Christian Seitz

Discovered in the 1920s, cytochrome bd is a terminal oxidase that has received renewed attention as a drug target since its atomic structure was first determined in 2016. Only found in prokaryotes, we study it here as a drug target for Mycobacterium tuberculosis (Mtb). Most previous drug discovery efforts toward cytochrome bd have involved analogues of the canonical substrate quinone, known as Aurachin D. Here, we report six new cytochrome bd inhibitor scaffolds determined from a computational screen and confirmed on target activity through in vitro testing. These scaffolds provide new avenues for lead optimization toward Mtb therapeutics.

Plants respond to increased CO₂ concentrations through stomatal closure, which can contribute to increased water use efficiency. Grasses display faster stomatal responses than eudicots due to dumbbell-shaped guard cells flanked by subsidiary cells working in opposition. However, forward genetic screening for stomatal CO₂ signal transduction mutants in grasses has yet to be reported. The grass model Brachypodium distachyon is closely related to agronomically important cereal crops, sharing largely collinear genomes. To gain insights into CO₂ control mechanisms of stomatal movements in grasses, we developed an unbiased forward genetic screen with an EMS-mutagenized Brachypodium distachyon M5 generation population using infrared imaging to identify plants with altered leaf temperatures at elevated CO₂. Among isolated mutants, a “chill1” mutant exhibited cooler leaf temperatures than wildtype Bd21-3 parent control plants after exposure to increased [CO₂]. chill1 plants showed strongly impaired high CO₂-induced stomatal closure despite retaining a robust abscisic acid-induced stomatal closing response. Through bulked segregant whole-genome-sequencing analyses followed by analyses of further backcrossed F4 generation plants and generation and characterization of sodium-azide and CRISPR-cas9 mutants, chill1 was mapped to a protein kinase, Mitogen-Activated Protein Kinase 5 (BdMPK5). The chill1 mutation impaired BdMPK5 protein-mediated CO₂/HCO₃^- sensing together with the High Temperature 1 (HT1) Raf-like kinase in vitro. Furthermore, AlphaFold2-directed structural modeling predicted that the identified BdMPK5-D90N chill1 mutant residue is located at the interface of BdMPK5 with the BdHT1 Raf-like kinase. BdMPK5 is a key signaling component that mediates CO₂-induced stomatal movements and is proposed to function as a component of the primary CO₂ sensor in grasses.

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following a peripheral nervous system injury. The low-density lipoprotein receptor-related protein-1 (LRP1) is significantly upregulated in SCs in response to acute injury, activating cJun and promoting SC survival. Matrix-metalloproteinase-9 (MMP-9) is an LRP1 ligand that binds LRP1 through its hemopexin domain (PEX) and activates SC survival signaling and migration. To identify novel peptide mimetics within the hemopexin domain of MMP-9, we examined the crystal structure of PEX, synthesized four peptides, and examined their potential to bind and activate LRP1. We demonstrate that a 22 amino acid peptide, peptide 2, was the only peptide that activated Akt and ERK1/2 signaling in SCs, similar to a glutathione s-transferase (GST)-fused holoprotein, GST-PEX. Intraneural injection of peptide 2, but not vehicle, into crush-injured sciatic nerves activated cJun greater than 2.5-fold in wild-type mice, supporting that peptide 2 can activate the SC repair signaling in vivo. Peptide 2 also bound to Fc-fusion proteins containing the ligand-binding motifs of LRP1, clusters of complement-like repeats (CCRII and CCRIV). Pulldown and computational studies of alanine mutants of peptide 2 showed that positively charged lysine and arginine amino acids within the peptide are critical for stability and binding to CCRII. Collectively, these studies demonstrate that a novel peptide derived from PEX can serve as an LRP1 agonist and possesses qualities previously associated with LRP1 binding and SC signaling in vitro and in vivo.

