Biology subjects

Clemons, W. M.

Publications and source records attributed to Clemons, W. M..

8 recordsLinked to original sources

bioRxiv2026

Rationally Engineered, Chemically Stable Tunicamycin Analogues Decouple DPAGT1 Inhibition from Non-Selective Toxicity

Tunicamycins are potent inhibitors of dolichyl-phosphate N-acetylglucosamine phosphotransferase (DPAGT1) but are unsuitable for therapeutic development due to non-selective cytotoxicity, acid-labile glycosidic linkages, and poor physicochemical properties. Although prior structural modifications reduced the promiscuous toxicity of tunicamycins, the intrinsic 11'-{beta}-1''- trehalose-type glycosidic linkage remains chemically unstable, limiting biological durability. Here, we report a rationally engineered scaffold-stabilization strategy in which the acid-labile linkage is replaced with a chemically robust cyclitol framework, enabling the concise synthesis of chemically stable and water-soluble tunicamycin analogues in only 12 synthetic steps. From this platform, TM-Cy-TBPA (4) was identified as a lead DPAGT1 inhibitor that potently suppresses the proliferation of breast cancer cells by inducing G2-phase arrest followed by apoptosis, while exhibiting minimal cytotoxicity toward nontransformed cells. The compound shows improved solubility, and favorable pharmacokinetic exposure. These results establish tunicamycin cyclitol analogues as a structurally distinct class of selective DPAGT1-targeted anticancer agents and demonstrate that stabilization of the glycosidic linkage is an effective strategy for enhancing pharmacological selectivity, improving in vivo performance, and simplifying the synthetic route.

biochemistry↗

bioRxiv2026

Structural basis of metalloid transport by the arsenite efflux pump ArsB

Bacteria resist toxic arsenite (AsIII) in their environments by actively pumping the metalloid out of the cell via efflux pumps such as ArsB. However, the mechanism of extrusion remains poorly understood, which hinders the development of engineered bioremediation strategies. We report high-resolution cryo-EM structures of ArsB from the arsenic-tolerant bacterium Leptospirillum ferriphilum. ArsB adopts an inverted two-fold repeat architecture resembling that of other ion transporter (IT) superfamily proteins. Structures determined in the presence of AsIII and antimonite (SbIII) reveal that the metalloid substrates interact with polar residues at the core of the transmembrane domain primarily via hydrogen bonding. Mutagenesis and in vivo functional assays support these interactions. Our ArsB structures represent an inward-facing conformation, where the metalloid-binding site is exposed to the cytoplasm, suitable for metalloid capture. Furthermore, we demonstrate that AsIII resistance conferred by ArsB varies with external pH, supporting that ArsB is a proton (H+)-coupled secondary transporter. Mutagenesis, in vivo functional assays, and pKa estimation imply that conserved aspartate residues near the metalloid-binding site likely mediate the H+-coupling mechanism. Our findings provide structural insights into metalloid recognition and H+/metalloid antiport in ArsB, laying a foundation for further elucidation of the molecular basis of toxic metalloid detoxification in bacteria.

biochemistry↗

bioRxiv2025

Nucleotide and metalloid-driven conformational changes in the arsenite efflux ATPase ArsA

A common mechanism of arsenic detoxification in bacteria is arsenite (AsIII) efflux facilitated by the ArsAB pump that couples metalloid transport to ATP hydrolysis. The cytoplasmic ATPase component, ArsA, binds and hydrolyzes ATP and facilitates the transfer of AsIII to the integral membrane transporter, ArsB. The underlying molecular mechanism of AsIII efflux by ArsAB remains unclear. ArsA is a member of the Intradimeric Walker A (IWA) family of ATPases that undergo dramatic nucleotide-dependent conformational changes to facilitate their respective biological functions. Similar conformational transitions in ArsA have been postulated to drive AsIII binding and transport via ArsB but have not been demonstrated. Here, we report multiple structures of ArsA determined by single-particle cryogenic electron microscopy in an open MgADP-bound state, open MgATP-bound state, and a distinct closed MgATP-bound state liganded to AsIII. Using X-ray absorption spectroscopy, we confirmed that AsIII coordinates three conserved cysteines at the metalloid-binding site of the closed state in a three-coordinate fashion. Coupled with biochemical characterization, our cryo-EM structures reveal key conformational changes in the ArsA catalytic cycle consistent with other members of the IWA family and provide the structural basis for allosteric activation of nucleotide hydrolysis by AsIII. This work enhances our understanding of how the ArsA catalytic cycle regulates metalloid efflux by ArsB.

biochemistry↗

bioRxiv2023

A feedback control mechanism governs the synthesis of lipid-linked precursors of the bacterial cell wall

