Polentarutti, M. | Search bioRxiv

bioRxiv2025

Structural bases for Nuclear Factor 1-X activation and DNA recognition. Prototypic insight into the NFI transcription factor family

Nuclear Factor I (NFI) proteins were first identified in adenovirus DNA replication and later as regulators of gene transcription, stem cell proliferation, and differentiation. They play key roles in development, cancer and congenital disorders. Within the NFI family, NFI-X is critical for neural stem cell biology, hematopoiesis, muscle development, muscular dystrophies and oncogenesis. Here, we present the first structural characterization of the NFI transcription factor, NFI-X, both alone and bound to its consensus palindromic DNA site. Our analyses reveal a novel, MH1-like fold within NFI-X DNA-binding domain (DBD) and identify crucial structural determinants for activity, such as a Zn{superscript 2} binding site, dimeric assembly, activation mechanism and DNA-binding specificity. Given the >95% sequence identity within the NFI DBDs, our structural data are prototypic for the entire family; a NFI Rosetta Stone that allows decoding a wealth of biochemical and functional data and provides a precise target for drug design in a wider disease context.

biochemistry↗

bioRxiv2018

Photosynthetic CO2 assimilation: the last gap in the structural proteome is closed

In land plants and algae, the Calvin-Benson (CB) cycle takes place in the chloroplast, a specialized organelle in which photosynthesis occurs. Thioredoxins (TRXs) are small ubiquitous proteins, known to harmonize the two stages of photosynthesis through a thiol-based mechanism. Among the 11 enzymes of the CB cycle, the TRX target phosphoribulokinase (PRK) has yet to be characterized at the atomic scale. To accomplish this goal, we determined the crystal structures of PRK from two model species: the green alga Chlamydomonas reinhardtii (CrPRK) and the land plant Arabidopsis thaliana (AtPRK). PRK is an elongated homodimer characterized by a large central {beta}-sheet of 18 strands, extending between two catalytic sites positioned at its edges. The electrostatic surface potential of the catalytic cavity has both a positive region suitable for binding the phosphate groups of substrates and an exposed negative region to attract positively charged TRX-f. In the catalytic cavity, the regulatory cysteines are 13 [A] apart and connected by a flexible region exclusive to photosynthetic eukaryotes--the clamp loop--which is believed to be essential for oxidation-induced structural rearrangements. Structural comparisons with prokaryotic and evolutionarily older PRKs revealed that both AtPRK and CrPRK have a strongly reduced dimer interface and increased number of random coiled regions, suggesting that a general loss in structural rigidity correlates with gains in TRX sensitivity during the molecular evolution of PRKs in eukaryotes. Significance StatementIn chloroplasts, five enzymes of the Calvin-Benson (CB) cycle are regulated by thioredoxins (TRXs). These enzymes have all been structurally characterized with the notable exception of phosphoribulokinase (PRK). Here, we determined the crystal structure of chloroplast PRK from two model photosynthetic organisms. Regulatory cysteines appear distant from each other and are linked by a long loop that is present only in plant-type PRKs and allows disulfide bond formation and subsequent conformational rearrangements. Structural comparisons with ancient PRKs indicate that the presence of flexible regions close to regulatory cysteines is a unique feature that is shared by TRX-dependent CB cycle enzymes, suggesting that the evolution of the PRK structure has resulted in a global increase in protein flexibility for photosynthetic eukaryotes.

plant biology↗