Biology subjects

Kloss, B.

Publications and source records attributed to Kloss, B..

7 recordsLinked to original sources

bioRxiv2025

Structure of an LGR dimer - an evolutionary predecessor of glycoprotein hormone receptors

The glycoprotein hormones of humans, produced in the pituitary and acting through receptors in the gonads to support reproduction and in the thyroid gland for metabolism, have co-evolved from invertebrate counterparts1,2. These hormones are heterodimeric cystine-knot proteins; and their receptors bind the cognate hormone at an extracellular domain and transmit the signal of this binding through a transmembrane domain that interacts with a heterotrimeric G protein. Structures determined for the human receptors as isolated for cryogenic electron microscopy (cryo-EM) are all monomeric3-6 despite compelling evidence for their functioning as dimers7-10. Here we describe the cryo-EM structure of the homologous receptor from a neuroendocrine pathway that promotes growth in a nematode11. This structure is an asymmetric dimer that can be activated by the hormone from that worm12, and it shares features especially like those of the thyroid stimulating hormone receptor (TSHR). When studied in the context of the human homologs, this dimer provides a structural explanation for the transactivation evident from functional complementation of binding-deficient and signaling-deficient receptors7, for the negative cooperativity in hormone action that is manifest in the 1:2 asymmetry of primary TSH:TSHR complexes8,9, and for switches in G-protein usage that occur as 2:2 complexes form9,10.

molecular biology↗

bioRxiv2024

Structural insights into terminal arabinosylation biosynthesis of the mycobacterial cell wall arabinan

The emergence of drug-resistant strains exacerbates the global challenge of tuberculosis caused by Mycobacterium tuberculosis (Mtb). Central to the pathogenicity of Mtb is its complex cell envelope, which serves as a barrier against both immune system and pharmacological attacks. Two key components of this envelope, arabinogalactan (AG) and lipoarabinomannan (LAM) are complex polysaccharides that contain integral arabinan domains important for cell wall structural and functional integrity. The arabinofuranosyltransferase AftB terminates the synthesis of these arabinan domains by catalyzing the addition of {beta}-(1[->]2)-linked terminal arabinofuranose residues. Here, we present the cryo-EM structures of Mycobacterium chubuense AftB in its apo and donor substrate analog-bound form, determined to 2.9 [A] and 3.4 [A] resolution, respectively. Our structures reveal that AftB has a GT-C fold transmembrane (TM) domain comprised of eleven TM helices and a periplasmic cap domain. AftB has an irregular tube-shaped cavity that bridges the two proposed substrate binding sites. By integrating structural analysis, biochemical assays, and molecular dynamics simulations, we elucidate the molecular basis of the reaction mechanism of AftB and propose a model for catalysis.

biochemistry↗

bioRxiv2024

Mechanistic studies of mycobacterial glycolipid biosynthesis by the mannosyltransferase PimE

Tuberculosis (TB), exceeded in mortality only by COVID-19 among global infectious diseases, is caused by Mycobacterium tuberculosis (Mtb). The pathogenicity of Mtb is largely attributed to its complex cell envelope, which includes a class of glycolipids called phosphatidyl-myo-inositol mannosides (PIMs), found uniquely in mycobacteria and its related corynebacterineae. These glycolipids maintain the integrity of the mycobacterial cell envelope, regulate its permeability, and mediate host-pathogen interactions. PIMs consist of a phosphatidyl-myo-inositol core decorated with one to six mannose residues and up to four acyl chains. The mannosyltransferase PimE catalyzes the transfer of the fifth PIM mannose residue from a polyprenyl phosphate-mannose (PPM) donor. This step in the biosynthesis of higher-order PIMs contributes to the proper assembly and function of the mycobacterial cell envelope; however, the structural basis for substrate recognition and the catalytic mechanism of PimE remain poorly understood. Here, we present the cryo-electron microscopy (cryo-EM) structures of PimE from Mycobacterium abscessus captured in its apo form and in a product-bound complex with the reaction product Ac1PIM5 and the by-product polyprenyl phosphate (PP), determined at 3.0 [A] and 3.5 [A], respectively. The structures reveal the active site within a distinctive binding cavity that accommodates both donor and acceptor substrates/products. Within the cavity, we identified residues involved in substrate coordination and catalysis, which we confirmed through in vitro enzymatic assays and further validated by in vivo complementation experiments. Molecular dynamics simulations were applied to identify the access pathways and the dynamics involved in substrate binding. Integrating structural, biochemical, genetic, and computational experiments, our study provides comprehensive insights into how PimE functions, opening potential avenues for development of novel anti-TB therapeutics.

biochemistry↗

bioRxiv2023

Structural and molecular basis of choline uptake into the brain by FLVCR2

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification, and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain has eluded the field for over fifty years. The MFS transporter FLVCR1 was recently determined to be a choline transporter, and while this protein is not highly expressed at the blood-brain barrier (BBB), its relative FLVCR2 is. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus, and embryonic lethality, but the physiological role of FLVCR2 is unknown. Here, we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in the inward- and outward-facing states using cryo-electron microscopy to 2.49 and 2.77 [A] resolution, respectively. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of neurotherapeutics into the brain.

biochemistry↗

bioRxiv2023

Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu

Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins. Here, we use native mass spectrometry (MS) in detergent micelles to uncover the patterns of oligomerization of the full-length SARS-CoV-2 envelope (E) protein, poliovirus VP4, and HIV Vpu. Our data suggest that the E protein is a specific dimer, VP4 is exclusively monomeric, and Vpu assembles into a polydisperse mixture of oligomers under these conditions. Overall, these results revealed the diversity in the oligomerization of viroporins, which has implications for mechanisms of their biological functions as well as their potential as therapeutic targets.

biophysics↗

bioRxiv2019

Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria

Mycobacterium tuberculosis causes tuberculosis, a disease that kills over one million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides - arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9 [A] resolution structure of M. abscessus AftD determined by single particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4 and 3.5 [A] structures of a mutant with impaired ACP binding reveal a conformational change that suggests the ACP may regulate AftD function. Using a conditional knock-out constructed in M. smegmatis, mutagenesis experiments confirm the essentiality of the putative active site and the ACP binding for AftD function.

biochemistry↗

bioRxiv2017

Structure-based analysis of CysZ-mediated cellular uptake of sulfate

Sulfur, most abundantly found in the environment as sulfate (SO42-), is an essential element in metabolites required by all living cells, including amino acids, co-factors and vitamins. Current understanding of the cellular delivery of SO42- at the molecular level is limited however. CysZ has been described as a SO42- permease, but its sequence family is without known structural precedent. Based on crystallographic structure information, SO42- binding and uptake experiments in cells and proteoliposomes, and single-channel conductance measurements, we provide insight into the molecular mechanism of CysZ-mediated translocation of SO42- across membranes. CysZ properties differ markedly from those of known transporters and ion channels. The structures display a hitherto unknown fold with dual topology, assembling in CysZ from Pseudomonas denitrificans as a trimer of antiparallel dimers in the membrane. CysZ structures from two other species recapitulate dimers from this assembly. Mutational studies highlight the functional relevance of conserved CysZ residues.

biophysics↗