Amie K. Boal

Structural biology of copper trafficking.

1.1. Background The use of copper in biological systems coincides with the advent of an oxygen atmosphere about 1.7 billion years ago. The presence of O2 both allowed the oxidation of insoluble Cu(I) to the more soluble and bioavailable Cu(II) and led to the requirement for a redox active metal with potentials in the 0-800 mV range. Not only did copper meet this need, but the oxidation of Fe(II) to the insoluble Fe(III) form rendered the use of iron more energetically expensive.1-5 As a result, copper plays a key role in many proteins that react with O2. Generally, O2-reactive centers are mononuclear (type 2), dinuclear (type 3), or trinuclear (type 2 and type 3). Well studied mononuclear copper enzymes include the monooxygenases dopamine-β-hydroxylase and peptidylglycine α-hydroxylating monooxygenase as well as oxidases that also contain organic cofactors, such as amine, galactose, and lysyl oxidases.6 Dinuclear copper proteins include the O2 carrier hemocyanin and enzymes like tyrosinase and catechol oxidase.7 Copper also plays a key role in numerous electron transfer proteins. Mononuclear type 1 (blue copper) centers are found in proteins such as plastocyanin and azurin.8 The multicopper oxidases like laccase, ascorbate oxidase, and ceruloplasmin contain both a catalytic trinuclear type 2/type 3 site and an electron transfer type 1 site.9,10 The classification of copper centers into types is derived from optical and electron paramagnetic resonance (EPR) spectroscopic properties, and there are some notable exceptions, including the cysteine-bridged dinuclear CuA electron transfer site in cytochrome c oxidase11 and nitrous oxide reductase, the tetranuclear catalytic CuZ center in nitrous oxide reductase,12 and the proposed catalytic copper center in particulate methane monooxygenase.13-15 The same redox properties that render copper useful in all these metalloproteins can lead to oxidative damage in cells. Reaction of Cu(I) with hydrogen peroxide and re-reduction of Cu(II) by superoxide via Fenton and Haber-Weiss chemistry yields hydroxyl radicals that can damage proteins, lipids, and nucleic acids.16 Thus, intracellular copper concentrations must be controlled such that copper ions are provided to essential enzymes, but do not accumulate to deleterious levels. In humans, deficiencies in copper metabolism are linked to diseases such as Menkes syndrome, Wilson disease, prion diseases, and Alzheimer’s disease.17 Several classes of proteins, including membrane transporters,18-20 metallochaperones,21,22 and metalloregulatory proteins,23,24 are implicated in copper homeostasis. These proteins have two functions. First, they ensure that copper is provided to the correct proteins and cellular compartments for necessary activities. Second, these proteins detoxify excess copper. Just as copper-containing proteins and enzymes are found in all kingdoms of life, members of these groups of homeostatic proteins are also widespread,5 and have been structurally and biochemically characterized from eukaryotes and prokaryotes.

DNA-Mediated Charge Transport in Redox Sensing and Signaling

The transport of charge through the DNA base-pair stack offers a route to carry out redox chemistry at a distance. Here we describe characteristics of this chemistry that have been elucidated and how this chemistry may be utilized within the cell. The shallow distance dependence associated with these redox reactions permits DNA-mediated signaling over long molecular distances in the genome and facilitates the activation of redox-sensitive transcription factors globally in response to oxidative stress. The long-range funneling of oxidative damage to sites of low oxidation potential in the genome also may provide a means of protection within the cell. Furthermore, the sensitivity of DNA charge transport to perturbations in base-pair stacking, as may arise with base lesions and mismatches, may be used as a route to scan the genome for damage as a first step in DNA repair. Thus, the ability of double-helical DNA in mediating redox chemistry at a distance provides a natural mechanism for redox sensing and signaling in the genome.

Crystal structures of cisplatin bound to a human copper chaperone.

Copper trafficking proteins, including the chaperone Atox1 and the P(1B)-type ATPase ATP7B, have been implicated in cellular resistance to the anticancer drug cisplatin. We have determined two crystal structures of cisplatin-Atox1 adducts that reveal platinum coordination by the conserved CXXC copper-binding motif. Direct interaction of cisplatin with this functionally relevant site has significant implications for understanding the molecular basis for resistance mediated by copper transport pathways.

