Structural elements required for deamidation of RhoA by cytotoxic necrotizing factor 1.
Cytotoxic necrotizing factor 1 (CNF1), a virulence factor expressed by pathogenic Escherichia coli, acts on Rho-GTPases and specifically deamidates a single glutamine residue (Gln-63 in RhoA) required for GTP hydrolysis. This modification constitutively activates the effector binding function of Rho-GTPases and eventually leads to their proteasome-mediated degradation. Previous structural investigation revealed that the CNF1 active site is located in a deep and narrow pocket and that the entrance to this pocket is formed by nine loop segments. We have examined the functional importance of five of these loops (2, 6, 7, 8, and 9) by deleting them individually. We find that deletion of proximally located loops 8 and 9 in the 32 kDa catalytic domain of CNF1 (CNF1-C) nearly or completely abolishes deamidation of RhoA in vitro, identifying a potential Rho-GTPase recognition site. Deletion of loop 7 causes protein folding errors, and deletion of loop 6 has a small effect on deamidation. In contrast, deletion of loop 2 is found to increase deamidation 5-7-fold, implying that this loop rearranges in binding RhoA. None of the loop deletions or wild-type CNF1-C is able to deamidate RhoA containing Asn-63 instead of Gln-63, suggesting that the fit between the toxin and its target is highly precise. In addition, we show that the specificity constant (k(cat)/K(m)) of CNF1-C for RhoA is 825 +/- 3 M(-1) s(-1). This modest value is consistent with the confining size of the active site pocket acting to exclude nonspecific targets but also limiting reactivity toward intended targets.
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0314, USA.
Lori
L
Buetow
Partho
P
Ghosh
D000577:Amides / Q000737:chemistry*; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D003603:Cytotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017384:Sequence Deletion; D013379:Substrate Specificity / Q000235:genetics; D013816:Thermodynamics; D020742:rhoA GTP-Binding Protein / Q000737:chemistry* / Q000378:metabolism
D000577:Amides; D001427:Bacterial Toxins; D003603:Cytotoxins; D029968:Escherichia coli Proteins; C060296:cytotoxic necrotizing factor type 1; D020742:rhoA GTP-Binding Protein