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Roles of structural plasticity in chaperone HdeA activity are revealed by 19F NMR.

ABSTRACT: HdeA, a minimal ATP-independent acid chaperone, is crucial for the survival of enteric pathogens as they transit the acidic (pH 1-3) environment of the stomach. Although protein disorder (unfolding) and structural plasticity have been elegantly linked to HdeA function, the details of the linkage are lacking. Here, we apply 19F NMR to reveal the structural transition associated with activation. We find that unfolding is necessary but not sufficient for activation. Multiple conformations are present in the functional state at low pH, but the partially folded conformation is essential for HdeA chaperone activity, and HdeA's intrinsic disulfide bond is required to maintain the partially folded conformation. The results show that both disorder and order are key to function. The ability of 19F NMR to reveal and quantify multiple conformational states makes it a powerful tool for studying other chaperones.

SUBMITTER: Zhai Z

PROVIDER: S-EPMC5975942 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Roles of structural plasticity in chaperone HdeA activity are revealed by <sup>19</sup>F NMR.

Zhai Zining Z Wu Qiong Q Zheng Wenwen W Liu Maili M Pielak Gary J GJ Li Conggang C

Chemical science 20151203 3

HdeA, a minimal ATP-independent acid chaperone, is crucial for the survival of enteric pathogens as they transit the acidic (pH 1-3) environment of the stomach. Although protein disorder (unfolding) and structural plasticity have been elegantly linked to HdeA function, the details of the linkage are lacking. Here, we apply <sup>19</sup>F NMR to reveal the structural transition associated with activation. We find that unfolding is necessary but not sufficient for activation. Multiple conformation ...[more]

PMID: 29910910

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Project description:Inhibitor discovery for protein-protein interactions has proven difficult due to the large protein surface areas and dynamic interfaces involved. This is particularly the case when targeting transcription-factor-protein interactions. To address this challenge, structural biology approaches for ligand discovery using X-ray crystallography, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy have had a significant impact on advancing small molecule inhibitors into the clinic, including the U.S. Food and Drug Administration approved drug, Venetoclax. Inspired by the protein-observed NMR approach using 1H-15N-HSQC NMR which detects chemical shift perturbations of 15N-labeled amides, we have applied a complementary protein-observed 19F NMR approach using 19F-labeled side-chains that are enriched at protein-protein-interaction interfaces. This protein-observed 19F NMR assay is abbreviated PrOF NMR to distinguish the experiment from the more commonly employed ligand-observed 19F NMR methods. In this Account, we describe our efforts using PrOF NMR as a ligand discovery tool, particularly for fragment-based ligand discovery (FBLD). We metabolically label the aromatic amino acids on proteins due to the enrichment of aromatic residues at protein interfaces. We choose the 19F nucleus due to its high signal sensitivity and the hyperresponsiveness of 19F to changes in chemical environment. Simultaneous labeling with two different types of fluorinated aromatic amino acids for PrOF NMR has also been achieved. We first describe the technical aspects of considering the application of PrOF NMR for characterizing native protein-protein interactions and for ligand screening. Several test cases are further described with a focus on a transcription factor coactivator interaction with the KIX domain of CBP/p300 and two epigenetic regulatory domains, the bromodomains of BRD4 and BPTF. Through these case studies, we highlight medicinal chemistry applications in FBLD, selectivity screens, structure-activity relationship (SAR) studies, and ligand deconstruction approaches. These studies have led to the discovery of some of the first inhibitors for BPTF and a novel inhibitor class for the N-terminal bromodomain of BRD4. The speed, ease of interpretation, and relatively low concentration of protein needed for NMR-based binding experiments affords a rapid, structural biology-based method to discover and characterize both native and new ligands for bromodomains, and it may find utility in the study of additional epigenetic proteins and transcription-factor-protein interactions.

Dataset Information

Roles of structural plasticity in chaperone HdeA activity are revealed by 19F NMR.

Publications

Roles of structural plasticity in chaperone HdeA activity are revealed by <sup>19</sup>F NMR.

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