TY - JOUR
T1 - Caveat mutator
T2 - Alanine substitutions for conserved amino acids in RNA ligase elicit unexpected rearrangements of the active site for lysine adenylylation
AU - Unciuleac, Mihaela Carmen
AU - Goldgur, Yehuda
AU - Shuman, Stewart
N1 - Publisher Copyright:
© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
PY - 2020/1/1
Y1 - 2020/1/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85085713986&partnerID=8YFLogxK
U2 - 10.1093/NAR/GKAA238
DO - 10.1093/NAR/GKAA238
M3 - Article
C2 - 32315072
AN - SCOPUS:85085713986
SN - 0305-1048
VL - 48
SP - 5603
EP - 5615
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 10
ER -