Discovered in the 1920s, cytochrome bd is a terminal oxidase that has received renewed attention as a drug target since its atomic structure was first solved in 2016. Only found in prokaryotes, we study it here as a drug target for Mycobacterium tuberculosis ( Mtb ). Most previous drug discovery efforts towards cytochrome bd have involved analogs of the canonical substrate quinone, known as Aurachin D. Here we report six new cytochrome bd inhibitor scaffolds determined from a computational screen totaling over one million molecules and confirmed on target activity through in vitro testing. These scaffolds provide new avenues for lead optimization towards Mtb therapeutics.

All protein simulations are conducted with varying degrees of simplification, oftentimes with unknown ramifications about how these simplifications affect the interpretability of the results. In this work, we investigated how protein glycosylation and lateral crowding effects modulate an array of properties characterizing the stability and dynamics of influenza neuraminidase. We constructed three systems: (1) glycosylated neuraminidase in a whole virion (i.e., crowded membrane) environment, (2) glycosylated neuraminidase in its own lipid bilayer, and (3) unglycosylated neuraminidase in its own lipid bilayer. We saw that glycans tend to stabilize the protein structure and reduce its conformational flexibility while restricting the solvent movement. Conversely, a crowded membrane environment encouraged exploration of the free energy landscape and a large-scale conformational change, while making the protein structure more compact. Understanding these effects informs what factors one must consider in attempting to recapture the desired level of physical accuracy.

All protein simulations are conducted with varying degrees of simplifications, oftentimes with unknown ramifications on how these simplifications affect the interpretability of the results. In this work we investigated how protein glycosylation and lateral crowding effects modulate an array of properties characterizing the stability and dynamics of influenza neuraminidase. We constructed three systems: 1) Glycosylated neuraminidase in a whole virion (i.e. crowded membrane) environment 2) Glycosylated neuraminidase in its own lipid bilayer 3) Unglycosylated neuraminidase in its own lipid bilayer. We saw that glycans tend to stabilize the protein structure and reduce its conformational flexibility while restricting solvent movement. Conversely, a crowded membrane environment encouraged exploration of the free energy landscape and a large scale conformational change while making the protein structure more compact. Understanding these effects informs what factors one must consider while attempting to recapture the desired level of physical accuracy. TOC figure: TOC. Schematic of the three systems used here. (A) 2009-H1N1-ungly is the unglycosylated, single protein NA system. (B) 2009-H1N1-gly is the glycosylated, single protein NA system. (C) 2009-H1N1-vir is the NA in the virion context.

The continuing rise in the atmospheric carbon dioxide (CO ₂ ) concentration causes stomatal closing, thus critically affecting transpirational water loss, photosynthesis, and plant growth. However, the primary CO ₂ sensor remains unknown. Here, we show that elevated CO ₂ triggers interaction of the MAP kinases MPK4/MPK12 with the HT1 protein kinase, thus inhibiting HT1 kinase activity. At low CO ₂ , HT1 phosphorylates and activates the downstream negatively regulating CBC1 kinase. Physiologically relevant HT1-mediated phosphorylation sites in CBC1 are identified. In a genetic screen, we identify dominant active HT1 mutants that cause insensitivity to elevated CO ₂ . Dominant HT1 mutants abrogate the CO ₂ /bicarbonate-induced MPK4/12-HT1 interaction and HT1 inhibition, which may be explained by a structural AlphaFold2- and Gaussian-accelerated dynamics-generated model. Unexpectedly, MAP kinase activity is not required for CO ₂ sensor function and CO ₂ -triggered HT1 inhibition and stomatal closing. The presented findings reveal that MPK4/12 and HT1 together constitute the long-sought primary stomatal CO ₂ /bicarbonate sensor upstream of the CBC1 kinase in plants.

Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction–modification and CRISPR–Cas systems¹. In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors^2–4. However, the principal composition and structure of this compartment remain unknown. Here we find that the bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.

Bacteria encode myriad defenses that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR/Cas systems. In response, one family of large bacteriophage employs a nucleus-like compartment to protect their replicating genomes by excluding host defense factors. However, the principle composition and structure of this compartment remain unknown. Here, we find that the bacteriophage nuclear shell assembles primarily from one protein, termed chimallin. Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin cooperatively self-assembles as a flexible sheet into closed micron-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.