Acid-Induced Activation of the Periplasmic Chaperone HdeA

Abstract

Enteric bacterial pathogens like Escherichia coli are able to survive passage through extremely acidic environments like the human stomach. This acid-resistance strongly depends on the activity of the small periplasmic chaperone HdeA. Inactive as a dimer at neutral pH, HdeA elegantly uses its own acid-induced unfolding and monomerization to become rapidly activated upon shift to low pH. In its active and intrinsically disordered monomeric state, HdeA tightly binds to acid-unfolded client proteins. Upon pH neutralization, HdeA slowly releases its client proteins, allowing them to refold.To better understand the acid-induced activation of HdeA, we sought to identify which of HdeA's acid-titratable residues were the key players in sensing environmental pH changes, and which residues' protonation would trigger HdeA's monomerization and unfolding.Using protein sequence alignments, constant pH molecular dynamics calculations and predictions of pKa values, we identified several residues that are likely involved in maintaining the inactive dimer conformation at neutral pH and causing the unfolding and monomerization events upon shift to low pH. We substituted these residues with alanines and examined their dimer stabilities at neutral pH, their pH midpoints of monomer-dimer transitions and unfolding, and their pH-dependent activities. We identified several HdeA variants that are activated at higher pH values compared to the wild-type protein and are significantly destabilized already at neutral pH. By combining two mutations, we were further able to generate an HdeA variant that shows chaperone activity at neutral pH (where wild-type HdeA is completely inactive), making it a constitutively active variant of a normally acid-activated chaperone.These mutants will help us to understand on a structural level which regions of HdeA need to be flexible or unstable for the protein to function, and will help to determine how pH-driven changes in HdeA flexibility drive its activation.