DNA distortion and specificity in a sequence-specific endonuclease.
ABSTRACT: Five new structures of the Q138F HincII enzyme bound to a total of three different DNA sequences and three different metal ions (Ca(2+), Mg(2+), and Mn(2+)) are presented. While previous structures were produced from soaking Ca(2+) into preformed Q138F HincII/DNA crystals, the new structures are derived from cocrystallization with Ca(2+), Mg(2+), or Mn(2+). The Mn(2)(+)-bound structure provides the first view of a product complex of Q138F HincII with cleaved DNA. Binding studies and a crystal structure show how Ca(2+) allows trapping of a Q138F HincII complex with noncognate DNA in a catalytically incompetent conformation. Many Q138F HincII/DNA structures show asymmetry, despite the binding of a symmetric substrate by a symmetric enzyme. The various complexes are fit into a model describing the different conformations of the DNA-bound enzyme and show how DNA conformational energetics determine DNA-cleavage rates by the Q138F HincII enzyme.
Project description:R.SwaI, a Type IIP restriction endonuclease, recognizes a palindromic eight base pair (bp) symmetric sequence, 5?-ATTTAAAT-3?, and cleaves that target at its center to generate blunt-ended DNA fragments. Here, we report three crystal structures of SwaI: unbound enzyme, a DNA-bound complex with calcium ions; and a DNA-bound, fully cleaved complex with magnesium ions. We compare these structures to two structurally similar ‘PD-D/ExK’ restriction endonucleases (EcoRV and HincII) that also generate blunt-ended products, and to a structurally distinct enzyme (the HNH endonuclease PacI) that also recognizes an 8-bp target site consisting solely of A:T base pairs. Binding by SwaI induces an extreme bend in the target sequence accompanied by un-pairing and re-ordering of its central A:T base pairs. This result is reminiscent of a more dramatic target deformation previously described for PacI, implying that long A:T-rich target sites might display structural or dynamic behaviors that play a significant role in endonuclease recognition and cleavage.
Project description:The nonheme diiron enzyme cyanobacterial aldehyde deformylating oxygenase, cADO, catalyzes the highly unusual deformylation of aliphatic aldehydes to alkanes and formate. We have determined crystal structures for the enzyme with a long-chain water-soluble aldehyde and medium-chain carboxylic acid bound to the active site. These structures delineate a hydrophobic channel that connects the solvent with the deeply buried active site and reveal a mode of substrate binding that is different from previously determined structures with long-chain fatty acids bound. The structures also identify a water channel leading to the active site that could facilitate the entry of protons required in the reaction. NMR studies examining 1-[(13)C]-octanal binding to cADO indicate that the enzyme binds the aldehyde form rather than the hydrated form. Lastly, the fortuitous cocrystallization of the metal-free form of the protein with aldehyde bound has revealed protein conformation changes that are involved in binding iron.
Project description:Human senescence marker protein 30 (SMP30), which functions enzymatically as a lactonase, hydrolyzes various carbohydrate lactones. The penultimate step in vitamin-C biosynthesis is catalyzed by this enzyme in nonprimate mammals. It has also been implicated as an organophosphate hydrolase, with the ability to hydrolyze diisopropyl phosphofluoridate and other nerve agents. SMP30 was originally identified as an aging marker protein, whose expression decreased androgen independently in aging cells. SMP30 is also referred to as regucalcin and has been suggested to have functions in calcium homeostasis. The crystal structure of the human enzyme has been solved from X-ray diffraction data collected to a resolution of 1.4 A. The protein has a 6-bladed beta-propeller fold, and it contains a single metal ion. Crystal structures have been solved with the metal site bound with either a Ca(2+) or a Zn(2+) atom. The catalytic role of the metal ion has been confirmed by mutagenesis of the metal coordinating residues. Kinetic studies using the substrate gluconolactone showed a k(cat) preference of divalent cations in the order Zn(2+) > Mn(2+) > Ca(2+) > Mg(2+). Notably, the Ca(2+) had a significantly higher value of K(d) compared to those of the other metal ions tested (566, 82, 7, and 0.6 mum for Ca(2+), Mg(2+), Zn(2+), and Mn(2+), respectively), suggesting that the Ca(2+)-bound form may be physiologically relevant for stressed cells with an elevated free calcium level.
