Biology subjects

Schmitz, K. R.

Publications and source records attributed to Schmitz, K. R..

8 recordsLinked to original sources

bioRxiv2026

Phosphoarginine modulates oligomerization and repressor activity of mycobacterial ClpC2

Phosphoarginine (pArg) modifications direct proteins for proteolytic destruction by ClpC1P1P2, an essential mycobacterial protease that has emerged as a promising antibacterial drug target against Mycobacterium tuberculosis. The broader regulatory landscape surrounding pArg is poorly understood. Here, we establish a mechanistic connection between pArg binding and the activity of ClpC2, a non-proteolytic transcriptional repressor with homology to the ClpC1 N-terminal domain. Biophysical studies reveal that ClpC2 forms concentration-dependent higher-order oligomers that bind cooperatively to operator sequences in the clpC2 promoter. A high-resolution crystal structure of the Streptomyces thermoviolaceus ClpC2 C-terminal domain reveals a conserved dimerization interface mediated by a C-terminal helix, which is sterically disrupted by pArg binding. Consequently, we find that binding of pArg, as well as some ClpC1-targeting antibiotics, disrupts ClpC2 oligomerization, dissociates ClpC2 from its operator DNA, and relieves transcriptional repression in vitro. Moreover, comparative analysis of clpC2 promoters with single versus dual operator sites predicts differences in regulatory sensitivity across mycobacterial species. Together, these findings establish ClpC2 as a pArg-responsive sensor capable of mechanistically linking elevated pArg levels to downstream transcriptional regulation.

biochemistry↗

bioRxiv2026

The E. coli DEAD box ATPase CsdA, is a NAD+ capped RNA binding protein

5' nicotinamide adenine dinucleotide (NAD+) caps are one of the most common metabolites derived non-canonical caps reported on bacterial RNAs. Multiple decapping proteins are known to regulate the stability of NAD+ capped transcripts. However, no other proteins have been identified that preferentially interact with these NAD+ caps, and mechanistic details of the cap-dependent recognition remain poorly understood. Using an affinity capture approach, we identified multiple E. coli proteins that selectively recognize NAD+ caps, including the ATP-dependent RNA helicase, CsdA. CsdA preferentially interacts directly with NAD+ capped RNAs and can discriminate between 5' NAD+ capped and 5' triphosphate end transcripts. Binding to NAD+ capped RNA versus 5' triphosphate RNA more greatly enhances the ATPase activity of CsdA and the presence of NAD+ caps on transcripts modulates the ability of CsdA to form RNA condensates. Furthermore, we find that CsdA enhances the decapping activity of the NADH hydrolase NudC, suggesting CsdA plays a role in regulating the degradation of NAD+ capped transcripts. CsdA is the first identified NAD+ cap reader protein and its preference for binding NAD+ capped RNA provides a mechanism by which E. coli cells link RNA stability to the identity of the 5' cap. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/730015v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@115a057org.highwire.dtl.DTLVardef@d1c2c6org.highwire.dtl.DTLVardef@14a6d2dorg.highwire.dtl.DTLVardef@145ccff_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Systematic Discovery of Novel Phosphoinositide-Binding Effectors in Legionella Reveals Conserved α-Helical Folds

Intracellular bacterial pathogens deploy effector proteins to hijack host membranes by targeting specific lipids. Phosphoinositides are key eukaryotic signaling molecules that govern membrane identity and dynamics, making them powerful levers for microbial interference. However, the bacterial repertoire of phosphoinositide-binding effectors remains largely undefined. In this study, we systematically identified such proteins using an expression library of 241 Legionella pneumophila effectors. We applied a combination of lipid bead pulldowns, mass spectrometry, colocalization with phosphoinositide biosensors, and protein-lipid overlay assays, leading to the validation of 18 new phosphoinositide-binding effectors. Structural predictions revealed that these proteins share compact alpha-helical folds distinct from canonical eukaryotic lipid-binding domains. Guided by this structural signature, we performed a computational screen across L. pneumophila, Coxiella burnetii, and Burkholderia pseudomallei. This analysis identified 15 additional effectors, which were experimentally validated as phosphoinositide binders. Altogether, we report 33 previously unrecognized bacterial phosphoinositide-binding effectors. While their lipid-binding profiles were diverse, many effectors showed a preference for compartments enriched in phosphatidylinositol 3-phosphate. These findings expand the known diversity of lipid-binding effectors produced by intracellular pathogens, identify new structural modules bacteria use for phosphoinositide recognition, and broaden our understanding of how pathogens exploit phosphoinositide signaling to manipulate host membranes. ImportanceIntracellular bacterial pathogens hijack host membrane trafficking by targeting phosphoinositides (PIPs), essential eukaryotic signaling lipids. Despite their importance in virulence, bacterial PIP-binding effectors remain largely uncharted because they lack recognizable sequence motifs. Using Legionella pneumophila as a model system, we systematically identified 30 novel PIP-binding effectors, doubling the known repertoire, and discovered that they employ compact -helical folds structurally distinct from canonical eukaryotic PIP-binding domains. Remarkably, these -helical modules are conserved in Coxiella burnetii and Burkholderia pseudomallei, revealing a convergent evolutionary strategy for PIP recognition across phylogenetically diverse pathogens. This work expands our understanding of bacterial lipid-targeting mechanisms and provides a structural blueprint for identifying virulence factors in emerging pathogens.

