Structure, function, and tethering of DNA-binding domains in σ54 transcriptional activators.

We compare the structure, activity, and linkage of DNA-binding domains (DBDs) from σ(54) transcriptional activators and discuss how the properties of the DBDs and the linker to the neighboring domain are affected by the overall properties and requirements of the full proteins. These transcriptional activators bind upstream of specific promoters that utilize σ(54)-polymerase. Upon receiving a signal the activators assemble into hexamers, which then, through adenosine triphosphate (ATP) hydrolysis, drive a conformational change in polymerase that enables transcription initiation. We present structures of the DBDs of activators nitrogen regulatory protein C 1 (NtrC1) and Nif-like homolog 2 (Nlh2) from the thermophile Aquifex aeolicus. The structures of these domains and their relationship to other parts of the activators are discussed. These structures are compared with previously determined structures of the DBDs of NtrC4, NtrC, ZraR, and factor for inversion stimulation. The N-terminal linkers that connect the DBDs to the central domains in NtrC1 and Nlh2 were studied and found to be unstructured. Additionally, a crystal structure of full-length NtrC1 was solved, but density of the DBDs was extremely weak, further indicating that the linker between ATPase and DBDs functions as a flexible tether. Flexible linking of ATPase and DBDs is likely necessary to allow assembly of the active hexameric ATPase ring. The comparison of this set of activators also shows clearly that strong dimerization of the DBD only occurs when other domains do not dimerize strongly.

Probing the conformation of a prion protein fibril with hydrogen exchange.

A fragment of the prion protein, PrP(89-143, P101L), bearing a mutation implicated in familial prion disease, forms fibrils that have been shown to induce prion disease when injected intracerebrally into transgenic mice expressing full-length PrP containing the P101L mutation. In this study, we utilize amide hydrogen exchange measurements to probe the organization of the peptide in its fibrillar form. We determined the extent of hydrogen exchange first by tandem proteolysis, liquid chromatography, and mass spectrometry (HXMS) and then by exchange-quenched NMR. Although single amide resolution is afforded by NMR measurements, HXMS is well suited to the study of natural prions because it does not require labeling with NMR active isotopes. Thus, natural prions obtained from infected animals can be compared with model systems such as PrP(89-143, P101L) studied here. In our study, we find two segments of sequence that display a high level of protection from exchange, residues 102-109 and 117-136. In addition, there is a region that displays exchange behavior consistent with the presence of a conformationally heterogeneous turn. We discuss our data with respect to several structural models proposed for infectious PrP aggregates and highlight HXMS as one of the few techniques well suited to studying natural prions.

Dearth of glutamate transporters contributes to striatal excitotoxicity.

Since excitotoxicity is hypothesized to contribute to cell death in Huntington's disease (HD), we examined the susceptibility of striatal and hippocampal neurons to glutamate-induced cell death. Striatal cultures were more susceptible to glutamate-triggered toxicity than sister hippocampal cultures. Dose-response curves were equivalent when secondary toxicity was blocked with application of the NMDA receptor antagonist, MK801, or enhanced with the pan-specific glutamate transport blocker, TBOA, following excitotoxin removal. TBOA failed to alter the dose-response characteristics of striatal excitotoxicity, ruling out reverse operation of glutamate transporters. Striatal cultures expressed less EAAC1 and less membrane-associated EAAC1, GLT1, and GLAST than hippocampal cultures. Antisense down-regulation of EAAC1 increased the sensitivity of hippocampal cultures to glutamate, indicating that this transporter can act as an important neuroprotectant. Thus, the relative expression levels of glutamate transporters, even in parts of the brain where they are considered adequately expressed, appear to influence the sensitivities of different neuronal populations to excitotoxicity.