Structural plasticity of an acid-activated chaperone allows promiscuous substrate binding

Abstract

HdeA has been shown to prevent acid-induced aggregation of proteins. With a mass of only 9.7 kDa, HdeA is one of the smallest chaperones known. Unlike other molecular chaperones, which are typically complex, multimeric ATP-dependent machines, HdeA is known to undergo an acid-induced dimer to monomer transition and functions at low pH as a disordered monomer without the need for energy factors. Thus, HdeA must possess features that allow it to bind substrates and regulate substrate affinity in a small and energy-independent package. To understand better how HdeA accomplishes this, we studied the conformational changes that accompany a shift to low pH and substrate binding. We find that the acid-induced partial unfolding and monomerization that lead to HdeA activation occur very rapidly (k >3.5 s ؊1 ). Activation exposes the hydrophobic dimer interface, which we found to be critical for substrate binding. We show by intramolecular FRET that the partially unfolded character of active HdeA allows the chaperone to adopt different conformations as required for the recognition and high-affinity binding of different substrate proteins. These efficient adaptations help to explain how a very small protein is rapidly activated and can bind a broad range of substrate proteins in a purely pH-regulated manner.HdeA ͉ periplasm ͉ posttranslational regulation

Protein refolding by pH-triggered chaperone binding and release

“…S1C). The sedimentation of the HdeA monomer is not consistent with a completely unfolded polypeptide as has been proposed (7,12), but rather is indicative of a fairly compact structure, consistent with our previous circular dichroism and fluorescence data suggesting that HdeA is only partially unfolded at low pH (8).…”

“…Light-scattering measurements indicate that HdeA suppresses MDH aggregation at low pH (Fig. 1A), as previously reported (8). In the absence of HdeA, pH neutralization causes MDH to rapidly aggregate, as indicated by the increase in the light-scattering signal immediately after the Author contributions: T.L.T., U.J., and J.C.B.…”

“…S2A, B). These observations underscore the importance of the rapid activation of HdeA for its optimal function (8). Binding of HdeA to MDH at low pH likely prevents the formation of off-pathway MDH intermediates or small aggregates, which rapidly form upon exposure to low pH, and have a strong tendency to aggregate rather than refold upon neutralization.…”

“…However, in contrast to most other proteins, which are inactivated upon pH-induced unfolding, partial unfolding and dissociation of HdeA into monomers causes the activation of its chaperone function (4,7). We recently reported that low pH induces the exposure of structurally plastic, high-affinity binding sites for unfolding proteins on HdeA, and enables it to effectively prevent protein aggregation in vitro (8). The partially unfolded and therefore highly flexible architecture of active HdeA monomers seems to contribute to HdeA's ability to adaptively bind to a wide range of different substrate proteins, some of which are much larger than the 9.7 kDa chaperone itself (8).…”

“…We recently reported that low pH induces the exposure of structurally plastic, high-affinity binding sites for unfolding proteins on HdeA, and enables it to effectively prevent protein aggregation in vitro (8). The partially unfolded and therefore highly flexible architecture of active HdeA monomers seems to contribute to HdeA's ability to adaptively bind to a wide range of different substrate proteins, some of which are much larger than the 9.7 kDa chaperone itself (8). Inactivation of HdeA is triggered by pH neutralization, which induces its refolding and dimerization (7).…”