Elucidation of structural changes involved in protein misfolding and amyloid formation is crucial for unraveling the molecular basis of amyloid formation. Here we report structural analyses of the amyloidogenic intermediate and amyloid aggregates of transthyretin using solution and solid-state nuclear magnetic resonance (NMR) spectroscopy. Our solution NMR results show that one of the two main β-sheet structures (CBEF β-sheet) is maintained in the aggregation-competent intermediate, while the other DAGH β-sheet is more flexible on millisecond time scales. Magic-angle-spinning solid-state NMR revealed that AB loop regions interacting with strand A in the DAGH β-sheet undergo conformational changes, leading to the destabilized DAGH β-sheet.

The σ subunits of bacterial RNA polymerase occur in many variant forms and confer promoter specificity to the holopolymerase. Members of the σ54 family of σ subunits require the action of a ‘transcriptional activator’ protein to open the promoter and initiate transcription. The activator proteins undergo regulated assembly from inactive dimers to hexamers that are active ATPases. These contact σ54 directly and, through ATP hydrolysis, drive a conformational change that enables promoter opening. σ54 activators use several different kinds of regulatory domains to respond to a wide variety of intracellular signals. One common regulatory module, the GAF domain, is used by σ54 activators to sense small-molecule ligands. The structural basis for GAF domain regulation in σ54 activators has not previously been reported. Here, we present crystal structures of GAF regulatory domains for Aquifex aeolicus σ54 activators NifA-like homolog (Nlh)2 and Nlh1 in three functional states—an ‘open’, ATPase-inactive state; a ‘closed’, ATPase-inactive state; and a ‘closed’, ligand-bound, ATPase-active state. We also present small-angle X-ray scattering data for Nlh2-linked GAF-ATPase domains in the inactive state. These GAF domain dimers regulate σ54 activator proteins by holding the ATPase domains in an inactive dimer conformation. Ligand binding of Nlh1 dramatically remodels the GAF domain dimer interface, disrupting the contacts with the ATPase domains. This mechanism has strong parallels to the response to phosphorylation in some two-component regulated σ54 activators. We describe a structural mechanism of GAF-mediated enzyme regulation that appears to be conserved among humans, plants, and bacteria.Graphical AbstractDownload high-res image (125KB)Download full-size imageHighlights► GAF domains regulate many different proteins by responding to small-molecule ligands. ► A σ54 activator GAF domain represses ATPase domain assembly using an extended helical pair. ► σ54 activator GAF domain binding pocket loops can open in the absence of ligand. ► Ligand binding changes the dimer interface between GAF domains that allows ATPase assembly. ► New activator GAF structures have provided insight into their detailed regulatory mechanism.

•Transcription initiation by bacterial σ54-polymerase requires an upstream activator.•The structure of an activator's DBD, with and without DNA, was solved.•The activator's DBD is homologous to a multipurpose protein, Fis.•Fis and NtrC4 contact DNA similarly, but Fis is more reliant on nonspecific contacts.•These subtle differences explain the proteins' divergent functions.Transcription initiation by bacterial σ54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain (DBD). The structurally characterized DBDs from activators all belong to the Fis (factor for inversion stimulation) family of helix–turn–helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DBD of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ54 activators. Two NtrC4-binding sites were identified upstream (− 145 and − 85 bp) from the start of the lpxC gene, which is responsible for the first committed step in lipid A biosynthesis. This is the first experimental evidence for σ54 regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the − 145-binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homolog, Fis. The greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base-specific contacts contributing to affinity than for Fis.Download high-res image (312KB)Download full-size image

•In free σ54, the AID (residues 1–50) is intrinsically disordered.•The AID becomes ordered upon core polymerase binding.•The AID alone binds transcriptional activators in their ATP state.•The AID binds the activator with native-like affinity.•σ54 residues 16–25 are the major contact region to the ATPase.Bacterial sigma factors are subunits of RNA polymerase that direct the holoenzyme to specific sets of promoters in the genome and are a central element of regulating transcription. Most polymerase holoenzymes open the promoter and initiate transcription rapidly after binding. However, polymerase containing the members of the σ54 family must be acted on by a transcriptional activator before DNA opening and initiation occur. A key domain in these transcriptional activators forms a hexameric AAA + ATPase that acts through conformational changes brought on by ATP hydrolysis. Contacts between the transcriptional activator and σ54 are primarily made through an N-terminal σ54 activator interacting domain (AID). To better understand this mechanism of bacterial transcription initiation, we characterized the σ54 AID by NMR spectroscopy and other biophysical methods and show that it is an intrinsically disordered domain in σ54 alone. We identified a minimal construct of the Aquifex aeolicus σ54 AID that consists of two predicted helices and retains native-like binding affinity for the transcriptional activator NtrC1. Using the NtrC1 ATPase domain, bound with the non-hydrolyzable ATP analog ADP-beryllium fluoride, we studied the NtrC1–σ54 AID complex using NMR spectroscopy. We show that the σ54 AID becomes structured after associating with the core loops of the transcriptional activators in their ATP state and that the primary site of the interaction is the first predicted helix. Understanding this complex, formed as the first step toward initiation, will help unravel the mechanism of σ54 bacterial transcription initiation.Download high-res image (112KB)Download full-size image