Project description:The three-dimensional X-ray crystal structure of the 'rare cutting' type II restriction endonuclease SgrAI bound to cognate DNA is presented. SgrAI forms a dimer bound to one duplex of DNA. Two Ca(2+) bind in the enzyme active site, with one ion at the interface between the protein and DNA, and the second bound distal from the DNA. These sites are differentially occupied by Mn(2+), with strong binding at the protein-DNA interface, but only partial occupancy of the distal site. The DNA remains uncleaved in the structures from crystals grown in the presence of either divalent cation. The structure of the dimer of SgrAI is similar to those of Cfr10I, Bse634I and NgoMIV, however no tetrameric structure of SgrAI is observed. DNA contacts to the central CCGG base pairs of the SgrAI canonical target sequence (CR|CCGGYG, | marks the site of cleavage) are found to be very similar to those in the NgoMIV/DNA structure (target sequence G|CCGGC). Specificity at the degenerate YR base pairs of the SgrAI sequence may occur via indirect readout using DNA distortion. Recognition of the outer GC base pairs occurs through a single contact to the G from an arginine side chain located in a region unique to SgrAI.
Project description:Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of cell function by responding specifically to micromolar concentrations of Ca(2+) in the presence of an ~1000-fold higher concentration of cellular Mg(2+). To explain the structural basis of metal ion binding specificity, we have determined the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg(2+), Mn(2+), and Zn(2+). In contrast to Ca(2+), which induces domain opening in CaM, octahedrally coordinated Mg(2+) and Mn(2+) stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn(2+) ions bind at the protein surface and do not compete with Ca(2+). The relative positions of bound Mg(2+) and Mn(2+) within the EF-hand loops are similar to those of Ca(2+); however, the Glu side chain at position 12 of the loop, whose bidentate interaction with Ca(2+) is critical for domain opening, does not bind directly to either Mn(2+) or Mg(2+), and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-hand ?-scaffold. The structures suggest that Mg(2+) contributes to the switching off of calmodulin activity and possibly other EF-hand proteins at the resting levels of Ca(2+). The Mg(2+)-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal-induced conformational change.
Project description:Cell death related nuclease 4 (CRN-4) is one of the apoptotic nucleases involved in DNA degradation in Caenorhabditis elegans. To understand how CRN-4 is involved in apoptotic DNA fragmentation, we analyzed CRN-4's biochemical properties, in vivo cell functions, and the crystal structures of CRN-4 in apo-form, Mn(2+)-bound active form, and Er(3+)-bound inactive form. CRN-4 is a dimeric nuclease with the optimal enzyme activity in cleaving double-stranded DNA in apoptotic salt conditions. Both mutational studies and the structures of the Mn(2+)-bound CRN-4 revealed the geometry of the functional nuclease active site in the N-terminal DEDDh domain. The C-terminal domain, termed the Zn-domain, contains basic surface residues ideal for nucleic acid recognition and is involved in DNA binding, as confirmed by deletion assays. Cell death analysis in C. elegans further demonstrated that both the nuclease active site and the Zn-domain are required for crn-4's function in apoptosis. Combining all of the data, we suggest a structural model where chromosomal DNA is bound at the Zn-domain and cleaved at the DEDDh nuclease domain in CRN-4 when the cell is undergoing apoptosis.
Project description:Linear chromosomes terminate in specialized nucleoprotein structures called telomeres, which are required for genomic stability and cellular proliferation. Telomeres end in an unusual 3' single-strand overhang that requires a special capping mechanism to prevent inappropriate recognition by the DNA damage machinery. In Schizosaccharomyces pombe, this protective function is mediated by the Pot1 protein, which binds specifically and with high affinity to telomeric ssDNA. We have characterized the thermodynamics and accommodation of both cognate and noncognate telomeric single-stranded DNA (ssDNA) sequences by Pot1pN, an autonomous ssDNA-binding domain (residues 1-187) found in full-length S. pombe Pot1. Direct calorimetric measurements of cognate telomeric ssDNA binding to Pot1pN show favorable enthalpy, unfavorable entropy, and a negative heat-capacity change. Thermodynamic analysis of the binding of noncognate telomeric ssDNA to Pot1pN resulted in unexpected changes in free energy, enthalpy, and entropy. Chemical-shift perturbation and structural analysis of these bound noncognate sequences show that these thermodynamic changes result from the structural rearrangement of both Pot1pN and the bound oligonucleotide. These data suggest that the ssDNA-binding interface is highly dynamic and, in addition to the conformation observed in the crystal structure of the Pot1pN/d(GGTTAC) complex, capable of adopting alternative thermodynamically equivalent conformations.
