Biology subjects

Kruse, A. C.

Publications and source records attributed to Kruse, A. C..

6 recordsLinked to original sources

bioRxiv2026

Structural basis for tetraspanin-dependent surface export and adhesive function of integrin α3β1

Integrin 3{beta}1 (ITG3{beta}1) is a member of an integrin subfamily that binds to laminin proteins and promotes attachment of epithelial cells to the basement membrane. ITG3{beta}1 forms a complex with the tetraspanin CD151, and loss-of-function mutations in both ITG3 and CD151 cause epidermolysis bullosa, a severe skin blistering disease resulting from a defect in basement membrane attachment. Here, we report the cryoEM structure of an ITG3{beta}1 complex with CD151 and show that mutation of CD151 at the binding interface disrupts complex formation in cells. Strikingly, CRISPR-mediated knockout of CD151 leads to a variably penetrant ITG3{beta}1 surface export defect that is restored by re-expression of wild-type but not interface-mutated CD151. Together, these studies define the molecular basis for binding of CD151 to ITG3{beta}1, and show that CD151 promotes ITG3{beta}1 surface export, providing a biochemical explanation for the CD151 loss-of-function phenotype in epidermolysis bullosa.

biochemistry↗

bioRxiv2018

An activation pathway governs cell wall polymerization by a bacterial morphogenic machine

Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall (CW) assembly. In Escherichia coli, alterations in a CW synthase component of the system called PBP2 were identified that overcome other inactivating defects. Rod system activity was stimulated in the suppressors in vivo, and purified synthase complexes with these changes showed more robust CW synthesis in vitro. Polymerization of the actin-like MreB component of the Rod system was also found to be enhanced in cells with the activated synthase. The results suggest an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both CW glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient CW assembly. An analogous activation pathway involving similar enzymatic components is likely responsible for controlling CW synthesis by the division machinery.

microbiology↗

bioRxiv2018

Structural basis for sigma-1 receptor ligand recognition

The {sigma}1 receptor is a poorly understood integral membrane protein expressed in most cells and tissues in the human body. It has been shown to modulate the activity of other membrane proteins such as ion channels and G protein-coupled receptors1-4, and ligands targeting the {sigma}1 receptor are currently in clinical trials for treatment of Alzheimers disease5, ischemic stroke6, and neuropathic pain7. Despite its importance, relatively little is known regarding {sigma}1 receptor function at the molecular level. Here, we present crystal structures of the human {sigma}1 receptor bound to the classical antagonists haloperidol and NE-100, as well as the agonist (+)-pentazocine, at crystallographic resolutions of 3.1 [A], 2.9 [A], and 3.1 [A] respectively. These structures reveal a unique binding pose for the agonist. The structures and accompanying molecular dynamics (MD) simulations demonstrate that the agonist induces subtle structural rearrangements in the receptor. In addition, we show that ligand binding and dissociation from {sigma}1 is a multistep process, with extraordinarily slow kinetics limited by receptor conformational change. We use MD simulations to reconstruct a ligand binding pathway that requires two major conformational changes. Taken together, these data provide a framework for understanding the molecular basis for agonist action at {sigma}1.

pharmacology and toxicology↗

bioRxiv2018

Evidence for the coupling of substrate recognition with transporter opening in MOP-family flippases

Bacteria produce a variety of surface-exposed polysaccharides important for cell integrity, biofilm formation, and evasion of the host immune response. Synthesis of these polymers often involves the assembly of monomer oligosaccharide units on the lipid carrier undecaprenyl-phosphate at the inner face of the cytoplasmic membrane. For many polymers, including cell wall peptidoglycan, the lipid-linked precursors must be transported across the membrane by flippases to facilitate polymerization at the membrane surface. Flippase activity for this class of polysaccharides is most often attributed to MOP (Multidrug/Oligosaccharidyllipid/Polysaccharide) family proteins. Little is known about how this ubiquitous class of transporters identifies and translocates its cognate precursor over the many different types of lipid-linked oligosaccharides produced by a given bacterial cell. To investigate the specificity determinants of MOP proteins, we selected for variants of the WzxC flippase involved in Escherichia coli capsule (colanic acid) synthesis that can substitute for the essential MurJ MOP-family protein and promote transport of cell wall peptidoglycan precursors. Variants with substitutions predicted to destabilize the inward-open conformation of WzxC lost substrate specificity and supported both capsule and peptidoglycan synthesis. Our results thus suggest that specific substrate recognition by a MOP transporter normally destabilizes the inward-open state, promoting transition to the outward-open conformation and concomitant substrate translocation. Furthermore, the ability of WzxC variants to suppress MurJ inactivation provides strong support for the designation of MurJ as the flippase for peptidoglycan precursors, the identity of which has been controversial.\n\nSIGNIFICANCEFrom cell walls in bacteria to protein glycosylation in eukaryotes, surface exposed polysaccharides are built on polyprenol-phosphate lipid carriers. Monomer units are typically assembled at the cytoplasmic face of the membrane and require translocation to the cell surface for polymerization/assembly. MOP-family proteins are a major class of transporters associated with this flippase activity. Despite their ubiquity and importance for cell surface biology, little is known about their transport mechanism. Here, we investigated substrate recognition by MOP transporters in bacteria. We present evidence that transport proceeds via destabilization of the inward-open state of the transporter by specific substrate binding thereby promoting a transition to the outward-open state and substrate release on the opposite face of the membrane.

microbiology↗

bioRxiv2018

Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli

The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein prior to its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data are only available for MurJ from the extremophile Thermosipho africanus. Here, we report the crystal structure of substrate-free MurJ from the Gram-negative model organism Escherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability of E. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Finally, through the use of sequence co-evolution analysis we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.

biochemistry↗

bioRxiv2017

Platform for rapid nanobody discovery in vitro

Camelid single-domain antibody fragments (\"nanobodies\") provide the remarkable specificity of antibodies within a single immunoglobulin VHH domain. This unique feature enables applications ranging from their use as biochemical tools to therapeutic agents. Virtually all nanobodies reported to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we developed a fully in vitro platform for nanobody discovery based on yeast surface display of a synthetic nanobody scaffold. This platform provides a facile and cost-effective method for rapidly isolating nanobodies targeting a diverse range of antigens. We provide a blueprint for identifying nanobodies starting from both purified and non-purified antigens, and in addition, we demonstrate application of the platform to discover rare conformationally-selective nanobodies to a lipid flippase and a G protein-coupled receptor. To facilitate broad deployment of this platform, we have made the library and all associated protocols publicly available.

biochemistry↗