Structural adaptation of vertebrate endonuclease G for 5-hydroxymethylcytosine recognition and function.

Modified DNA bases functionally distinguish the taxonomic forms of life-5-methylcytosine separates prokaryotes from eukaryotes and 5-hydroxymethylcytosine (5hmC) invertebrates from vertebrates. We demonstrate here that mouse endonuclease G (mEndoG) shows specificity for both 5hmC and Holliday junctions. The enzyme has higher affinity (>50-fold) for junctions over duplex DNAs. A 5hmC-modification shifts the position of the cut site and increases the rate of DNA cleavage in modified versus unmodified junctions. The crystal structure of mEndoG shows that a cysteine (Cys69) is positioned to recognize 5hmC through a thiol-hydroxyl hydrogen bond. Although this Cys is conserved from worms to mammals, a two amino acid deletion in the vertebrate relative to the invertebrate sequence unwinds an α-helix, placing the thiol of Cys69 into the mEndoG active site. Mutations of Cys69 with alanine or serine show 5hmC-specificity that mirrors the hydrogen bonding potential of the side chain (C-H < S-H < O-H). A second orthogonal DNA binding site identified in the mEndoG structure accommodates a second arm of a junction. Thus, the specificity of mEndoG for 5hmC and junctions derives from structural adaptations that distinguish the vertebrate from the invertebrate enzyme, thereby thereby supporting a role for 5hmC in recombination processes.

Hydrogen Bond Enhanced Halogen Bonds: A Synergistic Interaction in Chemistry and Biochemistry.

The halogen bond (XB) has become an important tool for molecular design in all areas of chemistry, including crystal and materials engineering and medicinal chemistry. Its similarity to the hydrogen bond (HB) makes the relationship between these interactions complex, at times competing against and other times orthogonal to each other. Recently, our two laboratories have independently reported and characterized a synergistic relationship, in which the XB is enhanced through direct intramolecular HBing to the electron-rich belt of the halogen. In one study, intramolecular HBing from an amine polarizes the iodopyridinium XB donors of a bidentate anion receptor. The resulting HB enhanced XB (or HBeXB) preorganizes and further augments the XB donors. Consequently, the affinity of the receptor for halogen anions was significantly increased. In a parallel study, a meta-chlorotyrosine was engineered into T4 lysozyme, resulting in a HBeXB that increased the thermal stability and activity of the enzyme at elevated temperatures. The crystal structure showed that the chlorine of the noncanonical amino acid formed a XB to the protein backbone, which augmented the HB of the wild-type enzyme. Calorimetric analysis resulted in an enthalpic contribution of this Cl-XB to the stability of the protein that was an order of magnitude greater than previously determined in biomolecules. Quantum mechanical (QM) calculations showed that rotating the hydroxyl group of the tyrosine to point toward rather than away from the halogen greatly increased its potential to serve as a XB donor, equivalent to what was observed experimentally. In sum, the two systems described here show that the HBeXB concept extends the range of interaction energies and geometries to be significantly greater than that of the XB alone. Additionally, surveys of structural databases indicate that the components for this interaction are already present in many existing molecular systems. The confluence of the independent studies from our two laboratories demonstrates the reach of the HBeXB across both chemistry and biochemistry and that intentional engineering of this enhanced interaction will extend the applications of XBs beyond these two initial examples.

Trapping intermediates in metal transfer reactions of the CusCBAF export pump of Escherichia coli.

Escherichia coli CusCBAF represents an important class of bacterial efflux pump exhibiting selectivity towards Cu(I) and Ag(I). The complex is comprised of three proteins: the CusA transmembrane pump, the CusB soluble adaptor protein, and the CusC outer-membrane pore, and additionally requires the periplasmic metallochaperone CusF. Here we used spectroscopic and kinetic tools to probe the mechanism of copper transfer between CusF and CusB using selenomethionine labeling of the metal-binding Met residues coupled to RFQ-XAS at the Se and Cu edges. The results indicate fast formation of a protein-protein complex followed by slower intra-complex metal transfer. An intermediate coordinated by ligands from each protein forms in 100 ms. Stopped-flow fluorescence of the capping CusF-W44 tryptophan that is quenched by metal transfer also supports this mechanism. The rate constants validate a process in which shared-ligand complex formation assists protein association, providing a driving force that raises the rate into the diffusion-limited regime.