Many bacterial surface glycans such as the peptidoglycan (PG) cell wall, O-antigens, and capsules are built from monomeric units linked to a polyprenyl lipid carrier. How this limiting lipid carrier is effectively distributed among competing pathways has remained unclear for some time. Here, we describe the isolation and characterization of hyperactive variants of Pseudomonas aeruginosa MraY, the essential and conserved enzyme catalyzing the formation of the first lipid-linked PG precursor called lipid I. These variants result in the elevated production of the final PG precursor lipid II in cells and are hyperactive in a purified system. Amino acid substitutions within the activated MraY variants unexpectedly map to a cavity on the extracellular side of the dimer interface, far from the active site. Our structural evidence and molecular dynamics simulations suggest that the cavity is a binding site for lipid II molecules that have been transported to the outer leaflet of the membrane. Overall, our results support a model in which excess externalized lipid II allosterically inhibits MraY, providing a feedback mechanism to prevent the sequestration of lipid carrier in the PG biogenesis pathway. MraY belongs to the broadly distributed polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase (PNPT) superfamily of enzymes. We therefore propose that similar feedback mechanisms may be widely employed to coordinate precursor supply with demand by polymerases, thereby optimizing the partitioning of lipid carriers between competing glycan biogenesis pathways.

microbiology↗

bioRxiv2020

Structural biologists, lets mind our colors

In structural biology, most figures of macromolecules are aimed at those well-versed in structure, requiring prior familiarity with scales and commonly used color schemes. Yet, as structural biology becomes democratized with the increasing pace of structure determination, the accessibility of structural data is paramount. Here, we identify three keys, and have written accompanying software plugins, for structural biologists to create figures truer to the hard-won data and clearer across different modes of color vision and to non-expert readers. O_LIUse perceptually uniform colormaps C_LIO_LIConsider readers with different modes of color vision C_LIO_LIBe explicit about scales and color usage C_LI

biochemistry↗

bioRxiv2020

The structure of the UDP-Glc/GlcNAc 4-epimerase from the human pathogen Campylobacter jejuni

Worldwide, the food-born pathogen Campylobacter jejuni is the leading bacterial source of human gastroenteritis. C. jejuni produces a variety of diverse cell-surface carbohydrates that are essential for pathogenicity. A critical component of these oligo- and polysaccharides is the sugar N-acetylgalactosamine (GalNAc). The sole source of this sugar is the epimerization of UDP-N-acetylglucosamine (GlcNAc), a reaction catalyzed by the enzyme UDP-GlcNAc 4-epimerase (Gne). This enzyme is unique among known bacterial epimerases in that it also catalyzes the equivalent reaction with the non-N-acetylated sugars. Understanding how CjGne catalyzes these various interconversions is critical to designing novel inhibitors of this enzyme. Here, to further the mechanistic understanding we present a 2.0[A] structure of CjGne with its NAD+ co-factor bound. Based on novel features found in the structure we perform a variety of biochemical studies to probe the mechanism and compare these results to another bifunctional epimerase, human GalE. We further show that ebselen, previously identified for inhibition of HsGalE, is active against CjGne, suggesting a route for antibiotic development.

microbiology↗

bioRxiv2019

The client-binding domain of the cochaperone SGTA/Sgt2 has a helical-hand structure that binds a short hydrophobic helix

The targeting and insertion of tail-anchored (TA) integral membrane proteins (IMP) into the correct membrane is critical for cellular homeostasis. The fungal protein Sgt2, and its human homolog SGTA, binds hydrophobic clients and is the entry point for targeting of ER-bound TA IMPs. Here we reveal molecular details that underlie the mechanism of Sgt2 binding to TA IMP clients. We establish that the Sgt2 C-terminal region is flexible but conserved and sufficient for client binding. A molecular model for this domain reveals a helical hand forming a hydrophobic groove, consistent with a higher affinity for TA IMP clients with hydrophobic faces and a minimal length of 11 residues. This work places Sgt2 into a broader family of TPR-containing co-chaperone proteins.

biochemistry↗

bioRxiv2017

Decoding sequence-level information to predict membrane protein expression

The expression of integral membrane proteins (IMPs) remains a major bottleneck in the characterization of this important protein class. IMP expression levels are currently unpredictable, which renders the pursuit of IMPs for structural and biophysical characterization challenging and inefficient. Experimental evidence demonstrates that changes within the nucleotide or amino-acid sequence for a given IMP can dramatically affect expression; yet these observations have not resulted in generalizable approaches to improved expression. Here, we develop a data-driven statistical predictor named IMProve, that, using only sequence information, increases the likelihood of selecting an IMP that expresses in E. coli. The IMProve model, trained on experimental data, combines a set of sequence-derived features resulting in an IMProve score, where higher values have a higher probability of success. The model is rigorously validated against a variety of independent datasets that contain a wide range of experimental outcomes from various IMP expression trials. The results demonstrate that use of the model can more than double the number of successfully expressed targets at any experimental scale. IMProve can immediately be used to identify favorable targets for characterization.

biophysics↗