Biomedical subjects

Lyudmila G Doudeva

Publications and source records attributed to Lyudmila G Doudeva.

5 recordsLinked to original sources

PubMed2006

Structural basis for sequence-dependent DNA cleavage by nonspecific endonucleases.

Nonspecific endonucleases hydrolyze DNA without sequence specificity but with sequence preference, however the structural basis for cleavage preference remains elusive. We show here that the nonspecific endonuclease ColE7 cleaves DNA with a preference for making nicks after (at 3'O-side) thymine bases but the periplasmic nuclease Vvn cleaves DNA more evenly with little sequence preference. The crystal structure of the 'preferred complex' of the nuclease domain of ColE7 bound to an 18 bp DNA with a thymine before the scissile phosphate had a more distorted DNA phosphate backbone than the backbones in the non-preferred complexes, so that the scissile phosphate was compositionally closer to the endonuclease active site resulting in more efficient DNA cleavage. On the other hand, in the crystal structure of Vvn in complex with a 16 bp DNA, the DNA phosphate backbone was similar and not distorted in comparison with that of a previously reported complex of Vvn with a different DNA sequence. Taken together these results suggest a general structural basis for the sequence-dependent DNA cleavage catalyzed by nonspecific endonucleases, indicating that nonspecific nucleases could induce DNA to deform to distinctive levels depending on the local sequence leading to different cleavage rates along the DNA chain.

Amino Acid Substitution↗

PubMed2006

Crystal structural analysis and metal-dependent stability and activity studies of the ColE7 endonuclease domain in complex with DNA/Zn2+ or inhibitor/Ni2+.

The nuclease domain of ColE7 (N-ColE7) contains an H-N-H motif that folds in a beta beta alpha-metal topology. Here we report the crystal structures of a Zn2+-bound N-ColE7 (H545E mutant) in complex with a 12-bp duplex DNA and a Ni2+-bound N-ColE7 in complex with the inhibitor Im7 at a resolution of 2.5 A and 2.0 A, respectively. Metal-dependent cleavage assays showed that N-ColE7 cleaves double-stranded DNA with a single metal ion cofactor, Ni2+, Mg2+, Mn2+, and Zn2+. ColE7 purified from Escherichia coli contains an endogenous zinc ion that was not replaced by Mg2+ at concentrations of <25 mM, indicating that zinc is the physiologically relevant metal ion in N-ColE7 in host E. coli. In the crystal structure of N-ColE7/DNA complex, the zinc ion is directly coordinated to three histidines and the DNA scissile phosphate in a tetrahedral geometry. In contrast, Ni2+ is bound in N-ColE7 in two different modes, to four ligands (three histidines and one phosphate ion), or to five ligands with an additional water molecule. These data suggest that the divalent metal ion in the His-metal finger motif can be coordinated to six ligands, such as Mg2+ in I-PpoI, Serratia nuclease and Vvn, five ligands or four ligands, such as Ni2+ or Zn2+ in ColE7. Universally, the metal ion in the His-metal finger motif is bound to the DNA scissile phosphate and serves three roles during hydrolysis: polarization of the P-O bond for nucleophilic attack, stabilization of the phosphoanion transition state and stabilization of the cleaved product.

Amino Acid Motifs↗

PubMed2005

High-resolution crystal structure of a truncated ColE7 translocation domain: implications for colicin transport across membranes.

