Molecular mechanisms of retinoid signalling and structural insights into a deep-sea bacterial disaggregase for novel therapeutic and biotechnological applications

Retinoids like all-trans retinoic acid (atRA) are the active metabolite forms of Vitamin A. Due to their highly diverse biological roles in humans, they have been implicated in a wide range of diseases including neurodegenerative disorders and some cancers. However, current retinoid-based treatments are broad-acting drugs, based on small modifications of atRA, and have adverse side eCects. A library of 25 novel synthetic retinoid analogues has been recently developed, with the potential for increased stability and specificity towards Cellular retinoic acid binding protein 2 (CRABP2), as well as the Retinoic acid receptors (RARs) and Retinoid X receptors (RXRs).

The relative binding affinities of these synthetic retinoids towards CRABP2 and the RARs were determined using a novel fluorescence competition assay. The resulting data demonstrate that the synthetic retinoids bind CRABP2, RAR⍺ and RARγ with nanomolar affinities, which revealed some of the key chemical functional groups that are responsible for these differences in binding affinity. Most of the synthetic retinoids exhibited dual affinity for RAR⍺ and RARγ, with limited selectivity between the RAR isotypes. The results also validated the use of the individual RAR isotypes as a good proxy for representing the true binding interactions of the synthetic retinoids with the biologically active RXR:RAR heterodimers. An orthogonal fluorescence-based assay was also developed to enable the high-throughput screening of synthetic retinoids in the future. In addition, four co-crystal structures of CRABP2 bound to the synthetic retinoids, MH21, DC526, DC527 and DC528, were solved using X-ray crystallography and reported to high resolution. These structures successfully validated the findings of the binding affinity assays and offered key insights into the similar binding modes of different, chemically distinct synthetic retinoids. These results have enabled a deeper understanding of the binding interactions between synthetic retinoids and CRABP2 or the RAR isotypes, which will be pivotal for the continued development of highly selective compounds as therapeutics for use in the treatment of a wide range of human diseases.

Molecular chaperones have been used extensively in biotechnology for soluble, high-yield protein expression. A novel ClpB disaggregase has been identified from the recently uncovered bacterial superphylum, Candidate Phyla Radiation (CPR). The first refined three-dimensional map of CPR-ClpB was reported to medium resolution, which showed a homo-hexamer in the classical two-tier, double-ring configuration with a central pore. These results lay the groundwork for future structural and functional studies of this novel disaggregase, with particular focus on its development for new biotechnological applications.