New insights into the molecular characteristics behind the function of Renilla luciferase.

Renilla Luciferase (RLuc) is a blue light emitter protein which can be applied as a valuable tool in medical diagnosis. But due to lack of the crystal structure of RLuc-ligand complex, the functional motions and catalytic mechanism of this enzyme remain largely unknown. In the present study, the active site properties and the ligand-receptor interactions of the native RLuc and its red-shifted light emitting variant (Super RLuc 8) were investigated using molecular docking approach, molecular dynamics (MD) analysis, and MM-PBSA method. The detailed analysis of the main clusters led to identifying a lid-like structure and its functional motions. Furthermore, an induced-fit mechanism is proposed where ligand-binding induces conformational changes of the active site. Our findings give an insight into the deeper understanding of RLuc conformational changes during binding steps and ligand-receptor pattern. Moreover, our work broaden our understanding of how active site geometry is adjusted to support the catalytic activity and red-shifted light emission in Super RLuc 8.

Molecular basis of thermostability enhancement of Renilla luciferase at higher temperatures by insertion of a disulfide bridge into the structure.

Renilla luciferase (RLuc), also known as Renilla-luciferin 2-monooxygenase, is a light producing enzyme used in many biotechnological applications such as bioreporters. However, its kinetics stability -especially at higher temperatures- is a limiting factor for developing thermostable bioreporters. The aim of this study was to improve the stability of super Renilla luciferase 8 (SRLuc 8) which is a red-emitter variety of RLuc at higher temperatures, by introduction of a disulfide bridge into its structure. In this study, the choice of the proper disulfide bond formation was based on computational methods and enzyme functionality (active site position) which is called geometric-functional method. N45 and A71 at the N-terminal of the enzyme were selected for directed evolution. The engineered luciferase was called C-SRLuc 8 and its activity and stability were assayed. The results indicated that the kinetic stability of C-SRLuc 8 increased significantly at 60°C to 70°C as compared to SRLuc 8; the residual activity of C-SRLuc 8 was approximately 20% after incubation at 65°C for 5min. Moreover, the enzyme activity decreased compared with SRLuc 8. The molecular basis of the structural changes was considered using molecular dynamics simulations and the results indicated that the N45C/A71C crosslink was involved in a hotspot foldon which seemed to be the rate-limiting step of conformational collapse at higher temperatures. The present study may provide an opportunity for the development of the next-generation of thermostable RLuc-based biosensors.

Poly(ADP-Ribose) Polymerase-1 Lacking Enzymatic Activity Is Not Compatible with Mouse Development.

Poly(ADP-ribose) polymerase-1 (PARP1) binds DNA lesions to catalyse poly(ADP-ribosyl)ation (PARylation) using NAD+ as a substrate. PARP1 plays multiple roles in cellular activities, including DNA repair, transcription, cell death, and chromatin remodelling. However, whether these functions are governed by the enzymatic activity or scaffolding function of PARP1 remains elusive. In this study, we inactivated in mice the enzymatic activity of PARP1 by truncating its C-terminus that is essential for ART catalysis (PARP1ΔC/ΔC, designated as PARP1-ΔC). The mutation caused embryonic lethality between embryonic day E8.5 and E13.5, in stark contrast to PARP1 complete knockout (PARP1-/-) mice, which are viable. Embryonic stem (ES) cell lines can be derived from PARP1ΔC/ΔC blastocysts, and these mutant ES cells can differentiate into all three germ layers, yet, with a high degree of cystic structures, indicating defects in epithelial cells. Intriguingly, PARP1-ΔC protein is expressed at very low levels compared to its full-length counterpart, suggesting a selective advantage for cell survival. Noticeably, PARP2 is particularly elevated and permanently present at the chromatin in PARP1-ΔC cells, indicating an engagement of PARP2 by non-enzymatic PARP1 protein at the chromatin. Surprisingly, the introduction of PARP1-ΔC mutation in adult mice did not impair their viability; yet, these mutant mice are hypersensitive to alkylating agents, similar to PARP1-/- mutant mice. Our study demonstrates that the catalytically inactive mutant of PARP1 causes the developmental block, plausibly involving PARP2 trapping.

The Enigmatic Function of PARP1: From PARylation Activity to PAR Readers.

Poly(ADP-ribosyl)ation (PARylation) is catalysed by poly(ADP-ribose) polymerases (PARPs, also known as ARTDs) and then rapidly removed by degrading enzymes. Poly(ADP-ribose) (PAR) is produced from PARylation and provides a delicate and spatiotemporal interaction scaffold for numerous target proteins. The PARylation system, consisting of PAR synthesizers and erasers and PAR itself and readers, plays diverse roles in the DNA damage response (DDR), DNA repair, transcription, replication, chromatin remodeling, metabolism, and cell death. Despite great efforts by scientists in biochemistry, cell and molecular biology, genetics, and pharmacology over the last five decades, the biology of PARPs and PARylation remains enigmatic. In this review, we summarize the current understanding of the biological function of PARP1 (ARTD1), the founding member of the PARP family, focusing on the inter-dependent or -independent nature of different functional domains of the PARP1 protein. We also discuss the readers of PAR, whose function may transduce signals and coordinate the cellular processes, which has recently emerged as a new research avenue for PARP biology. We aim to provide some perspective on how future research might disentangle the biology of PARylation by dissecting the structural and functional relationship of PARP1, a major effector of the PARPs family.