Structural Insights into the β‑Clasp and Dimerization Interface of Human Ribokinase

Alanine substitutions disrupting individual polar interactions were largely tolerated, preserving dimerization, near-wild-type activity, and thermal stability. However, disruption of the V122/V124-centered hydrophobic core reduced catalytic turnover with minimal effects on substrate affinity, indicating that the β-clasp primarily supports catalytic competence rather than substrate recognition. Progressive β3a deletions destabilized the dimer in a graded manner, yielding inactive, predominantly monomeric variants with noncooperative biphasic thermal unfolding transitions, although far-UV circular dichroism confirmed retention of the α/β fold.

Molecular dynamics simulations showed that disruptive variants increased β-clasp flexibility, weakened the Asp27-centered ribose-binding network, and reduced ribose retention. Together, these findings identify the β-clasp as a conformational hub coupling dimerization to lid positioning and productive phosphoryl transfer, establishing it as a potential allosteric target for regulation.