2026-10-13T08:50:22Z https://api.figshare.com/v2/oai

oai:figshare.com:article/33972937 2026-09-23T11:40:00Z category_1 category_4 category_14 category_19 category_873 category_734 category_135 portal_63 item_type_2 month_year_09_2026

Structural Insights into the β‑Clasp and Dimerization Interface of Human Ribokinase Juliana C. Ferreira (25096993) Adrian J. Villanueva (25096996) Samar Fadl (22909779) Zhandos Shegenov (25096999) Zeynep Nur Cinviz (25097002) Ozge Sensoy (1959838) Wael M. Rabeh (1850482) Biophysics Biochemistry Molecular Biology Pharmacology Chemical Sciences not elsewhere classified Biological Sciences not elsewhere classified Computational Biology productive phosphoryl transfer predominantly monomeric variants potential allosteric target human ribokinase ribokinase catalysis remains unclear bond networkthat forms integrated functional analyses clasp using structure core dimer scaffold reduced ribose retention clasp dimerization motif human rk stability β ‑ clasp thermal stability functional role clasp flexibility preserving dimerization yielding inactive type activity surrounding network substrate recognition substrate affinity step essential rk ), quantitative contribution phosphofructokinase b nucleotide biosynthesis minimal effects lid positioning largely tolerated ischemic injury hydrophobic interactions guided mutagenesis graded manner findings identify energy metabolism dimerization interface dependent phosphorylation conserved feature cell proliferation binding network although far Ribokinase (RK), a member of the phosphofructokinase B (PfkB) family, catalyzes the ATP-dependent phosphorylation of d-ribose to ribose-5-phosphate, a step essential for nucleotide biosynthesis and pentose phosphate pathway flux. Because these pathways support redox balance, energy metabolism, and cell proliferation, RK is relevant to cancer and ischemic injury. A conserved feature of PfkB enzymes is the β-clasp dimerization motif, an interlocking β-strand interface between protomers. However, its quantitative contribution to human RK stability and catalysis remains unclear. Here, we define the structural and functional role of the RK β-clasp using structure-guided mutagenesis and integrated functional analyses. The β-clasp contains two interaction layers: (1) a β3a–β7 backbone hydrogen-bond networkthat forms the core dimer scaffold and (2) a surrounding network of polar and hydrophobic interactions that reinforce the interface. 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. 2026-09-23T00:00:00Z Dataset Media 10.1021/acs.biochem.6c00373.s002 https://figshare.com/articles/media/Structural_Insights_into_the_Clasp_and_Dimerization_Interface_of_Human_Ribokinase/33972937 CC BY-NC 4.0