Structural Basis for Methyl Transfer by a Radical SAM Enzyme

An enzyme harnesses methyl donation and reductive cleavage of its cofactor within a single active site to methylate RNA. The radical S-adenosyl-l-methionine (SAM) enzymes RlmN and Cfr methylate 23S ribosomal RNA, modifying the C2 or C8 position of adenosine 2503. The methyl groups are installed by a two-step sequence involving initial methylation of a conserved Cys residue (RlmN Cys355) by SAM. Methyl transfer to the substrate requires reductive cleavage of a second equivalent of SAM. Crystal structures of RlmN and RlmN with SAM show that a single molecule of SAM coordinates the [4Fe-4S] cluster. Residue Cys355 is S-methylated and located proximal to the SAM methyl group, suggesting the SAM that is involved in the initial methyl transfer binds at the same site. Thus, RlmN accomplishes its complex reaction with structural economy, harnessing the two most important reactivities of SAM within a single site.

Biological Contexts for DNA Charge Transport Chemistry

Many experiments have now shown that double helical DNA can serve as a conduit for efficient charge transport (CT) reactions over long distances in vitro. These results prompt the consideration of biological roles for DNA-mediated CT. DNA CT has been demonstrated to occur in biologically relevant environments such as within the mitochondria and nuclei of HeLa cells as well as in isolated nucleosomes. In mitochondria, DNA damage that results from CT is funneled to a crucial regulatory element. Thus, DNA CT provides a strategy to funnel damage to particular sites in the genome. DNA CT might also be important in long-range signaling to DNA-bound proteins. Both DNA repair proteins, containing Fe-S clusters, and the transcription factor, p53, which is regulated through thiol-disulfide switches, can be oxidized from a distance through DNA-mediated CT. These observations highlight a means through which oxidative stress may be chemically signaled in the genome over long distances through CT from guanine radicals to DNA-bound proteins. Moreover, DNA-mediated CT may also play a role in signaling among DNA-binding proteins, as has been proposed as a mechanism for how DNA repair glycosylases more efficiently detect lesions inside the cell.

Structural Basis for Activation of Class Ib Ribonucleotide Reductase

Two Ways to Nucleotide Reduction Ribonucleotide reductases (RNRs) are essential for DNA synthesis and repair in all organisms, initiating nucleotide reduction through a free-radical mechanism. The class Ib RNRs are the primary aerobic RNRs for many human pathogens. NrdF, the class Ib RNR of Escherichia coli, can initiate nucleotide reduction through either a FeIII2-Y• or a MnIII2-Y• cofactor. Whereas the Fe-based cofactor can self-assemble, assembly of the Mn-based free radical requires a reduced flavoprotein, NrdI. Boal et al. (p. 1526, published online 5 August; see the Perspective by Sjöberg) have gained insight into the mechanism of cofactor activation by determining structures of MnII2-NrdF, FeII2-NrdF, and MnII2-NrdF in complex with reduced and oxidized NrdI. The structures show how a single protein, NrdF, can use two different oxidants to activate two different metallocofactors using distinct chemistries. A single protein activates two different metallofactors by distinct chemistries. The class Ib ribonucleotide reductase of Escherichia coli can initiate reduction of nucleotides to deoxynucleotides with either a MnIII2-tyrosyl radical (Y•) or a FeIII2-Y• cofactor in the NrdF subunit. Whereas FeIII2-Y• can self-assemble from FeII2-NrdF and O2, activation of MnII2-NrdF requires a reduced flavoprotein, NrdI, proposed to form the oxidant for cofactor assembly by reduction of O2. The crystal structures reported here of E. coli MnII2-NrdF and FeII2-NrdF reveal different coordination environments, suggesting distinct initial binding sites for the oxidants during cofactor activation. In the structures of MnII2-NrdF in complex with reduced and oxidized NrdI, a continuous channel connects the NrdI flavin cofactor to the NrdF MnII2 active site. Crystallographic detection of a putative peroxide in this channel supports the proposed mechanism of MnIII2-Y• cofactor assembly.

