Heat shock proteins (Hsps) play essential protective roles under stress conditions by preventing the formation of protein aggregates and degrading misfolded proteins. EcHsp31, the yedU (hchA) gene product, is a representative member of a family of chaperones that alleviates protein misfolding by interacting with early unfolding intermediates. The 1.6-Å crystal structure of the EcHsp31 dimer reveals a system of hydrophobic patches, canyons, and grooves, which may stabilize partially unfolded substrate. The presence of a well conserved, yet buried, triad in each two-domain subunit suggests a still unproven hydrolytic function of the protein. A flexible extended linker between the A and P domains may play a role in conformational flexibility and substrate binding. The ␣-␤ sandwich of the EcHsp31 monomer shows structural similarity to PhPI, a protease belonging to the DJ-1 superfamily. The structure-guided sequence alignment indicates that Hsp31 homologs can be divided in three classes based on variations in the P domain that dramatically affect both oligomerization and catalytic triad formation.heat shock protein ͉ Pyrococcus horikoshii protease I (PhPI) ͉ DJ-1 family

Two different stereoisomers of the dioxolane-linked gramicidin A (gA) channels were individually synthesized (the SS and RR dimers;. Science. 244:813-817). The structural differences between these dimers arise from different chiralities within the dioxolane linker. The SS dimer mimics the helicity and the inter- and intramolecular hydrogen bonding of the monomer-monomer association of gA's. In contrast, there is a significant disruption of the helicity and hydrogen bonding pattern of the ion channel in the RR dimer. Single ion channels formed by the SS and RR dimers in planar lipid bilayers have different proton transport properties. The lipid environment in which the different dimers are reconstituted also has significant effects on single-channel proton conductance (g(H)). g(H) in the SS dimer is about 2-4 times as large as in the RR. In phospholipid bilayers with 1 M [H(+)](bulk), the current-voltage (I-V) relationship of the SS dimer is sublinear. Under identical experimental conditions, the I-V plot of the RR dimer is supralinear (S-shaped). In glycerylmonooleate bilayers with 1 M [H(+)](bulk), both the SS and RR dimers have a supralinear I-V plot. Consistent with results previously published (. Biophys. J. 73:2489-2502), the SS dimer is stable in lipid bilayers and has fast closures. In contrast, the open state of the RR channel has closed states that can last a few seconds, and the channel eventually inactivates into a closed state in either phospholipid or glycerylmonooleate bilayers. It is concluded that the water dynamics inside the pore as related to proton wire transfer is significantly different in the RR and SS dimers. Different physical mechanisms that could account for this hypothesis are discussed. The gating of the synthetic gA dimers seems to depend on the conformation of the dioxolane link between gA's. The experimental results provide an important framework for a detailed investigation at the atomic level of proton conduction in different and relatively simple ion channel structures.

Based on co-crystal structures of human topoisomerase I with bound DNA, Lys 532 makes a minor groove contact with the strongly preferred thymidine residue at the site of covalent attachment (؊1 position). Replacement of Lys 532 with either arginine or alanine has essentially no effect on the sequence preference of the enzyme, indicating that this interaction is not required for the preference for a T at the ؊1 position. Although both the cleavage and religation activities of the K532R mutant enzyme are reduced, cleavage is reduced to a greater extent than religation. The reverse is true for the K532A mutant enzyme with religation so impaired that the nicked intermediate accumulates during plasmid relaxation assays. Consistent with the shift in the cleavage religation equilibrium toward cleavage for the K532A mutant enzyme, expression of the mutant enzyme in Saccharomyces cerevisiae is cytotoxic, and thus this mutant enzyme mimics the effects of the anticancer drug camptothecin. Cleavage assays with the mutant enzymes using an oligonucleotide containing a 5-bridging phosphorothiolate indicate that Lys 532 functions as a general acid during cleavage to protonate the leaving 5-oxygen. It is possible that the contact with the ؊1 base is important during catalysis to provide positional rigidity to the active site. The corresponding residues in the vaccinia virus topoisomerase and the tyrosine recombinases may have similar critical roles in catalysis.DNA topoisomerases are ubiquitous enzymes that solve topological problems generated by key nuclear processes such as DNA replication, transcription, recombination, DNA repair, chromatin assembly, and chromosome segregation by catalyzing the passage of individual DNA strands or double helices through one another. All cells contain two highly conserved classes of topoisomerases that are differentiated on the basis of their mechanistic and physical properties. The monomeric type I enzymes do not require high energy cofactors to change the helical state of DNA. They create a transient nick in one DNA strand through which the complementary strand is passed, leading to DNA relaxation (1, 2). The type II enzymes are multisubunit proteins that require ATP and modulate DNA topology by passing an intact helix through a transient doublestrand break they create in the same or different DNA (3). All type II topoisomerases and the type IA subfamily form transient covalent phosphodiester bonds with the 5Ј-end of broken DNA strands, whereas the type IB topoisomerases form a transient linkage to the 3Ј-end of the DNA (2).The type IB topoisomerase subfamily includes eukaryotic topoisomerase I, the topoisomerases encoded by vaccinia and other poxviruses, and some newly identified bacterial enzymes (1, 4). Recently, a structure of human topoisomerase I with a bound 22-bp DNA has been solved by x-ray crystallography (5, 6). Human topoisomerase I is a monomeric protein of 765 amino acids that binds to double-stranded DNA in a "clamp"-like manner with a preference for supercoiled DNA (7). Both ...

Heat shock proteins and proteases play a crucial role in cell survival under conditions of environmental stress. The heat shock protein Hsp31, produced by gene hchA at elevated temperatures in Escherichia coli, is a homodimeric protein consisting of a large A domain and a smaller P domain connected by a linker. Two catalytic triads are present per dimer, with the Cys and His contributed by the A domain and an Asp by the P domain. A new crystal Form II confirms the dimer and catalytic triad arrangement seen in the earlier crystal Form I. In addition, several loops exhibit increased flexibility compared to the previous Hsp31 dimer structure. In particular, loops D2 and D3 are intriguing because their mobility leads to the exposure of a sizable hydrophobic patch made up by surface areas of both subunits near the dimer interface. The residues creating this hydrophobic surface are completely conserved in the Hsp31 family. At the same time, access to the catalytic triad is increased. These observations lead to the hypothesis for the functioning of Hsp31 wherein loops D2 and D3 play a key role: first, at elevated temperatures, by becoming mobile and uncovering a large hydrophobic area that helps in binding to client proteins, and second, by removing the client protein from the hydrophobic patch when the temperature decreases and the loops adopt their low-temperature positions at the Hsp31 surface. The proposed mode of action of flexible loops in the functioning of Hsp31 may be a general principle employed by other chaperones.