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.


Supplementary Figures
: A random protein fragment assay of DHFR shows that inhibition is reading framedependent. (A) Histogram of enrichment (E) by in-frame (red) and out-of-frame (grey) fragments of ≤30 residues derived from the random fragmentation of E. coli DHFR. (B) Average enrichment per site due to in-frame fragments covering each residue, across the DHFR sequence (see Materials and Methods). Inhibitory peaks are indicated. Inset: overlay of per-site enrichment of in-frame and out-of-frame fragments. (C) DHFR crystallographic structure (PDB ID 7dfr) with N-and C-termini indicated, overlaid by the known refolding inhibitory fragment (left), alongside the C-terminal inhibitory peak (middle) and the N-terminal inhibitory peak (right) from the random fragment assay.

Figure S2: A protein fragment from interaction site 1 inhibits FtsZ polymerization in vitro. Left:
Results from measurements of FtsZ filament polymerization by 90° light scattering (Materials and Methods) in the presence of either a protein fragment from interaction site 1 (FtsZ residues 166-195) or a neutral control fragment (FtsZ residues 34-63). In each reaction, FtsZ was present at 5.06 µM and the peptide was present at 18.4 µM. Error bars: s.e.m. from replicate measurements (N = 4 for the interaction site 1 fragment, N = 3 for the neutral control fragment). The asterisk indicates p < 0.05. Right: Inhibitory fragment map of FtsZ, plotted as in Figure 2, overlaid with the interaction site 1 and neutral control fragments.   50S ribosomal subunit protein L7/L12 (RplL) is shown with a grey fill color; linear fits to the nonribosomal protein data are shown for the 30-residue and 50-residue data as dashed lines in corresponding colors. Note that the slopes are very similar. The faded grey dashed line near the 20residue fragment data is a shifted copy of the trendline for the 30-residue data, suggesting that a similar slope may also be reasonable for 20-residue fragments; however, among the proteins for which 20residue fragment libraries were generated, a leveling off is evident as the mean E enters the neutral regime (E ≈ 0), as expected, and there is insufficient sampling of mean E values below zero to fit these data directly.    Figure S1) attributable to each property is plotted, based on a nested ANOVA. Properties include protein fragment length and in-frame secondary structure features of the fragment-covered sequence region of the full-length gene; "fragment ID" designates fragment-specific effects not attributable to variation in any of the properties considered.

SI Text
Fragment mapping reveals promising target sites for inhibitor development: additional discussion DHFR is the target of the antibiotic trimethoprim, which binds at the enzymatic active site and inhibits catalysis (51). Our results suggest that inhibition of folding by compounds that bind to the N-terminal beta strand may be another approach to target this important enzyme. Another route would be welcome given the serious issue of trimethoprim resistance (52); due to the distinct mechanisms, a putative folding inhibitor would likely not be subject to the same resistance mutations.
A number of compounds inhibiting bacterial DNA gyrase, including the fluoroquinolones currently employed clinically, function by trapping the enzyme in a DNA cleavage complex (53). Other compounds, such as the aminocoumarins, interfere with ATPase activity (53). Our finding of an inhibitory fragment peak localized to the ends of the C-gate arms of GyrA, which form a homomeric head-to-head complex to close the gate, suggests that compounds targeting these Cgate closure sites would provide another means to inhibit gyrase. Such inhibitors would rely on a distinct binding mode from the aforementioned compounds, which bind at sites at or near the DNA gate or the ATPase site (53,54). In line with our findings, a monoclonal antibody against Mycobacterium smegmatis and Mycobacterium tuberculosis GyrA appears to bind at the same location as our fragments and prevent C-gate dimerization. Furthermore, the antibody epitope corresponds to E. coli GyrA residues 340-402 (55), fully overlapping with the inhibitory fragment peak centered around residue 386.
Single-stranded DNA binding protein (Ssb) is a desirable antibiotic target because inhibition should interfere with DNA replication and repair. Screening has therefore been performed for Ssb inhibitors (56)(57)(58), leading to the identification of numerous compounds; some of these were shown to interfere with critical interactions of eight conserved C-terminal residues with genome maintenance proteins (57,58). Unlike the case for FtsZ, we did not observe an inhibitory fragment peak mapping near this interaction-mediating C-terminal tail. Instead, we observed a strong peak mapping to the single alpha-helical element of this protein, suggesting a novel approach for inhibitor development: mimicking the interactions formed by the alpha helix in Ssb dimers to disrupt dimer and tetramer formation.
A further note regarding the transferability of inhibitory sites identified in E. coli to other bacteria: The range of bacteria potentially targetable based on these fragment scan results is extensive, given that protein-peptide interactions are based heavily on structural properties. Therefore, more distantly related orthologs retaining the relevant structural features are likely susceptible to the same types of inhibitors, especially those based directly on peptide fragments.