Guseman, A. J. | Search bioRxiv

bioRxiv2025

Tunable multivalent Fe(II)-based glycoassemblies as mimetics for native high-mannose glycans.

High Mannose Glycans (HMGs) play key roles in eukaryotic biology, regulating processes ranging from protein folding to host pathogen defense. Lectins have evolved to interact with these glycans through multivalent interactions facilitated by the multiple sugars displayed on glycans and via multiple binding sites on each lectin. Using Fe(II) iminopyridine complexes, we generated chemically defined multivalent glycan displays where the valency, arm length, and spatial display of mannose residues can be controlled via subcomponent synthesis. Due to its sensitivity towards the geometric display of mannose residues, monomeric Griffithsin (mGRFT) was utilized as a model lectin. Interactions between the Fe(II) glycan assemblies and mGRFT were characterized using biolayer interferometry (BLI), isothermal titration calorimetry (ITC), and NMR spectroscopy. Our results display a >1000-fold range in KD for Fe(II) iminopyridine complexes that can be tuned by factors such as saccharide tether length and number of sugars displayed. Through leveraging systematic molecular-level modifications, we demonstrate that tunable Fe(II) glycan assemblies can be used both as mimetics for high mannose glycans as well as competitive inhibitors for native glycan binding.

biophysics↗

bioRxiv2024

Cataract-prone variants of γD-crystallin populate a conformation with a partially unfolded N-terminal domain under native conditions

Human {gamma}D-crystallin, a monomeric protein abundant in the eye lens nucleus, must remain stably folded for an individuals entire lifetime to avoid aggregation and protein deposition-associated cataract formation. {gamma}D-crystallin contains two homologous domains, an N-terminal domain (NTD) and a C-terminal domain (CTD), which interact via a hydrophobic interface. A number of familial mutations in the gamma crystallin gene are linked to congenital early-onset cataract, most of which result in amino acid changes in the NTD. Several of these, such as V75D and W42R, are known to populate intermediates that, under partially denaturing conditions, possess a natively folded CTD and a completely unfolded NTD, with studies on W42R showing further evidence for a minor population of an intermediate under native conditions. We employed hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe the structural and energetic features of variants of {gamma}D-crystallin under both native and partially denaturing conditions. For V75D and W42R, we identify a species under native conditions that retains partial structure in the NTD and is structurally and energetically distinct from the intermediate populated under partially denaturing conditions. Residues at the NTD-CTD interface play crucial roles in stabilizing this intermediate, and disruption of interface contacts either by amino acid substitution or partial denaturation permits direct observation of two intermediates at the same time. The newly identified intermediate exposes hydrophobic amino acids that are buried in both the folded full-length protein and in the proteins stable isolated domains. Such non-native exposure of a hydrophobic patch may play an important role in cataract formation. Significance StatementHuman {gamma}D-crystallin, which plays a structural role in the eye lens, is a long-lived protein that must remain folded for an individuals entire lifetime to avoid aggregation and protein deposition - associated cataract formation. By using hydrogen-deuterium exchange mass spectrometry, we demonstrate that two cataract-associated variants of {gamma}D-crystallin populate an intermediate with partial structure along the interface between its two domains under native conditions. In these intermediates, hydrophobic amino acids that are normally buried in the N-terminal domains native folded structure become exposed, possibly leading to aggregation and cataract formation. Our findings illustrate the importance of studying a proteins energy landscapes under conditions that are close to physiological.

biophysics↗

bioRxiv2022

Targeting Spike Glycans to Inhibit SARS-CoV2 Viral Entry

SARS-CoV-2 Spike harbors glycans which function as ligands for lectins. Therefore, it should be possible to exploit lectins to target SARS-CoV-2 and inhibit cellular entry by binding glycans on the Spike protein. Burkholderia oklahomensis agglutinin (BOA) is an antiviral lectin that interacts with viral glycoproteins via N-linked high mannose glycans. Here, we show that BOA binds to the Spike protein and is a potent inhibitor of SARS-CoV-2 viral entry at nanomolar concentrations. Using a variety of biophysical tools, we demonstrate that the interaction is avidity driven and that BOA crosslinks the Spike protein into soluble aggregates. Furthermore, using virus neutralization assays, we demonstrate that BOA effectively inhibits all tested variants of concern as well as SARS-CoV 2003, establishing that glycan-targeting molecules have the potential to be pan-coronavirus inhibitors.

biochemistry↗