Physical interaction between bacterial heat shock protein (Hsp) 90 and Hsp70 chaperones mediates their cooperative action to refold denatured proteins.

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.

Insights into anti-termination regulation of the hut operon in Bacillus subtilis: importance of the dual RNA-binding surfaces of HutP.

The anti-termination protein, HutP, regulates the gene expression of the hut (histidine utilization) operon of Bacillus subtilis, by destabilizing the hut terminator RNA located upstream of the coding region encoding l-histidine degradation enzymes. On the basis of biochemical, in vivo and X-ray structural analyses, we now report that HutP uses its dual RNA-binding surfaces to access two XAG-rich regions (sites I and II) within the terminator RNA to mediate the destabilization process. In this process, HutP initiates destabilization at the 5'-end of its mRNA by binding to the first XAG-rich region (site I) and then accesses the second XAG-rich region (site II), located downstream of the stable G-C-rich segment of the terminator stem. By this action, HutP appears to disrupt the G-C-rich terminator stem, and thus prevents premature termination of transcription in the RNA segment preceding the regions encoding for the histidine degradation enzymes.

An RNA aptamer that distinguishes between closely related human influenza viruses and inhibits haemagglutinin-mediated membrane fusion.

Aptamers selected against various kinds of targets have shown remarkable specificity and affinity, similar to those displayed by antibodies to their antigens. To employ aptamers as genotyping reagents for the identification of pathogens and their strains, in vitro selections were carried out to find aptamers that specifically bind and distinguish the closely related human influenza A virus subtype H3N2. The selected aptamer, P30-10-16, binds specifically to the haemagglutinin (HA) region of the target strain A/Panama/2007/1999(H3N2) and failed to recognize other human influenza viruses, including another strain with the same subtype, H3N2. The aptamer displayed over 15-fold-higher affinity to the HA compared with the monoclonal antibody, and efficiently inhibited HA-mediated membrane fusion. These studies delineate the application of aptamers in the genotyping of viruses.

Selection of RNA aptamers against human influenza virus hemagglutinin using surface plasmon resonance.

Aptamers are functional nucleic acids possessing high affinity and specificity to their cognate ligands and are isolated from a library of nucleic acids by iterative rounds of selection and amplification. In the current study, we used surface plasmon resonance (Biacore) as an efficient methodology for selecting aptamers that bind to hemagglutinin (HA) of human influenza virus. This procedure allowed us to monitor and select the target-bound aptamers specifically and simultaneously. These studies not only yielded an aptamer that binds to the HA of influenza virus with high affinity but also revealed the consensus sequence, 5'-GUCGNCNU(N)(2-3)GUA-3, for HA recognition.

Structure of RNA aptamer complexed with an RNA-binding peptide of Tat with aid of residue-specific 13C, 15N labeling.

An RNA aptamer containing two binding sites of HIV Tat exhibits extremely high affinity to Tat. We have determined the structure of the aptamer complexed with an RNA-binding peptide of Tat. The analysis was made feasible by the use of several peptides in which a single arginine residue was specifically 13C, 15N-labeled. Residue specific labeling of the peptide enhanced the identification of intermolecular contacts, which are otherwise hard to identify due to spectral overlapping. The structure of the complex has revealed the origin of the high affinity of the aptamer to Tat.