Cell surface ß-lactamase recruitment: A facile selection to identify protein-protein interactions.

Protein-protein interactions (PPIs) are central to many cellular processes, and the identification of novel PPIs is a critical step in the discovery of protein therapeutics. Simple methods to identify naturally existing or laboratory evolved PPIs are therefore valuable research tools. We have developed a facile selection that links PPI-dependent ß-lactamase recruitment on the surface of Escherichia coli with resistance to ampicillin. Bacteria displaying a protein that forms a complex with a specific protein-ß-lactamase fusion are protected from ampicillin-dependent cell death. In contrast, bacteria that do not recruit ß-lactamase to the cell surface are killed by ampicillin. Given its simplicity and tunability, we anticipate this selection will be a valuable addition to the palette of methods for illuminating and interrogating PPIs.

Toxin-based screening of C-terminal tags in Escherichia coli reveals the exceptional potency of ssrA-like degrons.

All bacteria possess ATP-dependent proteases that destroy cytosolic proteins. These enzymes help cells mitigate proteotoxic stress, adapt to changing nutrient availability, regulate virulence phenotypes, and transition to pathogenic lifestyles. Moreover, ATP-dependent proteases have emerged as promising antibacterial and antivirulence targets in a variety of pathogens. The physiological roles of these proteases are largely defined by the complement of proteins that they degrade. Substrates are typically recognized in a highly selective manner, often via short unstructured sequences termed degrons. While a few degrons have been identified and rigorously characterized, we lack a systematic understanding of how proteases select valid degrons from the vast complexity of protein sequence space. Here, we describe a novel high-throughput screening approach in Escherichia coli that couples proteolysis of a protein toxin to cell survival. We used this method to screen a combinatorial library of C-terminal pentapeptide sequences for functionality as proteolytic degrons in wild type E. coli, and in strains lacking components of the ClpXP and ClpAP proteases. By examining the competitive enrichment of sequences over time, we found that about one percent of pentapeptide tags lead to toxin proteolysis. Interestingly, the most enriched degrons were ClpXP-dependent and highly similar to the ssrA tag, one of the most extensively characterized degrons in bacteria. Among ssrA-like sequences, we observed that specific upstream residues correlate with successful recognition. The lack of diversity among strongly enriched sequences suggests that ssrA-like tags comprise a uniquely potent class of short C-terminal degron in E. coli. Efficient proteolysis of substrates lacking such degrons likely requires adaptors or multivalent interactions. These findings broaden our understanding of the constraints that shape the bacterial proteolytic landscape. Our screening approach may be broadly applicable to probing aspects of proteolytic substrate selection in other bacterial systems.

Interactome Analysis Identifies MSMEI_3879 as a Substrate of Mycolicibacterium smegmatis ClpC1.

The prevalence of drug-resistant Mycobacterium tuberculosis infections has prompted extensive efforts to exploit new drug targets in this globally important pathogen. ClpC1, the unfoldase component of the essential ClpC1P1P2 protease, has emerged as one particularly promising antibacterial target. However, efforts to identify and characterize compounds that impinge on ClpC1 activity are constrained by our limited knowledge of Clp protease function and regulation. To expand our understanding of ClpC1 physiology, we employed a coimmunoprecipitation and mass spectrometry workflow to identify proteins that interact with ClpC1 in Mycolicibacterium smegmatis, a surrogate for M. tuberculosis. We identify a diverse panel of interaction partners, many of which coimmunoprecipitate with both the regulatory N-terminal domain and the ATPase core of ClpC1. Notably, our interactome analysis establishes MSMEI_3879, a truncated gene product unique to M. smegmatis, as a novel proteolytic substrate. Degradation of MSMEI_3879 by ClpC1P1P2 in vitro requires exposure of its N-terminal sequence, reinforcing the idea that ClpC1 selectively recognizes disordered motifs on substrates. Fluorescent substrates incorporating MSMEI_3879 may be useful in screening for novel ClpC1-targeting antibiotics to help address the challenge of M. tuberculosis drug resistance. IMPORTANCE Drug-resistant tuberculosis infections are a major challenge to global public health. Much effort has been invested in identifying new drug targets in the causative pathogen, Mycobacterium tuberculosis. One such target is the ClpC1 unfoldase. Compounds have been identified that kill M. tuberculosis by disrupting ClpC1 activity, yet the physiological function of ClpC1 in cells has remained poorly defined. Here, we identify interaction partners of ClpC1 in a model mycobacterium. By building a broader understanding of the role of this prospective drug target, we can more effectively develop compounds that inhibit its essential cellular activities.

Structure of HIV TAR in complex with a Lab-Evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.

Natural and lab-evolved proteins often recognize their RNA partners with exquisite affinity. Structural analysis of such complexes can offer valuable insight into sequence-selective recognition that can be exploited to alter biological function. Here, we describe the structure of a lab-evolved RNA recognition motif (RRM) bound to the HIV-1 trans-activation response (TAR) RNA element at 1.80 Å-resolution. The complex reveals a trio of arginines in an evolved ß2-ß3 loop penetrating deeply into the major groove to read conserved guanines while simultaneously forming cation-π and salt-bridge contacts. The observation that the evolved RRM engages TAR within a double-stranded stem is atypical compared to most RRMs. Mutagenesis, thermodynamic analysis and molecular dynamics validate the atypical binding mode and quantify molecular contributions that support the exceptionally tight binding of the TAR-protein complex (KD,App of 2.5 ± 0.1 nM). These findings led to the hypothesis that the ß2-ß3 loop can function as a standalone TAR-recognition module. Indeed, short constrained peptides comprising the ß2-ß3 loop still bind TAR (KD,App of 1.8 ± 0.5 µM) and significantly weaken TAR-dependent transcription. Our results provide a detailed understanding of TAR molecular recognition and reveal that a lab-evolved protein can be reduced to a minimal RNA-binding peptide.