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M-phase phosphoprotein 8 (MPP8) harbors an N-terminal chromodomain and a C-terminal ankyrin repeat domain. MPP8, via its chromodomain, binds histone H3 peptide tri- or di-methylated at lysine 9 (H3K9me3/H3K9me2) in submicromolar affinity. We determined the crystal structure of MPP8 chromodomain in complex with H3K9me3 peptide. MPP8 interacts with at least six histone H3 residues from glutamine 5 to serine 10, enabling its ability to distinguish lysine-9-containing peptide (QTARKS) from that of lysine 27 (KAARKS), both sharing the ARKS sequence. A partial hydrophobic cage with three aromatic residues (Phe59, Trp80 and Tyr83) and one aspartate (Asp87) encloses the methylated lysine 9. MPP8 has been reported to be phosphorylated in vivo, including the cage residue Tyr83 and the succeeding Thr84 and Ser85. Modeling a phosphate group onto the side-chain hydroxyl oxygen of Tyr83 suggests that the negatively charged phosphate group could enhance the binding of positively charged methyl-lysine or create a regulatory signal by allowing or inhibiting binding of other protein(s).

Dynamic histone lysine methylation involves the activities of modifying enzymes (writers), enzymes removing modifications (erasers), and readers of the histone code. One common feature of these activities is the recognition of lysines in methylated and unmethylated states, whether they are substrates, reaction products, or binding partners. We applied the concept of adding a lysine mimic to an established inhibitor (BIX-01294) of histone H3 lysine 9 methyltransferases G9a and G9a-like protein by including a 5-aminopentyloxy moiety, which is inserted into the target lysine-binding channel and becomes methylated by G9a-like protein, albeit slowly. The compound enhances its potency in vitro and reduces cell toxicity in vivo. We suggest that adding a lysine or methyl-lysine mimic should be considered in the design of small-molecule inhibitors for other methyl-lysine writers, erasers, and readers.

Circular permutation of Candida antarctica lipase B yields several enzyme variants with substantially increased catalytic activity. To better understand the structural and functional consequences of protein termini reorganization, we have applied protein engineering and x-ray crystallography to cp283, one of the most active hydrolase variants. Our initial investigation has focused on the role of an extended surface loop, created by linking the native N- and C-termini, on protein integrity. Incremental truncation of the loop partially compensates for observed losses in secondary structure and the permutants' temperature of unfolding. Unexpectedly, the improvements are accompanied by quaternary-structure changes from monomer to dimer. The crystal structures of one truncated variant (cp283 Delta 7) in the apo-form determined at 1.49 A resolution and with a bound phosphonate inhibitor at 1.69 A resolution confirmed the formation of a homodimer by swapping of the enzyme's 35-residue N-terminal region. Separately, the new protein termini at amino acid positions 282/283 convert the narrow access tunnel to the catalytic triad into a broad crevice for accelerated substrate entry and product exit while preserving the native active-site topology for optimal catalytic turnover.

The structure of the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase in complex with cognate DNA was determined at 1.89 Å resolution in the presence of S-adenosyl-l-homocysteine. DNA recognition and the dynamics of base-flipping were studied by site-directed mutagenesis, DNA methylation kinetics and fluorescence stopped-flow experiments. Our data illustrate the mechanism of coupling of DNA recognition and base-flipping. Contacts to the non-target strand in the second (3′) half of the GATC site are established by R124 to the fourth base-pair, and by L122 and P134 to the third base-pair. The aromatic ring of Y119 intercalates into the DNA between the second and third base-pairs, which is essential for base-flipping to occur. Compared to previous published structures of bacteriophage T4 Dam, three major new observations are made in E. coli Dam. (1) The first Gua is recognized by K9, removal of which abrogates the first base-pair recognition. (2) The flipped target Ade binds to the surface of EcoDam in the absence of S-adenosyl-l-methionine, which illustrates a possible intermediate in the base-flipping pathway. (3) The orphaned Thy residue displays structural flexibility by adopting an extrahelical or intrahelical position where it is in contact to N120.

Restriction endonucleases differ in their use of metal cofactors despite having remarkably similar folds for their catalytic regions. To explore this, we have characterized the interaction of endonuclease PvuII with the catalytically incompetent cation Ca2+. The structure of a glutaraldehyde-crosslinked crystal of the endonuclease PvuII-DNA complex, determined in the presence of Ca2+ at a pH of approximately 6.5, supports a two-metal mechanism of DNA cleavage by PvuII. The first Ca2+ position matches that found in all structurally examined endonucleases, while the second position is similar to that of EcoRV but is distinct from that of BamHI and BglI. The location of the second metal in PvuII, unlike that in BamHI/BglI, permits no direct interaction between the second metal and the O3′ oxygen leaving group. However, the interactions between the DNA scissile phosphate and the metals, the first metal and the attacking water, and the attacking water and DNA are the same in PvuII as they are in the two-metal models of BamHI and BglI, but are distinct from the proposed three-metal or the two-metal models of EcoRV.

The PvuII restriction endonuclease is a homodimer that recognizes and cleaves the DNA sequence 5′-CAGCTG-3′ in double-stranded DNA, and the structure of this enzyme has been reported. In the wild-type enzyme, Asp34 interacts with the internal guanine of the recognition sequence on the minor groove side. The Asp34 codon was altered to specify Gly (D34G), and in vitro studies have revealed that the D34G protein has lost binding specificity for the central G·C base-pairs, and that it cuts the canonical sequence with 10−4-fold reduced activity as compared to the wild-type enzyme. We have now determined the structure at 1.59 A resolution of the D34G PvuII endonuclease complexed with a 12 bp duplex deoxyoligonucleotide containing the cognate sequence. The D34G alteration results in several structural changes relative to wild-type protein/DNA complexes. First, the sugar moiety of the internal guanine changes from a C2′-endo to C3′-endo pucker while that of the 3′ guanine changes from C3′-endo to C2′-endo pucker. Second, the axial rise between the internal G·C base-pairs is reduced while that between the G·C and flanking base-pairs is expanded. Third, two distinct monomeric active sites are observed that we refer to as being “primed” and “unprimed” for phosphodiester bond cleavage. The primed and unprimed sites differ in the conformation of the Asp58 side-chain, and in the absence from unprimed sites of four networked water molecules. These water molecules, present in the primed site, have been implicated in the catalytic mechanism of this and other endonucleases; some of them can be replaced by the Mg2+ necessary for cleavage. Taken together, these structural changes imply that the Asp34 side-chains from the two subunits maintain a distinct conformation of its DNA substrate, properly situating the target backbone phosphates and indirectly manipulating the active sites. This provides some insight into how recognition of the specific DNA sequence is linked to catalysis by the highly specific restriction endonucleases, and reveals one way in which the structural conformation of the DNA is modulated coordinately with that of the PvuII protein.