Biology subjects

Kakuta, Y.

Publications and source records attributed to Kakuta, Y..

4 recordsLinked to original sources

bioRxiv2026

Hydration Energetics Shape Antibody Discrimination between Sulfotyrosine and Phosphotyrosine

Chemically similar post-translational modifications can mediate distinct biological functions, but how proteins distinguish between them remains unclear. Sulfotyrosine (sTyr) and phosphotyrosine (pTyr) exemplify this problem because they have similar sizes, local geometries, and electrostatic properties but function in different biological contexts. Here, we used the monoclonal antibody PSG2, which recognizes sTyr independently of the surrounding peptide sequence, to examine how a protein distinguishes these modifications. The crystal structure of PSG2 bound to an sTyr-containing peptide revealed a deep electropositive pocket with no modeled water molecules in direct contact with the sulfate group. Gas-phase density functional theory calculations favored pTyr over sTyr, showing that direct protein-ligand interactions alone are insufficient to explain PSG2 selectivity. Explicit first-shell hydration calculations showed that pTyr has a larger desolvation penalty than sTyr, and accounting for this difference reversed the calculated energetic order. Isothermal titration calorimetry showed favorable enthalpic and entropic contributions to sTyr binding, whereas no detectable heat signal was observed for pTyr. These results show that PSG2 distinguishes sTyr from pTyr through the balance between direct protein-ligand interactions and ligand desolvation.

biophysics↗

bioRxiv2026

Epithelial Glutamate Retention Protects against Colitis

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract that encompasses ulcerative colitis and Crohns disease. Here we identify the cystine/glutamate antiporter xCT as being markedly upregulated in the inflamed intestinal epithelium of patients with IBD. To clarify its functional contribution to disease pathogenesis, we performed genetic loss-of-function study and found that inhibition of xCT confers robust protection against dextran sulfate sodium (DSS)-induced colitis in mice. Intestinal epithelial cell (IEC)-specific deletion of xCT markedly attenuated colitis severity, demonstrating that epithelial xCT upregulation acts as a disease-exacerbating factor in IBD. Mechanistically, xCT deficiency preserved intracellular glutamate levels and protein polyglutamylation, thereby maintaining epithelial barrier integrity and protecting IECs from inflammatory injury. Consistently, pharmacological inhibition of glutamine synthetase, which increases intracellular glutamate, exerted a potent anti-inflammatory effect on the DSS-induced colitis. These findings identify intracellular glutamate retention in IECs as a previously unrecognized mechanism of epithelial protection and highlight both inhibition of xCT-dependent glutamate efflux and suppression of glutamine synthetase as potential therapeutic strategies for IBD.

molecular biology↗

bioRxiv2025

Formation of a μ-oxo nucleophile enables efficient hydrolysis by a trinuclear metal center in Family II inorganic pyrophosphatase

Efficient catalysis by metalloproteins relies on precise spatial arrangement of metal ions and active-site residues. Family II inorganic pyrophosphatase (PPase) from Shewanella species features a trinuclear metal center and displays higher catalytic activity than binuclear counterparts. Here we elucidate its hydrolytic mechanism using X-ray crystal structure-based extended X-ray absorption fine structure (XCS-EXAFS), site-directed mutagenesis, and density functional theory (DFT) calculations. We identify a catalytic -oxo nucleophile, formed via proton transfer from a bridging -hydroxide to Asp14 and subsequent hydrogen-bond rearrangement to Asp72, as the key species in SN2-type hydrolysis. This conversion defines the rate-limiting step with an activation barrier of 15.5 kcal/mol. Molecular orbital analysis reveals that the trinuclear cluster promotes -oxo formation, aligns the nucleophile for attack, and stabilizes the transition state. The side-chain rotation of the conserved Asp14 is crucial for catalysis. Our results highlight how metalloenzymes exploit geometric and electronic tuning to achieve high reactivity through evolutionarily optimized architectures.

biophysics↗

bioRxiv2025

Crystal structure of a thermophilic family II inorganic pyrophosphatase enabling high-temperature adaptation in Thermodesulfobacterium commune

Inorganic pyrophosphatases (PPases) are crucial for energy metabolism and classified into families with distinct metal ion requirements and structural features. This study is the first to successfully express, purify, and structurally characterize a thermophilic family II PPase isolated from Thermodesulfobacterium commune (TcPPase). TcPPase, which is optimally activated by Co2+ and Mn2+, required both the N- and C-terminal domains for complete catalytic function. Comparative structural analyses of psychrophilic and mesophilic homologs revealed that the enhanced thermal stability of TcPPase was due to its strong hydrophobic interactions, high proline content, dense hydrogen bond network, and additional salt bridges. These findings reveal the molecular basis for the thermal adaptation of family II PPases, providing valuable insights for thermostable enzyme engineering for biotechnological applications.

biochemistry↗