Homologous acetone carboxylases select Fe(II) or Mn(II) as the catalytic cofactor.

Acetone carboxylases (ACs) catalyze the metal- and ATP-dependent conversion of acetone and bicarbonate to form acetoacetate. Interestingly, two homologous ACs that have been biochemically characterized have been reported to have different metal complements, implicating different metal dependencies in catalysis. ACs from proteobacteria Xanthobacter autotrophicus and Aromatoleum aromaticum share 68% sequence identity but have been proposed to have different catalytic metals. In this work, the two ACs were expressed under the same conditions in Escherichia coli and were subjected to parallel chelation and reconstitution experiments with Mn(II) or Fe(II). Electron paramagnetic and Mössbauer spectroscopies identified signatures, respectively, of Mn(II) or Fe(II) bound at the active site. These experiments showed that the respective ACs, without the assistance of chaperones, second metal sites, or post-translational modifications facilitate correct metal incorporation, and despite the expected thermodynamic preference for Fe(II), each preferred a distinct metal. Catalysis was likewise associated uniquely with the cognate metal, though either could potentially serve the proposed Lewis acidic role. Subtle differences in the protein structure are implicated in serving as a selectivity filter for Mn(II) or Fe(II).IMPORTANCEThe Irving-Williams series refers to the predicted stabilities of transition metal complexes where the observed general stability for divalent first-row transition metal complexes increase across the row. Acetone carboxylases (ACs) use a coordinated divalent metal at their active site in the catalytic conversion of bicarbonate and acetone to form acetoacetate. Highly homologous ACs discriminate among different divalent metals at their active sites such that variations of the enzyme prefer Mn(II) over Fe(II), defying Irving-Williams-predicted behavior. Defining the determinants that promote metal discrimination within the first-row transition metals is of broad fundamental importance in understanding metal-mediated catalysis and metal catalyst design.

Yeast Rad52 is a homodecamer and possesses BRCA2-like bipartite Rad51 binding modes.

Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher how Rad52 functions using single-particle cryo-electron microscopy and biophysical approaches. We report that Rad52 is a homodecameric ring and each subunit possesses an ordered N-terminal and disordered C-terminal half. An intrinsic structural asymmetry is observed where a few of the C-terminal halves interact with the ordered ring. We describe two conserved charged patches in the C-terminal half that harbor Rad51 and RPA interacting motifs. Interactions between these patches regulate ssDNA binding. Surprisingly, Rad51 interacts with Rad52 at two different bindings sites: one within the positive patch in the disordered C-terminus and the other in the ordered ring. We propose that these features drive Rad51 nucleation onto a single position on the DNA to promote formation of uniform pre-synaptic Rad51 filaments in HR.

An Aurora B-RPA signaling axis secures chromosome segregation fidelity.

Errors in chromosome segregation underlie genomic instability associated with cancers. Resolution of replication and recombination intermediates and protection of vulnerable single-stranded DNA (ssDNA) intermediates during mitotic progression requires the ssDNA binding protein Replication Protein A (RPA). However, the mechanisms that regulate RPA specifically during unperturbed mitotic progression are poorly resolved. RPA is a heterotrimer composed of RPA70, RPA32 and RPA14 subunits and is predominantly regulated through hyperphosphorylation of RPA32 in response to DNA damage. Here, we have uncovered a mitosis-specific regulation of RPA by Aurora B kinase. Aurora B phosphorylates Ser-384 in the DNA binding domain B of the large RPA70 subunit and highlights a mode of regulation distinct from RPA32. Disruption of Ser-384 phosphorylation in RPA70 leads to defects in chromosome segregation with loss of viability and a feedback modulation of Aurora B activity. Phosphorylation at Ser-384 remodels the protein interaction domains of RPA. Furthermore, phosphorylation impairs RPA binding to DSS1 that likely suppresses homologous recombination during mitosis by preventing recruitment of DSS1-BRCA2 to exposed ssDNA. We showcase a critical Aurora B-RPA signaling axis in mitosis that is essential for maintaining genomic integrity.

Homodecameric Rad52 promotes single-position Rad51 nucleation in homologous recombination.

Homologous recombination (HR) is a pathway for the accurate repair of double-stranded DNA breaks. These breaks are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Saccharomyces cerevisiae Rad52 is a mediator protein that promotes HR by facilitating formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Canonically, Rad52 has been described to function by displacing RPA to promote Rad51 binding. However, in vitro, Rad51 readily forms a filament by displacing RPA in the absence of Rad52. Yet, in vivo, Rad52 is essential for HR. Here, we resolve how Rad52 functions as a mediator using single-particle cryo-electron microscopy and biophysical approaches. We show that Rad52 functions as a homodecamer and catalyzes single-position nucleation of Rad51. The N-terminal half of Rad52 is a well-ordered ring, while the C-terminal half is disordered. An intrinsic asymmetry within Rad52 is observed, where one or a few of the C-terminal halves interact with the ordered N-terminal ring. Within the C-terminal half, we identify two conserved charged patches that harbor the Rad51 and RPA interacting motifs. Interactions between these two charged patches regulate a ssDNA binding. These features drive Rad51 binding to a single position on the Rad52 decameric ring. We propose a Rad52 catalyzed single-position nucleation model for the formation of pre-synaptic Rad51 filaments in HR.

Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit.

Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

Rtt105 regulates RPA function by configurationally stapling the flexible domains.

Replication Protein A (RPA) is a heterotrimeric complex that binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins. When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting the facilitated exchange activity of RPA. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely binding to RPA-interacting proteins, while stabilizing RPA-ssDNA filaments at DNA lesion sites.

