Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O2 activation.

The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to Cα-formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)-FGE is functional in O2 activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor. We establish that the copper atom is coordinated by two active-site cysteine residues in a nearly linear geometry, supporting and extending prior biochemical and structural data. The active cuprous FGE complex was interrogated directly by X-ray absorption spectroscopy. These data unambiguously establish the configuration of the resting enzyme metal center and, importantly, reveal the formation of a three-coordinate tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calculations. Critically, inner-sphere substrate coordination turns on O2 activation at the copper center. These collective results provide a detailed mechanistic framework for understanding why nature chose this structurally unique monocopper active site to catalyze oxidase chemistry for sulfatase activation.

Development, Optimization, and Structural Characterization of an Efficient Peptide-Based Photoaffinity Cross-Linking Reaction for Generation of Homogeneous Conjugates from Wild-Type Antibodies.

Site-specific conjugation of small molecules to antibodies represents an attractive goal for the development of more homogeneous targeted therapies and diagnostics. Most site-specific conjugation strategies require modification or removal of antibody glycans or interchain disulfide bonds or engineering of an antibody mutant that bears a reactive handle. While such methods are effective, they complicate the process of preparing antibody conjugates and can negatively impact biological activity. Herein we report the development and detailed characterization of a robust photoaffinity cross-linking method for site-specific conjugation to fully glycosylated wild-type antibodies. The method employs a benzoylphenylalanine (Bpa) mutant of a previously described 13-residue peptide derived from phage display to bind tightly to the Fc domain; upon UV irradiation, the Bpa residue forms a diradical that reacts with the bound antibody. After the initial discovery of an effective Bpa mutant peptide and optimization of the reaction conditions to enable efficient conjugation without concomitant UV-induced photodamage of the antibody, we assessed the scope of the photoconjugation reaction across different human and nonhuman antibodies and antibody mutants. Next, the specific site of conjugation on a human antibody was characterized in detail by mass spectrometry experiments and at atomic resolution by X-ray crystallography. Finally, we adapted the photoconjugation method to attach a cytotoxic payload site-specifically to a wild-type antibody and showed that the resulting conjugate is both stable in plasma and as potent as a conventional antibody-drug conjugate in cells, portending well for future biological applications.

Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

Envisioning the dynamics and flexibility of Mre11-Rad50-Nbs1 complex to decipher its roles in DNA replication and repair.

The Mre11-Rad50-Nbs1 (MRN) complex is a dynamic macromolecular machine that acts in the first steps of DNA double strand break repair, and each of its components has intrinsic dynamics and flexibility properties that are directly linked with their functions. As a result, deciphering the functional structural biology of the MRN complex is driving novel and integrated technologies to define the dynamic structural biology of protein machinery interacting with DNA. Rad50 promotes dramatic long-range allostery through its coiled-coil and zinc-hook domains. Its ATPase activity drives dynamic transitions between monomeric and dimeric forms that can be modulated with mutants modifying the ATPase rate to control end joining versus resection activities. The biological functions of Mre11's dual endo- and exonuclease activities in repair pathway choice were enigmatic until recently, when they were unveiled by the development of specific nuclease inhibitors. Mre11 dimer flexibility, which may be regulated in cells to control MRN function, suggests new inhibitor design strategies for cancer intervention. Nbs1 has FHA and BRCT domains to bind multiple interaction partners that further regulate MRN. One of them, CtIP, modulates the Mre11 excision activity for homologous recombination repair. Overall, these combined properties suggest novel therapeutic strategies. Furthermore, they collectively help to explain how MRN regulates DNA repair pathway choice with implications for improving the design and analysis of cancer clinical trials that employ DNA damaging agents or target the DNA damage response.

The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once.

To avoid genome instability, DNA repair nucleases must precisely target the correct damaged substrate before they are licensed to incise. Damage identification is a challenge for all DNA damage response proteins, but especially for nucleases that cut the DNA and necessarily create a cleaved DNA repair intermediate, likely more toxic than the initial damage. How do these enzymes achieve exquisite specificity without specific sequence recognition or, in some cases, without a non-canonical DNA nucleotide? Combined structural, biochemical, and biological analyses of repair nucleases are revealing their molecular tools for damage verification and safeguarding against inadvertent incision. Surprisingly, these enzymes also often act on RNA, which deserves more attention. Here, we review protein-DNA structures for nucleases involved in replication, base excision repair, mismatch repair, double strand break repair (DSBR), and telomere maintenance: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). DNA and RNA structure-sensing nucleases are essential to life with roles in DNA replication, repair, and transcription. Increasingly these enzymes are employed as advanced tools for synthetic biology and as targets for cancer prognosis and interventions. Currently their structural biology is most fully illuminated for DNA repair, which is also essential to life. How DNA repair enzymes maintain genome fidelity is one of the DNA double helix secrets missed by James Watson and Francis Crick, that is only now being illuminated though structural biology and mutational analyses. Structures reveal motifs for repair nucleases and mechanisms whereby these enzymes follow the old carpenter adage: measure twice, cut once. Furthermore, to measure twice these nucleases act as molecular level transformers that typically reshape the DNA and sometimes themselves to achieve extraordinary specificity and efficiency.

