In silico investigation of molecular mechanisms underlying the function of NLuc and its variants

Bioluminescence, the emission of light by living organisms, results from chemiluminescent reactions facilitated by enzymes like luciferases. Among these, NanoLuc (NLuc) stands out due to its exceptional brightness, stability, and compact structure, making it a valuable tool in bioassays and imaging applications. NLuc is a 19.1 kDa monomeric enzyme derived from the deep-sea shrimp Oplophorus gracilirostris. Its structure comprises eleven antiparallel β-strands forming a β-barrel, capped by four α-helices. To enhance its versatility, NLuc has been engineered into split forms. Despite the advancements in split NLuc applications, several impediments exist that can be addressed to improve these systems. Recently the spectacular allosteric mechanisms of NLuc has been reported. Based on that, the enzyme exhibits homotropic negative allostery, where product binding to an allosteric site inhibits substrate binding at the catalytic site. Ongoing research into its structural dynamics and allosteric behaviors continues to expand its potential applications, while efforts to enhance the efficiency of its split forms aim to broaden its utility in complex biological assays. In this in silico assay, we performed dynamic simulations for both the various forms of apo-NLuc and the docking complex with the substrate and product. As a result, we clarify the sources of malfunctions in split NLuc and explore various aspects of split NLuc technologies. We also examine some hypotheses of NLuc mechanisms that display the complex behavior of this luciferase.