A finely balanced order-disorder equilibrium sculpts the folding-binding landscape of an antibiotic sequestering protein

Natarajan, L and De Sciscio, ML and Nardi, AN and Sekhar, A and Del Giudice, A and D'Abramo, M and Naganathan, AN (2024) A finely balanced order-disorder equilibrium sculpts the folding-binding landscape of an antibiotic sequestering protein. In: Proceedings of the National Academy of Sciences of the United States of America, 121 (20). e2318855121.

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Abstract

TipA, a MerR family transcription factor from Streptomyces lividans, promotes antibiotic resistance by sequestering broad-spectrum thiopeptide-based antibiotics, thus counteracting their inhibitory effect on ribosomes. TipAS, a minimal binding motif which is expressed as an isoform of TipA, harbors a partially disordered N-terminal subdomain that folds upon binding multiple antibiotics. The extent and nature of the underlying molecular heterogeneity in TipAS that shapes its promiscuous folding-function landscape is an open question and is critical for understanding antibiotic-sequestration mechanisms. Here, combining equilibrium and time-resolved experiments, statistical modeling, and simulations, we show that the TipAS native ensemble exhibits a pre-equilibrium between binding-incompetent and binding-competent substates, with the fully folded state appearing only as an excited state under physiological conditions. The binding-competent state characterized by a partially structured N-terminal subdomain loses structure progressively in the physiological range of temperatures, swells on temperature increase, and displays slow conformational exchange across multiple conformations. Binding to the bactericidal antibiotic thiostrepton follows a combination of induced-fit and conformational-selection-like mechanisms, via partial binding and concomitant stabilization of the binding-competent substate. These ensemble features are evolutionarily conserved across orthologs from select bacteria that infect humans, underscoring the functional role of partial disorder in the native ensemble of antibiotic-sequestering proteins belonging to the MerR family.

Item Type:	Journal Article
Publication:	Proceedings of the National Academy of Sciences of the United States of America
Additional Information:	The copyright for this article belongs to
Keywords:	antiinfective agent; bacterial protein; protein binding; transcription factor, chemistry; genetics; metabolism; molecular model; protein conformation; protein folding; Streptomyces lividans, Anti-Bacterial Agents; Bacterial Proteins; Models, Molecular; Protein Binding; Protein Conformation; Protein Folding; Streptomyces lividans; Transcription Factors
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	04 Jun 2024 07:26
Last Modified:	04 Jun 2024 07:26
URI:	https://eprints.iisc.ac.in/id/eprint/85107

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