A catalytic dyad modulates conformational change in the CO2-fixing flavoenzyme 2-ketopropyl coenzyme M oxidoreductase/carboxylase.

2-Ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a member of the flavin and cysteine disulfide containing oxidoreductase family (DSOR) that catalyzes the unique reaction between atmospheric CO2 and a ketone/enolate nucleophile to generate acetoacetate. However, the mechanism of this reaction is not well understood. Here, we present evidence that 2-KPCC, in contrast to the well-characterized DSOR enzyme glutathione reductase, undergoes conformational changes during catalysis. Using a suite of biophysical techniques including limited proteolysis, differential scanning fluorimetry, and native mass spectrometry in the presence of substrates and inhibitors, we observed conformational differences between different ligand-bound 2-KPCC species within the catalytic cycle. Analysis of site-specific amino acid variants indicated that 2-KPCC-defining residues, Phe501-His506, within the active site are important for transducing these ligand induced conformational changes. We propose that these conformational changes promote substrate discrimination between H+ and CO2 to favor the metabolically preferred carboxylation product, acetoacetate.

Hydrogen-deuterium exchange reveals a dynamic DNA-binding map of replication protein A.

Replication protein A (RPA) binds to single-stranded DNA (ssDNA) and interacts with over three dozen enzymes and serves as a recruitment hub to coordinate most DNA metabolic processes. RPA binds ssDNA utilizing multiple oligosaccharide/oligonucleotide binding domains and based on their individual DNA binding affinities are classified as high versus low-affinity DNA-binding domains (DBDs). However, recent evidence suggests that the DNA-binding dynamics of DBDs better define their roles. Utilizing hydrogen-deuterium exchange mass spectrometry (HDX-MS), we assessed the ssDNA-driven dynamics of the individual domains of human RPA. As expected, ssDNA binding shows HDX changes in DBDs A, B, C, D and E. However, DBD-A and DBD-B are dynamic and do not show robust DNA-dependent protection. DBD-C displays the most extensive changes in HDX, suggesting a major role in stabilizing RPA on ssDNA. Slower allosteric changes transpire in the protein-protein interaction domains and linker regions, and thus do not directly interact with ssDNA. Within a dynamics-based model for RPA, we propose that DBD-A and -B act as the dynamic half and DBD-C, -D and -E function as the less-dynamic half. Thus, segments of ssDNA buried under the dynamic half are likely more readily accessible to RPA-interacting proteins.

The flexible N-terminus of BchL autoinhibits activity through interaction with its [4Fe-4S] cluster and released upon ATP binding.

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide to chlorophyllide, catalyzed by dark-operative protochlorophyllide oxidoreductase. Dark-operative protochlorophyllide oxidoreductase contains two [4Fe-4S]-containing component proteins (BchL and BchNB) that assemble upon ATP binding to BchL to coordinate electron transfer and protochlorophyllide reduction. But the precise nature of the ATP-induced conformational changes is poorly understood. We present a crystal structure of BchL in the nucleotide-free form where a conserved, flexible region in the N-terminus masks the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions in this region produce a hyperactive enzyme complex, suggesting a role for the N-terminus in autoinhibition. Hydrogen-deuterium exchange mass spectrometry shows that ATP binding to BchL produces specific conformational changes leading to release of the flexible N-terminus from the docking interface. The release also promotes changes within the local environment surrounding the [4Fe-4S] cluster and promotes BchL-complex formation with BchNB. A key patch of amino acids, Asp-Phe-Asp (the 'DFD patch'), situated at the mouth of the BchL ATP-binding pocket promotes intersubunit cross stabilization of the two subunits. A linked BchL dimer with one defective ATP-binding site does not support protochlorophyllide reduction, illustrating nucleotide binding to both subunits as a prerequisite for the intersubunit cross stabilization. The masking of the [4Fe-4S] cluster by the flexible N-terminal region and the associated inhibition of the activity is a novel mechanism of regulation in metalloproteins. Such mechanisms are possibly an adaptation to the anaerobic nature of eubacterial cells with poor tolerance for oxygen.

The reactive form of a C-S bond-cleaving, CO2-fixing flavoenzyme.

NADPH: 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a bacterial disulfide oxidoreductase (DSOR) that, uniquely in this family, catalyzes CO2 fixation. 2-KPCC differs from other DSORs by having a phenylalanine that replaces a conserved histidine, which in typical DSORs is essential for stabilizing the reduced, reactive form of the active site. Here, using site-directed mutagenesis and stopped-flow kinetics, we examined the reactive form of 2-KPCC and its single turnover reactions with a suicide substrate and CO2 The reductive half-reaction of 2-KPCC was kinetically and spectroscopically similar to that of a typical DSOR, GSH reductase, in which the active-site histidine had been replaced with an alanine. However, the reduced, reactive form of 2-KPCC was distinct from those typical DSORs. In the absence of the histidine, the flavin and disulfide moieties were no longer coupled via a covalent or charge transfer interaction as in typical DSORs. Similar to thioredoxins, the pKa between 7.5 and 8.1 that controls reactivity appeared to be due to a single proton shared between the cysteines of the dithiol, which effectively stabilizes the attacking cysteine sulfide and renders it capable of breaking the strong C-S bond of the substrate. The lack of a histidine protected 2-KPCC's reactive intermediate from unwanted protonation; however, without its input as a catalytic acid-base, the oxidative half-reaction where carboxylation takes place was remarkably slow, limiting the overall reaction rate. We conclude that stringent regulation of protons in the DSOR active site supports C-S bond cleavage and selectivity for CO2 fixation.