Structural mechanism of ubiquitin and NEDD8 deamidation catalyzed by bacterial effectors that induce macrophage-specific apoptosis

Abstract

Targeting eukaryotic proteins for deamidation modification is increasingly appreciated as a general bacterial virulence mechanism. Here, we present an atomic view of how a bacterial deamidase effector, cycle-inhibiting factor homolog in Burkholderia pseudomallei (CHBP), recognizes its host targets, ubiquitin (Ub) and Ub-like neural precursor cell expressed, developmentally down-regulated 8 (NEDD8), and catalyzes site-specific deamidation. Crystal structures of CHBP-Ub/NEDD8 complexes show that Ub and NEDD8 are similarly cradled by a large cleft in CHBP with four contacting surfaces. The pattern of Ub/NEDD8 recognition by CHBP resembles that by the E1 activation enzyme, which critically involves the Lys-11 surface in Ub/NEDD8. Close examination of the papain-like catalytic center reveals structural determinants of CHBP being an obligate glutamine deamidase. Molecular-dynamics simulation identifies Gln-31/ Glu-31 of Ub/NEDD8 as one key determinant of CHBP substrate preference for NEDD8. Inspired by the idea of using the unique bacterial activity as a tool, we further discover that CHBP-catalyzed NEDD8 deamidation triggers macrophage-specific apoptosis, which predicts a previously unknown macrophage-specific proapoptotic signal that is negatively regulated by neddylation-mediated protein ubiquitination/degradation.Cullin neddylation | type III secretion system | enteropathogenic E. coli | transglutamination | MLN4924 P rotein deamidation refers to hydrolysis of the side-chain carboxamide of a glutamine or rarely an asparagine. The related transamidation involves sequential deamidation and incorporation of a lysine ε-amine, resulting in cross-linking of two polypeptides (1). Humans have eight structurally related transglutaminases (TG1-7 and factor XIII) that regulate many biological processes. TGs can catalyze deamidation in the absence of a proper amine. Other promiscuous or dedicated enzymes capable of performing deamidation are also present in humans (2).Deamidation is more widely used in microorganisms. In bacterial chemosensory system, CheB from Escherichia coli/Salmonella enterica (3) and CheD from most nonenteric chemotactic bacteria (4) catalyze site-specific deamidation of chemotaxis receptors. Many secreted bacterial toxins also have the deamidase activity. Cytotoxic necrotizing factor 1/2 (CNF1/2) from certain virulent E. coli strains deamidate Gln-63/61 in Rho GTPases, rendering constitutively active GTPases and altered actin cytoskeleton (5, 6). Pasteurella multocida toxin (PMT) activates heterotrimeric G proteins by deamidating a conserved Gln in Gα (7). Cycle-inhibiting factor (Cif) from enteropathogenic E. coli (EPEC) and Cif homolog in Burkholderia pseudomallei (CHBP), both delivered into host cells through the type III secretion system (TTSS), induce a cytopathic effect of cell cycle arrest and actin stress fiber formation on epithelial cells. The Cif/CHBP family deamidates a conserved Gln-40 in host ubiquitin (Ub) and Ub-like protein (UBL) neural precursor cell expressed, developmentally downregu...

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

“…Additionally, the cocrystal structure of CHBP and ubiquitin has also been determined (71). The structures of these complexes reveal an extensive binding interface between Cif proteins and their ubiquitin/NEDD8 substrate (Fig.…”

“…To date, recombinant Cif Ec , Cif Bp , and Cif Yp have all been shown to deamidate Gln40 of NEDD8 in vitro (14,71). Ectopic expression of the Gln40Glu NEDD8 variant in HeLa cells phenocopies either the ectopic expression of Cifs or the effects of Cif during bacterial infection, demonstrating that this activity is responsible for the Cif-mediated cytopathic phenotype.…”

“…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).…”

“…Cifs target residue Gln40 in NEDD8 and ubiquitin with exquisite selectivity (neither Gln39 nor Gln41 of NEDD8 is a target for deamidation) (14,71). To date, recombinant Cif Ec , Cif Bp , and Cif Yp have all been shown to deamidate Gln40 of NEDD8 in vitro (14,71).…”