Interrogating the Essential Bacterial Cell Division Protein FtsQ with Fragments Using Target Immobilized NMR Screening (TINS).

The divisome is a large protein complex that regulates bacterial cell division and therefore represents an attractive target for novel antibacterial drugs. In this study, we report on the ligandability of FtsQ, which is considered a key component of the divisome. For this, the soluble periplasmic domain of Escherichia coli FtsQ was immobilized and used to screen a library of 1501 low molecular weight (< 300 Da), synthetic compounds for those that interact with the protein. A primary screen was performed using target immobilized NMR screening (TINS) and yielded 72 hits. Subsequently, these hits were validated in an orthogonal assay. At first, we aimed to do this using surface plasmon resonance (SPR), but the lack of positive control hampered optimization of the experiment. Alternatively, a two-dimensional heteronuclear single quantum coherence (HSQC) NMR spectrum of FtsQ was obtained and used to validate these hits by chemical shift perturbation (CSP) experiments. This resulted in the identification of three fragments with weak affinity for the periplasmic domain of FtsQ, arguing that the ligandability of FtsQ is low. While this indicates that developing high affinity ligands for FtsQ is far from straightforward, the identified hit fragments can help to further interrogate FtsQ interactions.

Structural Analysis of the Interaction between the Bacterial Cell Division Proteins FtsQ and FtsB.

Most bacteria and archaea use the tubulin homologue FtsZ as its central organizer of cell division. In Gram-negative Escherichia coli bacteria, FtsZ recruits cytosolic, transmembrane, periplasmic, and outer membrane proteins, assembling the divisome that facilitates bacterial cell division. One such divisome component, FtsQ, a bitopic membrane protein with a globular domain in the periplasm, has been shown to interact with many other divisome proteins. Despite its otherwise unknown function, it has been shown to be a major divisome interaction hub. Here, we investigated the interactions of FtsQ with FtsB and FtsL, two small bitopic membrane proteins that act immediately downstream of FtsQ. We show in biochemical assays that the periplasmic domains of E. coli FtsB and FtsL interact with FtsQ, but not with each other. Our crystal structure of FtsB bound to the ß domain of FtsQ shows that only residues 64 to 87 of FtsB interact with FtsQ. A synthetic peptide comprising those 24 FtsB residues recapitulates the FtsQ-FtsB interactions. Protein deletions and structure-guided mutant analyses validate the structure. Furthermore, the same structure-guided mutants show cell division defects in vivo that are consistent with our structure of the FtsQ-FtsB complex that shows their interactions as they occur during cell division. Our work provides intricate details of the interactions within the divisome and also provides a tantalizing view of a highly conserved protein interaction in the periplasm of bacteria that is an excellent target for cell division inhibitor searches.IMPORTANCE In most bacteria and archaea, filaments of FtsZ protein organize cell division. FtsZ forms a ring structure at the division site and starts the recruitment of 10 to 20 downstream proteins that together form a multiprotein complex termed the divisome. The divisome is thought to facilitate many of the steps required to make two cells out of one. FtsQ and FtsB are part of the divisome, with FtsQ being a central hub, interacting with most of the other divisome components. Here we show for the first time in detail how FtsQ interacts with its downstream partner FtsB and show that mutations that disturb the interface between the two proteins effectively inhibit cell division.

The Soluble Periplasmic Domains of Escherichia coli Cell Division Proteins FtsQ/FtsB/FtsL Form a Trimeric Complex with Submicromolar Affinity.

Cell division in Escherichia coli involves a set of essential proteins that assembles at midcell to form the so-called divisome. The divisome regulates the invagination of the inner membrane, cell wall synthesis, and inward growth of the outer membrane. One of the divisome proteins, FtsQ, plays a central but enigmatic role in cell division. This protein associates with FtsB and FtsL, which, like FtsQ, are bitopic inner membrane proteins with a large periplasmic domain (denoted FtsQp, FtsBp, and FtsLp) that is indispensable for the function of each protein. Considering the vital nature and accessible location of the FtsQBL complex, it is an attractive target for protein-protein interaction inhibitors intended to block bacterial cell division. In this study, we expressed FtsQp, FtsBp, and FtsLp individually and in combination. Upon co-expression, FtsQp was co-purified with FtsBp and FtsLp from E. coli extracts as a stable trimeric complex. FtsBp was also shown to interact with FtsQp in the absence of FtsLp albeit with lower affinity. Interactions were mapped at the C terminus of the respective domains by site-specific cross-linking. The binding affinity and 1:1:1 stoichiometry of the FtsQpBpLp complex and the FtsQpBp subcomplex were determined in complementary surface plasmon resonance, analytical ultracentrifugation, and native mass spectrometry experiments.

Fine-mapping the contact sites of the Escherichia coli cell division proteins FtsB and FtsL on the FtsQ protein.

Escherichia coli cell division is effected by a large assembly of proteins called the divisome, of which a subcomplex consisting of three bitopic inner membrane proteins, FtsQ, FtsB, and FtsL, is an essential part. These three proteins, hypothesized to link cytoplasmic to periplasmic events during cell division, contain large periplasmic domains that are of major importance for function and complex formation. The essential nature of this subcomplex, its low abundance, and its multiple interactions with key divisome components in the relatively accessible periplasm make it an attractive target for the development of protein-protein interaction inhibitors. Although the crystal structure of the periplasmic domain of FtsQ has been solved, the structure of the FtsQBL complex is unknown, with only very crude indications of the interactions in this complex. In this study, we used in vivo site-specific photo cross-linking to probe the surface of the FtsQ periplasmic domain for its interaction interfaces with FtsB and FtsL. An interaction hot spot for FtsB was identified around residue Ser-250 in the C-terminal region of FtsQ and a membrane-proximal interaction region for both proteins around residue Lys-59. Sequence alignment revealed a consensus motif overlapping with the C-terminal interaction hot spot, underlining the importance of this region in FtsQ. The identification of contact sites in the FtsQBL complex will guide future development of interaction inhibitors that block cell division.

Chemical subtleties in small-molecule modulation of peptide receptor function: the case of CXCR3 biaryl-type ligands.

The G protein-coupled chemokine receptor CXCR3 plays a role in numerous inflammatory events. The endogenous ligands for the chemokine receptors are peptides, but in this study we disclose small-molecule ligands that are able to activate CXCR3. A class of biaryl-type compounds that is assembled by convenient synthetic routes is described as a new class of CXCR3 agonists. Intriguingly, structure-activity relationship and structure-function relationship studies reveal that subtle chemical modifications on the outer aryl ring (e.g., either the size or position of a halogen atom) result in a full spectrum of agonist efficacies on CXCR3. Quantum mechanics calculations and nuclear Overhauser effect spectroscopy NMR studies suggest that the biaryl dihedral angle and the electronic nature of ortho-substituents play an important role in determining agonist efficacies. Compounds 38 (VUF11222) and 39 (VUF11418) are the first reported nonpeptidomimetic agonists on CXCR3, rendering them highly useful chemical tools for detailed assessment of CXCR3 activation as well as for studying downstream CXCR3 signaling.