The Rad50 hook domain regulates DNA damage signaling and tumorigenesis.

The Mre11 complex (Mre11, Rad50, and Nbs1) is a central component of the DNA damage response (DDR), governing both double-strand break repair and DDR signaling. Rad50 contains a highly conserved Zn(2+)-dependent homodimerization interface, the Rad50 hook domain. Mutations that inactivate the hook domain produce a null phenotype. In this study, we analyzed mutants with reduced hook domain function in an effort to stratify hook-dependent Mre11 complex functions. One of these alleles, Rad50(46), conferred reduced Zn(2+) affinity and dimerization efficiency. Homozygous Rad50(46/46) mutations were lethal in mice. However, in the presence of wild-type Rad50, Rad50(46) exerted a dominant gain-of-function phenotype associated with chronic DDR signaling. At the organismal level, Rad50(+/46) exhibited hydrocephalus, liver tumorigenesis, and defects in primitive hematopoietic and gametogenic cells. These outcomes were dependent on ATM, as all phenotypes were mitigated in Rad50(+/46) Atm(+/-) mice. These data reveal that the murine Rad50 hook domain strongly influences Mre11 complex-dependent DDR signaling, tissue homeostasis, and tumorigenesis.

Structural and functional characterization of interactions involving the Tfb1 subunit of TFIIH and the NER factor Rad2.

The general transcription factor IIH (TFIIH) plays crucial roles in transcription as part of the pre-initiation complex (PIC) and in DNA repair as part of the nucleotide excision repair (NER) machinery. During NER, TFIIH recruits the 3'-endonuclease Rad2 to damaged DNA. In this manuscript, we functionally and structurally characterized the interaction between the Tfb1 subunit of TFIIH and Rad2. We show that deletion of either the PH domain of Tfb1 (Tfb1PH) or several segments of the Rad2 spacer region yield yeast with enhanced sensitivity to UV irradiation. Isothermal titration calorimetry studies demonstrate that two acidic segments of the Rad2 spacer bind to Tfb1PH with nanomolar affinity. Structure determination of a Rad2-Tfb1PH complex indicates that Rad2 binds to TFIIH using a similar motif as TFIIEα uses to bind TFIIH in the PIC. Together, these results provide a mechanistic bridge between the role of TFIIH in transcription and DNA repair.

Structure-based design of a potent artificial transactivation domain based on p53.

Malfunctions in transcriptional regulation are associated with a number of critical human diseases. As a result, there is considerable interest in designing artificial transcription activators (ATAs) that specifically control genes linked to human diseases. Like native transcriptional activator proteins, an ATA must minimally contain a DNA-binding domain (DBD) and a transactivation domain (TAD) and, although there are several reliable methods for designing artificial DBDs, designing artificial TADs has proven difficult. In this manuscript, we present a structure-based strategy for designing short peptides containing natural amino acids that function as artificial TADs. Using a segment of the TAD of p53 as the scaffolding, modifications are introduced to increase the helical propensity of the peptides. The most active artificial TAD, termed E-Cap-(LL), is a 13-mer peptide that contains four key residues from p53, an N-capping motif and a dileucine hydrophobic bridge. In vitro analysis demonstrates that E-Cap-(LL) interacts with several known p53 target proteins, while in vivo studies in a yeast model system show that it is a 20-fold more potent transcriptional activator than the native p53-13 peptide. These results demonstrate that structure-based design represents a promising approach for developing artificial TADs that can be combined with artificial DBDs to create potent and specific ATAs.

Crystal structures of the organomercurial lyase MerB in its free and mercury-bound forms: insights into the mechanism of methylmercury degradation.

Bacteria resistant to methylmercury utilize two enzymes (MerA and MerB) to degrade methylmercury to the less toxic elemental mercury. The crucial step is the cleavage of the carbon-mercury bond of methylmercury by the organomercurial lyase (MerB). In this study, we determined high resolution crystal structures of MerB in both the free (1.76-A resolution) and mercury-bound (1.64-A resolution) states. The crystal structure of free MerB is very similar to the NMR structure, but important differences are observed when comparing the two structures. In the crystal structure, an amino-terminal alpha-helix that is not present in the NMR structure makes contact with the core region adjacent to the catalytic site. This interaction between the amino-terminal helix and the core serves to bury the active site of MerB. The crystal structures also provide detailed insights into the mechanism of carbon-mercury bond cleavage by MerB. The structures demonstrate that two conserved cysteines (Cys-96 and Cys-159) play a role in substrate binding, carbon-mercury bond cleavage, and controlled product (ionic mercury) release. In addition, the structures establish that an aspartic acid (Asp-99) in the active site plays a crucial role in the proton transfer step required for the cleavage of the carbon-mercury bond. These findings are an important step in understanding the mechanism of carbon-mercury bond cleavage by MerB.