Redox signaling between DNA repair proteins for efficient lesion detection

Base excision repair (BER) enzymes maintain the integrity of the genome, and in humans, BER mutations are associated with cancer. Given the remarkable sensitivity of DNA-mediated charge transport (CT) to mismatched and damaged base pairs, we have proposed that DNA repair glycosylases (EndoIII and MutY) containing a redox-active [4Fe4S] cluster could use DNA CT in signaling one another to search cooperatively for damage in the genome. Here, we examine this model, where we estimate that electron transfers over a few hundred base pairs are sufficient for rapid interrogation of the full genome. Using atomic force microscopy, we found a redistribution of repair proteins onto DNA strands containing a single base mismatch, consistent with our model for CT scanning. We also demonstrated in Escherichia coli a cooperativity between EndoIII and MutY that is predicted by the CT scanning model. This relationship does not require the enzymatic activity of the glycosylase. Y82A EndoIII, a mutation that renders the protein deficient in DNA-mediated CT, however, inhibits cooperativity between MutY and EndoIII. These results illustrate how repair proteins might efficiently locate DNA lesions and point to a biological role for DNA-mediated CT within the cell.

Mechanism of the C5 stereoinversion reaction in the biosynthesis of carbapenem antibiotics

Carbapenems Through the Looking Glass The carbapenem class of antibiotics is a critical weapon in the ongoing fight against drug-resistant bacteria. Microbial biosynthesis of these compounds, which contain a strained β-lactam ring motif, proceeds via a precursor that has the wrong configuration at one of the ring carbons. Chang et al. (p. 1140) combined x-ray crystallography with multiple spectroscopic probes to map out the mechanism by which the CarC enzyme inverts the precursor configuration to its mirror image. Crystallography and spectroscopy detail a key mechanistic step in the microbial biosynthesis of an important antibiotic class. The bicyclic β-lactam/2-pyrrolidine precursor to all carbapenem antibiotics is biosynthesized by attachment of a carboxymethylene unit to C5 of l-proline followed by β-lactam ring closure. Carbapenem synthase (CarC), an Fe(II) and 2-(oxo)glutarate (Fe/2OG)–dependent oxygenase, then inverts the C5 configuration. Here we report the structure of CarC in complex with its substrate and biophysical dissection of its reaction to reveal the stereoinversion mechanism. An Fe(IV)-oxo intermediate abstracts the hydrogen (H•) from C5, and tyrosine 165, a residue not visualized in the published structures of CarC lacking bound substrate, donates H• to the opposite face of the resultant radical. The reaction oxidizes the Fe(II) cofactor to Fe(III), limiting wild-type CarC to one turnover, but substitution of the H•-donating tyrosine disables stereoinversion and confers to CarC the capacity for catalytic substrate oxidation.

Crystallographic capture of a radical S-adenosylmethionine enzyme in the act of modifying tRNA

An RNA methylase caught in the act RNA methylation is important in RNA function and in antibiotic resistance. The RNA methylase RlmN is a dual-specificity enzyme that can act on ribosomal and transfer RNA (tRNA). RlmN is a radical S-adenosylmethionine (SAM) enzyme, which produces a protein/RNA cross-linked intermediate. Schwalm et al. determined the structure of RlmN cross-linked to a tRNA substrate and found that the enzyme recognizes the overall shape of the tRNA. Then it remodels the anticodon region to access the base that it methylates. The remodeling activity is likely to be key to the enzymes dual specificity. Science, this issue p. 309 An x-ray structure shows how a dual-specificity RNA methylase recognizes its RNA substrate. RlmN is a dual-specificity RNA methylase that modifies C2 of adenosine 2503 (A2503) in 23S rRNA and C2 of adenosine 37 (A37) in several Escherichia coli transfer RNAs (tRNAs). A related methylase, Cfr, modifies C8 of A2503 via a similar mechanism, conferring resistance to multiple classes of antibiotics. Here, we report the x-ray structure of a key intermediate in the RlmN reaction, in which a Cys118→Ala variant of the protein is cross-linked to a tRNAGlu substrate through the terminal methylene carbon of a formerly methylcysteinyl residue and C2 of A37. RlmN contacts the entire length of tRNAGlu, accessing A37 by using an induced-fit strategy that completely unfolds the tRNA anticodon stem-loop, which is likely critical for recognition of both tRNA and ribosomal RNA substrates.

Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers

X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.