Biochemical and Structural Characterization of Divergent Members of the Protein Kinase Family

The protein kinase family of enzymes catalyzes the transfer of the γ phosphate from an ATP molecule onto protein substrates. Using bioinformatic approaches, proteins with barely recognizable similarity to the known kinases can be found. Divergent catalytic motifs and sequence insertions allow such enzymes to catalyze unexpected reactions. In this work, I characterize two processes catalyzed by distant members of the protein kinase family: SidJ-catalyzed Glutamylation, and NiRAN-catalyzed RNA capping. A Legionella pneumophila effector SidJ adopts a kinase fold yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ubiquitin ligases. SidJ becomes activated upon translocation into the host cell and uses cellular ATP and glutamic acid to inactivate another bacterial effector family, the SidE all-in-one ubiquitin ligases. By determining cryo-EM structures of the SidJ-SidE reaction intermediates, I show that SidJ uses its kinase-like active site to adenylate an active site Glu in SidE, forming a stable acyl adenylate intermediate complex. Subsequently, a secondary active site formed by unique insertions in the kinase catalytic loop binds the reaction intermediate, and positions glutamate for peptide bond formation. A similar reaction is catalyzed by SdjA, a SidJ paralog in the L. pneumophila genome. I show that SdjA has distinct substrate specificity from that of SidJ and identify structural motifs responsible for the difference. SARS-CoV-2 is a positive-sense single-stranded RNA virus, the causative agent of the COVID 19 pandemic. The RNA genome of SARS-CoV-2 is protected by a 5′ cap structure, allowing the virus to avoid the cellular immune response, and enables translation of its genes. Although the enzymatic machinery decorating the cap with methyl groups was characterized, how the core cap structure is made was not known. I found, along with another student Gina Park, that the virus uses a kinase-like NiRAN domain attached to its RNA-dependent RNA polymerase (nsp12) to transfer its transcripts onto the N-terminus of the viral nsp9 protein in a RNAylation reaction. The NiRAN domain then transfers the RNA chain from the RNA-nsp9 species onto a GDP molecule, forming the core cap structure, GpppA. I characterized the reaction by performing cryo-EM analysis of the replication/transcription complex with nsp9 bound to the NiRAN active site.

Author dc:creator

• Osinski, Adam Krzysztof

Contributors dc:contributor

• Bai, Xiaochen
• Luo, Xuelian
• Henne, W. Mike
• Tagliabracci, Vincent S.

• Bacterial Proteins
• Protein Kinases
• SARS-CoV-2
• Viral Proteins
• Virus Replication

Language dc:language

en

Identifier

1482732354

OAI identifier oai:identifier

oai:utswmed-ir.tdl.org:2152.5/10443