Structure of SgK223 pseudokinase reveals novel mechanisms of homotypic and heterotypic association

Abstract

The mammalian pseudokinase SgK223, and its structurally related homologue SgK269, are oncogenic scaffolds that nucleate the assembly of specific signalling complexes and regulate tyrosine kinase signalling. Both SgK223 and SgK269 form homo- and hetero-oligomers, a mechanism that underpins a diversity of signalling outputs. However, mechanistic insights into SgK223 and SgK269 homo- and heterotypic association are lacking. Here we present the crystal structure of SgK223 pseudokinase domain and its adjacent N- and C-terminal helices. The structure reveals how the N- and C-regulatory helices engage in a novel fold to mediate the assembly of a high-affinity dimer. In addition, we identified regulatory interfaces on the pseudokinase domain required for the self-assembly of large open-ended oligomers. This study highlights the diversity in how the kinase fold mediates non-catalytic functions and provides mechanistic insights into how the assembly of these two oncogenic scaffolds is achieved in order to regulate signalling output.

Emerging concepts in pseudoenzyme classification, evolution, and signaling

“…Pseudokinase domains of guanylyl cyclase-A and guanylyl cyclase-B regulate activity of the tandem guanylyl cyclase domains (81) Molecular switch Phosphorylation of the MLKL pseudokinase domain triggers exposure of the executioner four-helix bundle domain and cell death (47,72) Protein interaction domain MLKL pseudokinase domain is regulated by binding to the RIPK3 kinase domain and HSP90:Cdc37 co-chaperones (73,82) Scaffold for assembly of signaling complexes TRIB pseudokinases nucleate assembly of a complex between a substrate (such as C/EBP) and the E3 ubiquitin ligase COP1, whose intrasubcellular localization is controlled by Tribbles 1 TRIB1 (74)(75)(76)83) (73,84) SgK223 (Pragmin)/SgK269 (PEAK1) forms higher-order signaling assemblies that include Src-family kinases (73,(84)(85)(86)(87)(88) Fundamental metabolic regulators of isoprenoid lipid production UbiB pseudoenzyme family adopts an (inactive?) atypical protein kinase-like fold found in bacteria, archaea, and eukaryotes; human mitochondrial ADCK3 pseudokinase binds nucleotides such as ADP but can be re-engineered into an ATP-dependent autophosphorylating enzyme; also relevant to the yeast Coq8p ATPase (69,89) Pseudo-histidine kinase Protein interaction domain The first of these mechanisms, for which an increasing number of examples are available in the protein kinase, phosphatase, and ubiquitin signaling literature, all retain clear "enzyme-like" overall folds.…”

Dimerization of the Pragmin Pseudo-Kinase Regulates Protein Tyrosine Phosphorylation

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Structure 26, 545-554.e1-e4; April 3, 2018) During the course of preparing our work for submission, two papers that are relevant to our research were published. Patel et al (2017) dealt with the crystal structure of SGK223, the human ortholog of Pragmin, and Ha and Boggon (2018) examined the crystal structure of SGK269. Both papers demonstrate similar dimerization interfaces and domain organizations as our own crystal structure.

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Nucleotide binding, evolutionary insights and interaction partners of the pseudokinase Unc-51-like kinase 4

“…Pseudokinases are present in all major groups of the human kinome and across diverse species (Kwon et al, 2019). The structural features of pseudokinases are accordingly diverse, sharing many regulatory mechanisms observed in canonical kinases (Ha and Boggon, 2018;Jura et al, 2009;Patel et al, 2017;Scheeff et al, 2009;Shrestha et al, 2020;Zeqiraj et al, 2009b).…”