Claire Stewart

All Publications

• Structural basis for selective thymidine binding by the Borrelia burgdorferi substrate-binding protein BmpA. The Journal of biological chemistry Liu, Q., Nun Ez, V. A., Fernandez, D., Stewart, C. J., Sharaf, N. G. 2026: 113206

  Abstract

  BmpA is a putative substrate-binding protein from Borrelia burgdorferi, the causative agent of Lyme disease, an organism with limited metabolic capacity that relies on salvage pathways rather than de novo nucleotide biosynthesis. Here, we determine the crystal structure of BmpA to a resolution of 2.6 Å, revealing a conserved substrate-binding protein fold with a deeply buried nucleoside-binding pocket. Using microscale thermophoresis, we show that BmpA binds thymidine with high affinity followed by cytidine and adenosine, whereas binding to ribose, guanosine, inosine, and uridine was not detected. Structure-guided mutagenesis further demonstrates that two conserved aromatic residues (Phe27 and Phe176) are essential for thymidine recognition, as alanine substitution at either position abolishes detectable binding. Additionally, a Foldseek-based structural homology search identified related proteins across diverse bacterial and archaeal species that share a conserved overall fold and binding-site architecture despite low sequence similarity, consistent with an evolutionarily conserved scaffold that can accommodate distinct nucleoside ligands. Together, our work illustrates how conserved binding protein architectures enable selective nucleoside acquisition and provides a foundation for understanding nutrient uptake strategies in organisms with reduced genomes.

  View details for DOI 10.1016/j.jbc.2026.113206

  View details for PubMedID 42208901

• Crowding beyond excluded volume: A tale of two dimers PROTEIN SCIENCE Olgenblum, G. I., Stewart, C. J., Redvanly, T. W., Young, O. M., Lauzier, F., Hazlett, S., Wang, S., Rockcliffe, D. A., Parnham, S., Pielak, G. J., Harries, D. 2025; 34 (4): e70062

  Abstract

  Protein-protein interactions are modulated by their environment. High macromolecular solute concentrations crowd proteins and shift equilibria between protein monomers and their assemblies. We aim to understand the mechanism of crowding by elucidating the molecular-level interactions that determine dimer stability. Using 19F-NMR spectroscopy, we studied the effects of various polyethylene glycols (PEGs) on the equilibrium thermodynamics of two protein complexes: a side-by-side and a domain-swap dimer. Analysis using our mean-field crowding model shows that, contrary to classic crowding theories, PEGs destabilize both dimers through enthalpic interactions between PEG and the monomers. The enthalpic destabilization becomes more dominant with increasing PEG concentration because the reduction in PEG mesh size with concentration diminishes the stabilizing effect of excluded volume interactions. Additionally, the partially folded domain-swap monomers fold in the presence of PEG, contributing to dimer stabilization at low PEG concentrations. Our results reveal that polymers crowd protein complexes through multiple conjoined mechanisms, impacting both their stability and oligomeric state.

  View details for DOI 10.1002/pro.70062

  View details for Web of Science ID 001445948400001

  View details for PubMedID 40095390

  View details for PubMedCentralID PMC11912439

• Expression, purification, and characterization of diacylated Lipo-YcjN from Escherichia coli. The Journal of biological chemistry Trevino, M. A., Amankwah, K. A., Fernandez, D., Weston, S. A., Stewart, C. J., Gallardo, J. M., Shahgholi, M., Sharaf, N. G. 2024: 107853

  Abstract

  YcjN is a putative substrate binding protein expressed from a cluster of genes involved in carbohydrate import and metabolism in Escherichia coli. Here, we determine the crystal structure of YcjN to a resolution of 1.95 A, revealing that its three-dimensional structure is similar to substrate binding proteins in subcluster D-I, which includes the well-characterized maltose binding protein (MBP). Furthermore, we found that recombinant overexpression of YcjN results in the formation of a lipidated form of YcjN that is posttranslationally diacylated at cysteine 21. Comparisons of size-exclusion chromatography profiles and dynamic light scattering measurements of lipidated and non-lipidated YcjN proteins suggest that lipidated YcjN aggregates in solution via its lipid moiety. Additionally, bioinformatic analysis indicates that YcjN-like proteins may exist in both Bacteria and Archaea, potentially in both lipidated and non-lipidated forms. Together, our results provide a better understanding of the aggregation properties of recombinantly expressed bacterial lipoproteins in solution and establish a foundation for future studies that aim to elucidate the role of these proteins in bacterial physiology.

  View details for DOI 10.1016/j.jbc.2024.107853

  View details for PubMedID 39362470