Moorman, C. C. | bioRxiv Science

bioRxiv2026

Snapshots from the Catalytic Landscape of Chalcone Isomerase

Chalcone isomerase (CHI) catalyzes the cyclization of 3-ring scaffolds of flavonoids, a class of plant-based natural products important for nutrition and disease prevention. A persistent question has been whether the enzyme uses dynamics to facilitate conformational rearrangements of substrates within the active site. To help resolve this question, CHI was crystallized with phloretin, a flexible substrate analogue that cannot undergo cyclization. The crystal structure possesses eight protein molecules per asymmetric unit, revealing different active site conformations that accommodate different bound conformers of phloretin. Together, the structural snapshots depict a series of coordinated, dynamic chemical interactions that lower barriers to substrate rearrangements approaching bond formation. Differential scanning fluorimetry combined with mutational analysis and enzyme kinetics further confirm that phloretin binds to the enzyme active site and that it acts as a competitive inhibitor of CHI. Together these findings answer outstanding questions about the flexibility and dynamics of CHI catalysis, information that may be useful for future biosynthetic design and enzyme engineering goals. Overall, this work supports a catalytic model in which the CHI enzyme operates as a dynamic ensemble of structures necessary to facilitate catalytic substrate rearrangements.

biochemistry↗

bioRxiv2024

Structural and functional analysis of cancer-associated missense variants in the retinoblastoma protein (Rb) pocket domain

The retinoblastoma tumor suppressor (Rb) is a multifunctional protein that primarily regulates the cell cycle but also has roles in cellular differentiation, DNA damage response and apoptosis. The loss of Rb is a key event in the development or progression of many cancers. Essential functions of Rb occur through its pocket domain, which is necessary for regulating binding interactions with E2F transcription factors and transcription repressors that bind via an LxCxE motif. The pocket domain is the most highly-conserved region of the multidomain protein, as well as the most frequent site of mutations. To understand what effects cancer missense mutations have on Rbs pocket domain, we used fluorescence polarization and differential scanning fluorimetry to quantify changes, caused by 75 cancer-associated missense variants, to E2F transactivation domain (E2FTD) binding, LxCxE binding, and changes to the thermostability of the protein. We find that 43% of the missense variants we tested reduce Rb-E2FTD binding. Many of these variants are not located at the E2F binding site, yet they destabilize the fold of the protein and show temperature-sensitive binding effects. We also find that 21% of tested mutations reduce LxCxE binding, and several mutations selectively disrupt either E2FTD or LxCxE binding. Protein X-ray crystallography of four missense variants reveals how different mutations destabilize the protein fold and inhibit E2FTD or LxCxE binding. Taken together, this work provides the first understanding of the multiple ways through which stability, structure and function of Rbs pocket domain is altered by a large number of missense mutations seen in cancer.

biochemistry↗