Secondary-structure switch regulates the substrate binding of a YopJ family acetyltransferase

Abstract

The Yersinia outer protein J (YopJ) family effectors are widely deployed through the type III secretion system by both plant and animal pathogens. As non-canonical acetyltransferases, the enzymatic activities of YopJ family effectors are allosterically activated by the eukaryote-specific ligand inositol hexaphosphate (InsP6). However, the underpinning molecular mechanism remains undefined. Here we present the crystal structure of apo-PopP2, a YopJ family member secreted by the plant pathogen Ralstonia solanacearum. Structural comparison of apo-PopP2 with the InsP6-bound PopP2 reveals a substantial conformational readjustment centered in the substrate-binding site. Combining biochemical and computational analyses, we further identify a mechanism by which the association of InsP6 with PopP2 induces an α-helix-to-β-strand transition in the catalytic core, resulting in stabilization of the substrate recognition helix in the target protein binding site. Together, our study uncovers the molecular basis governing InsP6-mediated allosteric regulation of YopJ family acetyltransferases and further expands the paradigm of fold-switching proteins.

From prediction to function: Current practices and challenges towards the functional characterization of type III effectors

“…Eventually, the apo crystal structure of RipP2 or RipP2 in complex with IP 6 , acetyl-CoA, and the WRKY domain from RRS1-R was determined. This figure provides an overview of the results found by Deslandes et al (2003) , Tasset et al (2010) , Le Roux et al (2015) , Xiou et al (2015) , Zhang et al (2017) , Xia et al (2021) , and Huh (2022) .…”

“…In the case of RipP2, the homology with acetyltransferases and the link with the RRS1-R protein in Arabidopsis has led to the eventual uncovering of the trans- and autoacetylation function of RipP2 ( Figure 4 ; Deslandes et al, 2003 ; Tasset et al, 2010 ). This finding was mined for further identification of the RipP2 catalytic triad, for mapping the acetyl-receiving residues of host WRKY transcription factors, and elucidation of the apo crystal structure of RipP2 and RipP2 in complex with IP 6 , acetyl-CoA, and the WRKY domain from RRS1-R ( Le Roux et al, 2015 ; Xiou et al, 2015 ; Zhang et al, 2017 ; Xia et al, 2021 ). In turn, available structural data might be helpful for homology modelling and functional characterization of a different effector protein.…”

“…So, the structure of Pto DC3000 T3E AvrPto has been solved in solution by nucleic magnetic resonance (NMR) spectroscopy ( Lee et al, 2004 ) and the crystal structure of AvrB was determined by single wavelength anomalous dispersion (SAD; Wulf et al, 2004 ). For the RSSC, only the crystal structures of RipP2 (apo or in complex) have been described ( Xia et al, 2021 ). Complexed protein structures are also interesting for the characterization of the protein function.…”

Fluid protein fold space and its implications

“…[56] The C-terminal region of the apo protein assumes an α-helix that switches to a β-sheet upon binding inositol hexaphosphate (InsP6), [57] a small molecule host factor abundant in most eukaryotic cells, including plants. [56] Importantly, this InsP6-induced fold switch activates PopP2's acetyltransferase activity by relaying the InsP6-binding signal from a regulatory domain to PopP2's substrate-binding helix. [56] The energetics of single folders, equilibrium fold switchers, and triggered fold switchersdiffer from one another (Figure 2C [28] ).…”

“…[56] Importantly, this InsP6-induced fold switch activates PopP2's acetyltransferase activity by relaying the InsP6-binding signal from a regulatory domain to PopP2's substrate-binding helix. [56] The energetics of single folders, equilibrium fold switchers, and triggered fold switchersdiffer from one another (Figure 2C [28] ). The energy landscape of single-fold proteins has one deep energy well.…”