6VTE

Naegleria gruberi RNA Ligase K170M mutant with AMP and Mn

Summary for 6VTE

• Entry DOI: 10.2210/pdb6vte/pdb
• Descriptor: RNA ligase, ADENOSINE MONOPHOSPHATE, MANGANESE (II) ION, ... (4 entities in total)
• Functional Keywords: rna repair, adenylyltransferase, ligase
• Biological source: Naegleria gruberi (Amoeba)
• Total number of polymer chains: 1
• Total formula weight: 39066.01

Authors

Unciuleac, M.C.,Goldgur, Y.,Shuman, S. (deposition date: 2020-02-12, release date: 2020-04-08, Last modification date: 2023-10-11)

Primary citation

Unciuleac, M.C.,Goldgur, Y.,Shuman, S.
Caveat mutator: alanine substitutions for conserved amino acids in RNA ligase elicit unexpected rearrangements of the active site for lysine adenylylation.
Nucleic Acids Res., 48:5603-5615, 2020

PubMed Abstract: Naegleria gruberi RNA ligase (NgrRnl) exemplifies the Rnl5 family of adenosine triphosphate (ATP)-dependent polynucleotide ligases that seal 3'-OH RNA strands in the context of 3'-OH/5'-PO4 nicked duplexes. Like all classic ligases, NgrRnl forms a covalent lysyl-AMP intermediate. A two-metal mechanism of lysine adenylylation was established via a crystal structure of the NgrRnl•ATP•(Mn2+)2 Michaelis complex. Here we conducted an alanine scan of active site constituents that engage the ATP phosphates and the metal cofactors. We then determined crystal structures of ligase-defective NgrRnl-Ala mutants in complexes with ATP/Mn2+. The unexpected findings were that mutations K170A, E227A, K326A and R149A (none of which impacted overall enzyme structure) triggered adverse secondary changes in the active site entailing dislocations of the ATP phosphates, altered contacts to ATP, and variations in the numbers and positions of the metal ions that perverted the active sites into off-pathway states incompatible with lysine adenylylation. Each alanine mutation elicited a distinctive off-pathway distortion of the ligase active site. Our results illuminate a surprising plasticity of the ligase active site in its interactions with ATP and metals. More broadly, they underscore a valuable caveat when interpreting mutational data in the course of enzyme structure-function studies.

PubMed: 32315072
DOI: 10.1093/nar/gkaa238

Experimental method

X-RAY DIFFRACTION (2.1 Å)