ColE7 is a nuclease-type colicin released from Escherichia coli to kill sensitive bacterial cells by degrading the nucleic acid molecules in their cytoplasm. ColE7 is classified as one of the group A colicins, since the N-terminal translocation domain (T-domain) of the nuclease-type colicins interact with specific membrane-bound or periplasmic Tol proteins during protein import. Here, we show that if the N-terminal tail of ColE7 is deleted, ColE7 (residues 63-576) loses its bactericidal activity against E.coli. Moreover, TolB protein interacts directly with the T-domain of ColE7 (residues 1-316), but not with the N-terminal deleted T-domain (residues 60-316), as detected by co-immunoprecipitation experiments, confirming that the N-terminal tail is required for ColE7 interactions with TolB. The crystal structure of the N-terminal tail deleted ColE7 T-domain was determined by the multi-wavelength anomalous dispersion method at a resolution of 1.7 angstroms. The structure of the ColE7 T-domain superimposes well with the T-domain of ColE3 and TR-domain of ColB, a group A Tol-dependent colicin and a group B TonB-dependent colicin, respectively. The structural resemblance of group A and B colicins implies that the two groups of colicins may share a mechanistic connection during cellular import.

Amino Acid Sequence↗

PubMed2002

The crystal structure of the nuclease domain of colicin E7 suggests a mechanism for binding to double-stranded DNA by the H-N-H endonucleases.

The bacterial toxin ColE7 contains an H-N-H endonuclease domain (nuclease ColE7) that digests cellular DNA or RNA non-specifically in target cells, leading to cell death. In the host cell, protein Im7 forms a complex with ColE7 to inhibit its nuclease activity. Here, we present the crystal structure of the unbound nuclease ColE7 at a resolution of 2.1A. Structural comparison between the unbound and bound nuclease ColE7 in complex with Im7, suggests that Im7 is not an allosteric inhibitor that induces backbone conformational changes in nuclease ColE7, but rather one that inhibits by blocking the substrate-binding site. There were two nuclease ColE7 molecules in the P1 unit cell in crystals and they appeared as a dimer related to each other by a non-crystallographic dyad symmetry. Gel-filtration and cross-linking experiments confirmed that nuclease ColE7 indeed formed dimers in solution and that the dimeric conformation was more favored in the presence of double-stranded DNA. Structural comparison of nuclease ColE7 with the His-Cys box homing endonuclease I-PpoI further demonstrated that H-N-H motifs in dimeric nuclease ColE7 were oriented in a manner very similar to that of the betabetaalpha-fold of the active sites found in dimeric I-PpoI. A mechanism for the binding of double-stranded DNA by dimeric H-N-H nuclease ColE7 is suggested.

Amino Acid Motifs↗

PubMed2002

Metal ions and phosphate binding in the H-N-H motif: crystal structures of the nuclease domain of ColE7/Im7 in complex with a phosphate ion and different divalent metal ions.

H-N-H is a motif found in the nuclease domain of a subfamily of bacteria toxins, including colicin E7, that are capable of cleaving DNA nonspecifically. This H-N-H motif has also been identified in a subfamily of homing endonucleases, which cleave DNA site specifically. To better understand the role of metal ions in the H-N-H motif during DNA hydrolysis, we crystallized the nuclease domain of colicin E7 (nuclease-ColE7) in complex with its inhibitor Im7 in two different crystal forms, and we resolved the structures of EDTA-treated, Zn(2+)-bound and Mn(2+)-bound complexes in the presence of phosphate ions at resolutions of 2.6 A to 2.0 A. This study offers the first determination of the structure of a metal-free and substrate-free enzyme in the H-N-H family. The H-N-H motif contains two antiparallel beta-strands linked to a C-terminal alpha-helix, with a divalent metal ion located in the center. Here we show that the metal-binding sites in the center of the H-N-H motif, for the EDTA-treated and Mg(2+)-soaked complex crystals, were occupied by water molecules, indicating that an alkaline earth metal ion does not reside in the same position as a transition metal ion in the H-N-H motif. However, a Zn(2+) or Mn(2+) ions were observed in the center of the H-N-H motif in cases of Zn(2+) or Mn(2+)-soaked crystals, as confirmed in anomalous difference maps. A phosphate ion was found to bridge between the divalent transition metal ion and His545. Based on these structures and structural comparisons with other nucleases, we suggest a functional role for the divalent transition metal ion in the H-N-H motif in stabilizing the phosphoanion in the transition state during hydrolysis.

Amino Acid Motifs↗