Tandem zinc finger (ZF) proteins are the largest and most rapidly diverging family of DNA-binding transcription regulators in mammals. ZFP568 represses a transcript of placental-specific insulin like growth factor 2 (Igf2-P0) in mice. ZFP568 binds a 24-base pair sequence-specific element upstream of Igf2-P0 via the eleven-ZF array. Both DNA and protein conformations deviate from the conventional one finger-three bases recognition, with individual ZFs contacting 2, 3, or 4 bases and recognizing thymine on the opposite strand. These interactions arise from a shortened minor groove caused by an AT-rich stretch, suggesting adaptability of ZF arrays to sequence variations. Despite conservation in mammals, mutations at Igf2 and ZFP568 reduce their binding affinity in chimpanzee and humans. Our studies provide important insights into the evolutionary and structural dynamics of ZF-DNA interactions that play a key role in mammalian development and evolution. Evolutionary and structure-function dynamics of zinc finger-DNA interactions reveal unconventional recognition codes and co-evolution of ZFP568 and its target gene Igf2 in mammals.
M phase phosphoprotein 8 (MPP8) harbors a 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 (H3K9me3/2) 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, 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 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).
PHF2 belongs to a class of α-ketoglutarate-Fe2+–dependent dioxygenases. PHF2 harbors a plant homeodomain (PHD) and a Jumonji domain. PHF2, via its PHD domain, binds Lys4-trimethylated histone 3 (H3K4me3) in sub micromolar affinity, and has been reported to have demethylase activity of mono-methylated lysine 9 of histone 3 (H3K9me1) in vivo. However, we did not detect demethylase activity for PHF2 Jumonji domain (with and without its linked PHD) in the context of histone peptides. We determined the crystal structures of PHF2 Jumonji domain in the absence and presence of additional exogenous metal ions. Only when exposed to high metal concentration (50 mM), Fe2+ or Ni2+ was soaked into the preformed crystals and bound by six ligands in an octahedral coordination. The side-chains of H249, D251, and the two oxygen atoms of N-oxalylglycine (an analog of α-ketoglutarate) provide four coordinations in the equatorial plane, while the hydroxyl oxygen atom of Y321 and one water molecule provide the two axial coordinations as the fifth and sixth ligands, respectively. The metal binding site in PHF2 closely resembles the Fe2+ sites in other Jumonji domains examined, with one important difference, that a tyrosine (Y321 of PHF2) replaces a histidine as the fifth ligand. However, neither Y321H mutation nor high metal concentration renders PHF2 an active demethylase on histone peptides. Both wild type and Y321H mutant bind Ni2+ with approximately equal affinity of 50 µM. We propose there must be other regulatory factors required for the enzymatic activity of PHF2 in vivo, or perhaps PHF2 acts on non-histone substrates. Furthermore, PHF2 shares significant sequence homolog throughout the entire region, including the above-mentioned tyrosine at the corresponding iron-binding position, with that of Schizosaccharomyces pombe Epe1, which plays essential role in heterochromatin function but also has no known enzymatic activity.
by
John Horton;
Xu Liu;
Molly Gale;
Lizhen Wu;
John R. Shanks;
Xing Zhang;
Philip J. Webber;
Joshua S.K. Bell;
Stephen C Kales;
Bryan T Mott;
Ganesha Rai;
Daniel J Jansen;
Mark J Henderson;
Daniel J Urban;
Matthew D Hall;
Anton Simeonov;
David J Maloney;
Margaret A. Johns;
Haian Fu;
Ajit Jadhav;
Paula Vertino;
Qin Yan;
Xiaodong Cheng
The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases removes methyl groups from methylated lysine 4 of histone H3. Accumulating evidence supports a role for KDM5 family members as oncogenic drivers. We compare the in vitro inhibitory properties and binding affinity of ten diverse compounds with all four family members, and present the crystal structures of the KDM5A-linked Jumonji domain in complex with eight of these inhibitors in the presence of Mn(II). All eight inhibitors structurally examined occupy the binding site of α-ketoglutarate, but differ in their specific binding interactions, including the number of ligands involved in metal coordination. We also observed inhibitor-induced conformational changes in KDM5A, particularly those residues involved in the binding of α-ketoglutarate, the anticipated peptide substrate, and intramolecular interactions. We discuss how particular chemical moieties contribute to inhibitor potency and suggest strategies that might be utilized in the successful design of selective and potent epigenetic inhibitors.
Combinatorial readout of multiple covalent histone modifications is poorly understood. We provide insights into how an activating histone mark, in combination with linked repressive marks, is differentially ‘read’ by two related human demethylases, PHF8 and KIAA1718 (also known as JHDM1D). Both enzymes harbor a plant homeodomain (PHD) that binds Lys4-trimethylated histone 3 (H3K4me3) and a jumonji domain that demethylates either H3K9me2 or H3K27me2. The presence of H3K4me3 on the same peptide as H3K9me2 makes the doubly methylated peptide a markedly better substrate of PHF8, whereas the presence of H3K4me3 has the opposite effect, diminishing the H3K9me2 demethylase activity of KIAA1718 without adversely affecting its H3K27me2 activity. The difference in substrate specificity between the two is explained by PHF8 adopting a bent conformation, allowing each of its domains to engage its respective target, whereas KIAA1718 adopts an extended conformation, which prevents its access to H3K9me2 by its jumonji domain when its PHD engages H3K4me3.
Histone modifications have important roles in transcriptional control, mitosis and heterochromatin formation. G9a and G9a-like protein (GLP) are euchromatin-associated methyltransferases that repress transcription by mono- and dimethylating histone H3 at Lys9 (H3K9). Here we demonstrate that the ankyrin repeat domains of G9a and GLP bind with strong preference to N-terminal H3 peptides containing mono- or dimethyl K9. X-ray crystallography revealed the basis for recognition of the methylated lysine by a partial hydrophobic cage with three tryptophans and one acidic residue. Substitution of key residues in the cage eliminated the H3 tail interaction. Hence, G9a and GLP contain a new type of methyllysine binding module (the ankyrin repeat domains) and are the first examples of protein (histone) methyltransferases harboring in a single polypeptide the activities that generate and read the same epigenetic mark.
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.
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 (cp283Δ7) in the apo-form determined at 1.49Å resolution and with a bound phosphonate inhibitor at 1.69Å 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.
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 (GLP) by including a 5-aminopentyloxy moiety, which is inserted into the target lysine-binding channel and becomes methylated by GLP, albeit slowly. The compound enhanced its potency in vitro and reduced cell toxicity in vivo. We suggest that adding a lysine or methyllysine mimic should be considered in the design of small molecule inhibitors for other methyl-lysine writers, erasers and readers.