microbiology↗

bioRxiv2024

ClpS directs degradation of primary N-end rule substrates in Mycolicibacterium smegmatis

Drug-resistant tuberculosis infections are a major threat to global public health. The essential mycobacterial ClpC1P1P2 protease has received attention as a prospective target for novel antibacterial therapeutics. However, efforts to probe its function in cells are constrained by our limited knowledge of its physiological proteolytic repertoire. Here, we interrogate the role of mycobacterial ClpS in directing N-end rule proteolysis by ClpC1P1P2 in Mycolicibacterium smegmatis. Binding assays demonstrate that mycobacterial ClpS binds canonical primary N-degrons (Leu, Phe, Tyr, Trp) with moderate affinity. N-degron binding restricts the conformational flexibility of a loop adjacent to the ClpS N-degron binding pocket and strengthens ClpS*ClpC1 binding affinity [~]30-fold, providing a mechanism for cells to prioritize N-end rule proteolysis when substrates are abundant. Proteolytic reporter assays in M. smegmatis confirm degradation of substrates bearing primary N-degrons, but suggest that secondary N-degrons are absence in mycobacteria. This work expands our understanding of the mycobacterial N-end rule pathway and identifies ClpS as a critical component for substrate specificity, providing insights that may support the development of improved Clp protease inhibitors.

biochemistry↗

bioRxiv2024

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.

biochemistry↗

bioRxiv2023

Bioinformatic identification of ClpI, a distinct class of Clp unfoldases in Actinomycetota

All clades of bacteria possess Hsp100/Clp family unfoldase enzymes that contribute to aspects of protein quality control. In Actinomycetota, these include ClpB, which functions as an independent chaperone and disaggregase, and ClpC, which cooperates with the ClpP1P2 peptidase to carry out regulated proteolysis of client proteins. We initially sought to algorithmically catalog Clp unfoldase orthologs from Actinomycetota into ClpB and ClpC categories. In the process, we uncovered a phylogenetically distinct third group of double-ringed Clp enzymes, which we term ClpI. ClpI enzymes are architecturally similar to ClpB and ClpC, with intact ATPase modules and motifs associated with substrate unfolding and translation. While ClpI possess an M-domain similar in length to that of ClpC, their N-terminal domain is more variable than the strongly conserved N-terminal domain of ClpC. Surprisingly, we identified separate sets of ClpI sequences that possess or lack the LGF-motifs required for stable assembly with ClpP1P2. In species where they occur, we suggest that ClpI enzymes provides additional pathways and points of regulatory control over protein quality control programs, supplementing the conserved roles of ClpB and ClpC.

bioinformatics↗

bioRxiv2022

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 mycobacterial drug targets. ClpC1, the unfoldase component of the essential ClpC1P1P2 protease, has emerged as one particularly promising antibacterial target. However, efforts to identify and characterize ClpC1-targeting compounds are constrained by our limited knowledge of Clp protease function and regulation. To expand our understanding of ClpC1 physiology, we employed a co-immunoprecipitation and mass spectrometry workflow to identify proteins that interact with ClpC1 in Mycolicibacterium smegmatis, a relative of M. tuberculosis. We identify a diverse panel of interaction partners, many of which make co-immunoprecipitate with both the regulatory N-terminal domain and the ATPase core of ClpC1. Notably, our interactome analysis identifies MSMEI_3879, a truncated gene product unique to M. smegmatis, as a novel proteolytic substrate. Degradation of MSMEI_3879 by ClpC1P1P2 in vitro requires an exposed N-terminal sequence, reinforcing the idea that ClpC1 selectively recognizes disordered motifs. 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.

biochemistry↗

bioRxiv2021

Identification of diverse targets of arginine phosphorylation in Mycolicibacterium smegmatis by shotgun proteomics

Tuberculosis is a leading cause of worldwide infectious mortality. The prevalence of multidrug-resistant Mycobacterium tuberculosis (Mtb) infections drives an urgent need to exploit new drug targets. One such target is the ATP-dependent protease ClpC1P1P2, which is strictly essential for viability. However, few proteolytic substrates of mycobacterial ClpC1P1P2 have been identified to date. Recent studies in Bacillus subtilis have shown that the orthologous ClpCP protease recognizes proteolytic substrates bearing post-translational arginine phosphorylation. While several lines of evidence suggest that ClpC1P1P2 is similarly capable of recognizing phosphoarginine-bearing proteins, the existence of phosphoarginine modifications in mycobacteria has remained in question. Here, we confirm the presence of post-translational phosphoarginine modifications in Mycolicibacterium smegmatis (Msm), a nonpathogenic surrogate of Mtb. Using a phosphopeptide enrichment workflow coupled with shotgun phosphoproteomics, we identify arginine phosphosites on several functionally diverse targets within the Msm proteome. Interestingly, phosphoarginine modifications are not upregulated by heat stress, suggesting divergent roles in mycobacteria and Bacillus. Our findings provide new evidence supporting the existence of phosphoarginine-mediated proteolysis by ClpC1P1P2 in mycobacteria and other actinobacterial species.

microbiology↗