cofactor in the same scaffold (7,8). These recent developments highlight the complexity of correctly identifying the metals that make up native metallocofactors. While the metal preferences of some primary coordination motifs are well known and distinct, others ar...