A FRET-Based Method for Probing the Conformational Behavior of an Intrinsically Disordered Repeat Domain fromBordetella pertussisAdenylate Cyclase

Abstract

A better understanding of the conformational changes exhibited by intrinsically disordered proteins is necessary as we continue to unravel their myriad biological functions. In repeats in toxin (RTX) domains, calcium binding triggers the natively unstructured domain to adopt a beta roll structure. Here we present an in vitro Forster resonance energy transfer (FRET)-based method for the investigation of the conformational behavior of an RTX domain from the Bordetella pertussis adenylate cyclase consisting of nine repeat units. Equilibrium and stopped-flow FRET between fluorescent proteins, attached to the termini of the domain, were measured in an analysis of the end-to-end distance changes in the RTX domain. The method was complemented with circular dichroism spectroscopy, tryptophan fluorescence, and bis-ANS dye binding. High ionic strength was observed to decrease the calcium affinity of the RTX domain. A truncation and single amino acid mutations yielded insights into the structural determinants of beta roll formation. Mutating the conserved Asp residue in one of the nine repeats significantly reduced the affinity of the domains for calcium ions. Removal of the sequences flanking the repeat domain prevented folding, but replacing them with fluorescent proteins restored the conformational behavior, suggesting an entropic stabilization. The FRET-based method is a useful technique that complements other low-resolution techniques for investigating the dynamic conformational behavior of the RTX domain and other intrinsically disordered protein domains.

Monitoring the conformational changes of an intrinsically disordered peptide using a quartz crystal microbalance

“…15 Using a Forster resonance energy transfer (FRET) construct, we previously reported a salt dependency on the binding affinity of the RTX-C construct. 24 The lack of a salt dependency when tracking conformational changes in the cap via tryptophan fluorescence suggests that the RTX region is primarily responsible for the salt dependency of the binding affinity. Next, we sought to determine if calcium-dependent conformational changes of the RTX domain could be detected by QCM.…”

“…This is similar to the repeated folding and unfolding of the beta roll reported in solution. 24 To determine whether the change is specific to calcium or an ionic effect, the experiment was repeated using 10 mM MgCl 2 . No change in resistance and only a small ($2 Hz) change in frequency was observed [ Fig.…”

“…This supports the conclusion that the signal change is due to beta roll formation and is consistent with the observation of calcium-specific binding of the beta roll in solution. 24 Next, we tested the uncapped cys-RTX and RTXcys constructs. We hypothesized that cys-RTX would not be calcium responsive, as immobilization occurs on the N-terminus, while RTX-cys could be responsive, due to an entropic stabilization effect by the mass of the surface at the C-terminus.…”

Calcium, Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

“…These motifs constitute the main Ca 2ϩ binding sites of the protein (16). The RTX motifs are intrinsically disordered in the absence of calcium (17)(18)(19)(20). The intrinsic disorder predictors (21)(22)(23)(24) (Fig.…”

Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin

“…The C-terminal part of the molecule (from position 900 to 1706) is involved in toxin binding to a specific cellular receptor (CD11b/ CD18). This domain consists of ϳ40 copies of a glycine and aspartate-rich nonapeptide repeat (residues 913-1613) (7)(8)(9)(10)(11)(12)(13)(14) characteristic of a large family of bacterial cytolysins known as RTX (repeat-in-toxin) toxins (15)(16)(17).…”