Project description:Human DNA polymerase ? (pol?) inserts, albeit slowly, T opposite the carcinogenic lesion O6-methylguanine (O6MeG) ?30-fold more frequently than C. To gain insight into this promutagenic process, we solved four ternary structures of pol? with an incoming dCTP or dTTP analogue base-paired with O6MeG in the presence of active-site Mg(2+) or Mn(2+). The Mg(2+)-bound structures show that both the O6MeG·dCTP/dTTP-Mg(2+) complexes adopt an open protein conformation, staggered base pair, and one active-site metal ion. The Mn(2+)-bound structures reveal that, whereas the O6Me·dCTP-Mn(2+) complex assumes the similar altered conformation, the O6MeG·dTTP-Mn(2+) complex adopts a catalytically competent state with a closed protein conformation and pseudo-Watson-Crick base pair. On the basis of these observations, we conclude that pol? slows nucleotide incorporation opposite O6MeG by inducing an altered conformation suboptimal for catalysis and promotes mutagenic replication by allowing Watson-Crick-mode for O6MeG·T but not for O6MeG·C in the enzyme active site. The O6MeG·dTTP-Mn(2+) ternary structure, which represents the first structure of mismatched pol? ternary complex with a closed protein conformation and coplanar base pair, the first structure of pseudo-Watson-Crick O6MeG·T formed in the active site of a DNA polymerase, and a rare, if not the first, example of metal-dependent conformational activation of a DNA polymerase, indicate that catalytic metal-ion coordination is utilized as a kinetic checkpoint by pol? and is crucial for the conformational activation of pol?. Overall, our structural studies not only explain the promutagenic pol? catalysis across O6MeG but also provide new insights into the replication fidelity of pol?.
Project description:Two genes, coding for the HincII from Haemophilus influenzae Rc restriction-modification system, were cloned and expressed in Escherichia coli RR1. Their DNA sequences were determined. The HincII methylase (M.HincII) gene was 1,506 base pairs (bp) long, corresponding to a protein of 502 amino acid residues (Mr = 55,330). The HincII endonuclease (R.HincII) gene was 774 bp long, corresponding to a protein of 258 amino acid residues (Mr = 28,490). The amino acid residues predicted from the R.HincII and the N-terminal amino acid sequence of the enzyme found by analysis were identical. These methylase and endonuclease genes overlapped by 1 bp on the H. influenzae Rc chromosomal DNA. The clone, named E. coli RR1-Hinc, overproduced R.HincII. The R.HincII activity of this clone was 1,000-fold that from H. influenzae Rc. The amino acid sequence of M.HincII was compared with the sequences of four other adenine-specific type II methylases. Important homology was found between tne M.HincII and these other methylases.
Project description:The oxygen-evolving complex of Photosystem II (PS II) in green plants and algae contains a cluster of four manganese atoms in the active site, which catalyzes the photoinduced oxidation of water to dioxygen. Along with Mn, calcium and chloride ions are necessary cofactors for proper functioning of the complex. A key unresolved question is whether Ca is close to the Mn cluster, within about 3.5 Å. To further test and verify this finding, we substituted strontium for Ca and probed from the Sr point-of-view for any nearby Mn. Sr has been shown to replace Ca and still maintain enzyme activity (about 40% of normal rate). The extended X-ray absorption fine structure (EXAFS) of Sr-PS II probes the local environment around the Sr cofactor to detect any nearby Mn. We focused on the functional Sr by removing nonessential, loosely bound Sr in the protein environment. For comparison, an inactive sample was prepared by treating the intact PS II with hydroxylamine to disrupt the Mn cluster and to produce nonfunctional enzyme. Sr EXAFS results indicate major differences in the phase and amplitude between the functional (intact) and nonfunctional (NH2OH-treated) samples. In intact samples, the Fourier transform of the Sr EXAFS shows a peak that is missing in inactive samples. This Fourier peak II is best simulated by two Mn neighbors at a distance of 3.5 Å. Thus, with X-ray absorption studies on Sr-reconstituted PS II, we confirm the proximity of Ca (Sr) cofactor to the Mn cluster and show that the active site is a Mn-Ca heteronuclear cluster.