Mapping functional regions of essential bacterial proteins with dominant-negative protein fragments

Significance

Peptide fragments derived from protein sequences can inhibit interactions of their parental proteins, providing tools to study protein function in vivo. Here, we employed a massively parallel assay to measure inhibition of Escherichia coli growth by fragments tiling the sequences of 10 of its essential proteins. We leveraged these data to decipher principles of fragment-based inhibition, demonstrating that parental protein concentration drives activity and characterizing how fragment length interplays with activity and specificity. We employed statistical analysis to parse the roles of biophysical properties in fragment-to-fragment variation, finding that the specific characteristics of each fragment largely drive its inhibitory activity. These results advance our understanding of protein interactions in vivo and have implications for the rational design of peptide inhibitors.

Abstract

Massively parallel measurements of dominant-negative inhibition by protein fragments have been used to map protein interaction sites and discover peptide inhibitors. However, the underlying principles governing fragment-based inhibition have thus far remained unclear. Here, we adapted a high-throughput inhibitory fragment assay for use in Escherichia coli, applying it to a set of 10 essential proteins. This approach yielded single amino acid resolution maps of inhibitory activity, with peaks localized to functionally important interaction sites, including oligomerization interfaces and folding contacts. Leveraging these data, we performed a systematic analysis to uncover principles of fragment-based inhibition. We determined a robust negative correlation between susceptibility to inhibition and cellular protein concentration, demonstrating that inhibitory fragments likely act primarily by titrating native protein interactions. We also characterized a series of trade-offs related to fragment length, showing that shorter peptides allow higher-resolution mapping but suffer from lower inhibitory activity. We employed an unsupervised statistical analysis to show that the inhibitory activities of protein fragments are largely driven not by generic properties such as charge, hydrophobicity, and secondary structure, but by the more specific characteristics of their bespoke macromolecular interactions. Overall, this work demonstrates fundamental characteristics of inhibitory protein fragment function and provides a foundation for understanding and controlling protein interactions in vivo.