The nucleotide excision repair pathway corrects many structurally unrelated DNA lesions. Damage recognition in bacteria is performed by UvrA, a member of the ABC ATPase superfamily whose functional form is a dimer with four nucleotide-binding domains (NBDs), two per protomer. In the 3.2 A structure of UvrA from Bacillus stearothermophilus, we observe that the nucleotide-binding sites are formed in an intramolecular fashion and are not at the dimer interface as is typically found in other ABC ATPases. UvrA also harbors two unique domains; we show that one of these is required for interaction with UvrB, its partner in lesion recognition. In addition, UvrA contains three zinc modules, the number and ligand sphere of which differ from previously published models. Structural analysis, biochemical experiments, surface electrostatics, and sequence conservation form the basis for models of ATP-modulated dimerization, UvrA-UvrB interaction, and DNA binding during the search for lesions.
Molecular dynamics simulations of alpha-lytic protease (alphaLP) alone and complexed with its pro region (PRO) are performed to understand the origin of its high unfolding (and folding) barrier when it is alone and how the pro region lowers this barrier. At room temperature, alphaLP exhibits lower dynamic fluctuations than alpha-chymotrypsin. Simulation of PRO alone led to reorientation of its N terminal helix and collapse to a more compact state. A model for the uncleaved proenzyme was built and found to be stable in the time scale of the simulations. Energetic analysis suggests that the origin of strain in the uncleaved proenzyme compared with the cleaved complex is in the intramolecular backbone electrostatic interactions of the cleaved strand. In high temperature simulations, the interaction of the long beta hairpin of the enzyme with the C terminal beta sheet of PRO is among the most stable in the complex and a likely "nucleation site" for folding. In the course of unfolding, the C terminal tail of PRO is sometimes observed to intervene between the long hairpin and the aspartate loop of the enzyme, perhaps thereby lowering the energy barrier for separation of the two hairpins. Tighter interactions at the interface between the enzyme and its pro region are also occasionally observed, providing an additional mechanism for unfolding catalysis. Simulations of a mutant enzyme where the buried ion pair residues R102 and D142 were replaced by W and L, respectively, did not display any distinguishable behavior compared with the wild type.
Molecular dynamics simulations of α‐lytic protease (αLP) alone and complexed with its pro region (PRO) are performed to understand the origin of its high unfolding (and folding) barrier when it is alone and how the pro region lowers this barrier. At room temperature, αLP exhibits lower dynamic fluctuations than α‐chymotrypsin. Simulation of PRO alone led to reorientation of its N terminal helix and collapse to a more compact state. A model for the uncleaved proenzyme was built and found to be stable in the time scale of the simulations. Energetic analysis suggests that the origin of strain in the uncleaved proenzyme compared with the cleaved complex is in the intramolecular backbone electrostatic interactions of the cleaved strand. In high temperature simulations, the interaction of the long beta hairpin of the enzyme with the C terminal beta sheet of PRO is among the most stable in the complex and a likely “nucleation site” for folding. In the course of unfolding, the C terminal tail of PRO is sometimes observed to intervene between the long hairpin and the aspartate loop of the enzyme, perhaps thereby lowering the energy barrier for separation of the two hairpins. Tighter interactions at the interface between the enzyme and its pro region are also occasionally observed, providing an additional mechanism for unfolding catalysis. Simulations of a mutant enzyme where the buried ion pair residues R102 and D142 were replaced by W and L, respectively, did not display any distinguishable behavior compared with the wild type. Proteins 2000;41:21–32. © 2000 Wiley‐Liss, Inc.
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