The molecular basis of ubiquitin-like protein NEDD8 deamidation by the bacterial effector protein Cif

Abstract

The cycle inhibiting factors (Cifs) are a family of translocated effector proteins, found in diverse pathogenic bacteria, that interfere with the host cell cycle by catalyzing the deamidation of a specific glutamine residue (Gln40) in NEDD8 and the related protein ubiquitin. This modification prevents recycling of neddylated cullin-RING ligases, leading to stabilization of various cullin-RING ligase targets, and also prevents polyubiquitin chain formation. Here, we report the crystal structures of two Cif/NEDD8 complexes, revealing a conserved molecular interface that defines enzyme/substrate recognition. Mutation of residues forming the interface suggests that shape complementarity, rather than specific individual interactions, is a critical feature for complex formation. We show that Cifs from diverse bacteria bind NEDD8 in vitro and conclude that they will all interact with their substrates in the same way. The "occluding loop" in Cif gates access to Gln40 by forcing a conformational change in the C terminus of NEDD8. We used native PAGE to follow the activity of Cif from the human pathogen Yersinia pseudotuberculosis and selected variants, and the position of Gln40 in the active site has allowed us to propose a catalytic mechanism for these enzymes.bacterial pathogenesis | cyclomodulins | host cell manipulation | structural biology | type III secreted effector proteins

Structural and enzymatic characterization of a host-specificity determinant fromSalmonella

“…For example, E. coli-encoded Cif functions as a deamidase, using a Cys-His-Gln catalytic core to deamidate a critical glutamine in the ubiquitin-like NEDD8 protein (Hsu et al, 2008;Cui et al, 2010). Through binding to and deamidating NEDD8, Cif effectively prevents the E3 ligase activity of neddylated CRL complexes, resulting in cell-cycle arrest, the formation of stress fibers and host apoptosis (Cui et al, 2010;Merlet et al, 2009;Rabut & Peter, 2008;Saha & Deshaies, 2008;Crow et al, 2012;Taieb et al, 2011;Jubelin et al, 2010;Yao et al, 2012). Another effector, Yersinia pestis YopJ, employs its cysteine-dependent triad, His-Glu-Cys, to acetylate serine and threonine residues on MAPK kinases and the IB kinase complex, inhibiting both MAPK signaling and activation of the NFB pathway, respectively (Mukherjee et al, 2007;Ding et al, 1996).…”

What a Difference a Dalton Makes: Bacterial Virulence Factors Modulate Eukaryotic Host Cell Signaling Systems via Deamidation

“…Interestingly, it is the occluding loop in Cifs that appears to be responsible for forcing this reorientation during substrate binding. Displacement of the flexible C-terminal tail is likely important for substrate recognition and for orienting Gln40 in the Cif catalytic pocket (71,78). Consistent with this, deletion of the C-terminal domain of either ubiquitin or NEDD8 diminished deamidation by Cifs (76).…”

“…There is very little change in the structures of Cifs bound to their substrates compared to the uncomplexed states (71,76,78). However, a significant reorientation of the flexible C-terminal tail of NEDD8 and ubiquitin is observed upon binding by Cifs.…”

“…The crystal structures of CHYP, CHPL, and CHBP have been determined in complex with NEDD8 (71,78). Additionally, the cocrystal structure of CHBP and ubiquitin has also been determined (71).…”

“…CHBP has a preference for NEDD8 as a substrate (14), and CHYP deamidation of NEDD8 is 10-fold more efficient than deamidation of ubiquitin (78). Molecular dynamic studies revealed that this efficiency difference may be due to decreased flexibility of NEDD8 in complex with Cif compared to ubiquitin (71).…”