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Published on: December 17, 2013
Cryo-EM Detection of AMPylated Histidine Implies Covalent Catalysis in AMPylation Mediated by a Bacterial Effector
Zhengrui Zhang1, Rishi Patel1, Zhao-Qing Luo2
1Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
Abstract:
AMPylation is a post-translational modification (PTM) whereby adenosine monophosphate (AMP) from adenosine triphosphate (ATP) is transferred onto protein hydroxyl groups of serine, threonine, or tyrosine. Recently, an actin-dependent AMPylase namely LnaB from the bacterial pathogen Legionella pneumophila was found to AMPylate phosphate groups of phosphoribosylated ubiquitin and Src family kinases. LnaB represents an evolutionarily distinct family of AMPylases with conserved active site Ser-His-Glu residues. Here, we capture the structure of the LnaB-actin complex in a putative intermediate state via single-particle cryogenic electron microscopy (cryo-EM) and find that the catalytic histidine of LnaB is covalently attached to AMP through a phosphoramidate linkage at the Nδ1 atom. This observation provides direct structural evidence of histidine AMPylation as a PTM and implies the possibility of covalent catalysis in LnaB-mediated AMPylation, a mechanism distinct from known AMPylases. Subsequent biochemical studies confirm the observed AMP binding site and provide additional insights into the catalytic properties of LnaB. Together, our work highlights the power of cryo-EM in capturing labile PTMs and transient species during enzymatic reactions, while opening new avenues of mechanistic investigation into the LnaB family.
Insights
This study reveals histidine AMPylation, a novel post-translational modification (PTM) in the bacterial enzyme LnaB. Cryo-EM and biochemical data uncover a unique covalent catalysis mechanism for AMPylation.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- AMPylation is a post-translational modification (PTM) involving the transfer of adenosine monophosphate (AMP) from adenosine triphosphate (ATP) to protein hydroxyl groups.
- LnaB, an actin-dependent AMPylase from Legionella pneumophila, represents a distinct family of AMPylases and has been shown to modify phosphoribosylated ubiquitin and Src family kinases.
Purpose of the Study:
- To elucidate the structure and catalytic mechanism of the LnaB-actin complex.
- To provide structural evidence for histidine AMPylation as a novel post-translational modification.
- To investigate the unique catalytic properties of the LnaB enzyme family.
Main Methods:
- Single-particle cryogenic electron microscopy (cryo-EM) to capture the LnaB-actin complex structure.
- Biochemical assays to confirm AMP binding and characterize catalytic properties.
Main Results:
- The cryo-EM structure revealed a putative intermediate state where the catalytic histidine of LnaB is covalently attached to AMP via a phosphoramidate linkage.
- This provides direct structural evidence for histidine AMPylation as a PTM.
- Biochemical studies confirmed the AMP binding site and LnaB's catalytic characteristics.
Conclusions:
- The study demonstrates histidine AMPylation as a novel PTM and suggests a distinct covalent catalytic mechanism for LnaB.
- Cryo-EM is a powerful tool for capturing transient states in enzymatic reactions.
- This work opens new avenues for mechanistic studies of the LnaB AMPylase family.
