Mechanisms of Yersinia YopO kinase substrate specificity

Wei Lin Lee,Robert C Robinson,Sheena Wee,Kunchithapadam Swaminathan,Pavithra Singaravelu,Jonathan M Grimes,Jayantha Gunaratne,Khay Chun Ang,Bo Xue

doi:10.1038/srep39998

Abstract

Yersinia bacteria cause a range of human diseases, including yersiniosis, Far East scarlet-like fever and the plague. Yersiniae modulate and evade host immune defences through injection of Yersinia outer proteins (Yops) into phagocytic cells. One of the Yops, YopO (also known as YpkA) obstructs phagocytosis through disrupting actin filament regulation processes - inhibiting polymerization-promoting signaling through sequestration of Rac/Rho family GTPases and by using monomeric actin as bait to recruit and phosphorylate host actin-regulating proteins. Here we set out to identify mechanisms of specificity in protein phosphorylation by YopO that would clarify its effects on cytoskeleton disruption. We report the MgADP structure of Yersinia enterocolitica YopO in complex with actin, which reveals its active site architecture. Using a proteome-wide kinase-interacting substrate screening (KISS) method, we identified that YopO phosphorylates a wide range of actin-modulating proteins and located their phosphorylation sites by mass spectrometry. Using artificial substrates we clarified YopO’s substrate length requirements and its phosphorylation consensus sequence. These findings provide fresh insight into the mechanism of the YopO kinase and demonstrate that YopO executes a specific strategy targeting actin-modulating proteins, across multiple functionalities, to compete for control of their native phospho-signaling, thus hampering the cytoskeletal processes required for macrophage phagocytosis.

Highlights

Pathogenic Yersinia species give rise to a number of human diseases
We report the X-ray structure of MgADP YopO:actin complex, which reveals that YopO is most structurally similar to mammalian p21-activated kinases (PAKs)
MgADP binding to YopO stabilizes the active site of the kinase domain via interactions with the P-loop and its surrounding residues

Summary

Results

Exemplified by the low sequence identity, different hydrophobic residues line the adenine-binding pocket in comparison to PAK1 and MST3, while the overall active site architecture of the kinase domain of YopO is structurally conserved (Supplementary Fig. 2c). We performed disorder prediction (PONDR) for the YopO substrates and identified that the regions in which the phosphorylation sites are present are predicted to be disordered (Supplementary Fig. 8) This is consistent with structural models constructed between YopO, actin and actin-binding proteins[16], in that only substrate regions that can access the catalytic cleft flanked between the kinase domain and the GDI domain become phosphorylated. We hypothesize that the propensity for phosphorylation depends on the ability of the substrate to stretch from the actin-binding site to the catalytic cleft of the kinase As such we designed artificial substrates to determine the length requirement for presentation to the kinase domain (Fig. 5 and Supplementary Table 4). These patterns suggest that generally, YopO prefers basic or hydrophobic residues both up and downstream of the phosphorylation site

Discussion

Author Contributions

Additional Information