TY - JOUR
T1 - Structure of an essential Type IV pilus biogenesis protein provides insights into pilus and type II secretion systems
AU - Yamagata, Atsushi
AU - Milgotina, Ekaterina
AU - Scanlon, Karen
AU - Craig, Lisa
AU - Tainer, John A.
AU - Donnenberg, Michael S.
N1 - Funding Information:
We thank the staff of ALS beamline 5.0.1 for diffraction facilities. This work was supported by National Institutes of Health awards AI22160 (J.A.T.) and AI37606 (M.S.D). A.Y. was supported in part by a Skaggs Institute of Chemical Biology fellowship .
PY - 2012/5/25
Y1 - 2012/5/25
N2 - Type IV pili (T4Ps) are long cell surface filaments, essential for microcolony formation, tissue adherence, motility, transformation, and virulence by human pathogens. The enteropathogenic Escherichia coli bundle-forming pilus is a prototypic T4P assembled and powered by BfpD, a conserved GspE secretion superfamily ATPase held by inner-membrane proteins BfpC and BfpE, a GspF-family membrane protein. Although the T4P assembly machinery shares similarity with type II secretion (T2S) systems, the structural biochemistry of the T4P machine has been obscure. Here, we report the crystal structure of the two-domain BfpC cytoplasmic region (N-BfpC), responsible for binding to ATPase BfpD and membrane protein BfpE. The N-BfpC structure reveals a prominent central cleft between two α/β-domains. Despite negligible sequence similarity, N-BfpC resembles PilM, a cytoplasmic T4P biogenesis protein. Yet surprisingly, N-BfpC has far greater structural similarity to T2S component EpsL, with which it also shares virtually no sequence identity. The C-terminus of the cytoplasmic domain, which leads to the transmembrane segment not present in the crystal structure, exits N-BfpC at a positively charged surface that most likely interacts with the inner membrane, positioning its central cleft for interactions with other Bfp components. Point mutations in surface-exposed N-BfpC residues predicted to be critical for interactions among BfpC, BfpE, and BfpD disrupt pilus biogenesis without precluding interactions with BfpE and BfpD and without affecting BfpD ATPase activity. These results illuminate the relationships between T4P biogenesis and T2S systems, imply that subtle changes in component residue interactions can have profound effects on function and pathogenesis, and suggest that T4P systems may be disrupted by inhibitors that do not preclude component assembly.
AB - Type IV pili (T4Ps) are long cell surface filaments, essential for microcolony formation, tissue adherence, motility, transformation, and virulence by human pathogens. The enteropathogenic Escherichia coli bundle-forming pilus is a prototypic T4P assembled and powered by BfpD, a conserved GspE secretion superfamily ATPase held by inner-membrane proteins BfpC and BfpE, a GspF-family membrane protein. Although the T4P assembly machinery shares similarity with type II secretion (T2S) systems, the structural biochemistry of the T4P machine has been obscure. Here, we report the crystal structure of the two-domain BfpC cytoplasmic region (N-BfpC), responsible for binding to ATPase BfpD and membrane protein BfpE. The N-BfpC structure reveals a prominent central cleft between two α/β-domains. Despite negligible sequence similarity, N-BfpC resembles PilM, a cytoplasmic T4P biogenesis protein. Yet surprisingly, N-BfpC has far greater structural similarity to T2S component EpsL, with which it also shares virtually no sequence identity. The C-terminus of the cytoplasmic domain, which leads to the transmembrane segment not present in the crystal structure, exits N-BfpC at a positively charged surface that most likely interacts with the inner membrane, positioning its central cleft for interactions with other Bfp components. Point mutations in surface-exposed N-BfpC residues predicted to be critical for interactions among BfpC, BfpE, and BfpD disrupt pilus biogenesis without precluding interactions with BfpE and BfpD and without affecting BfpD ATPase activity. These results illuminate the relationships between T4P biogenesis and T2S systems, imply that subtle changes in component residue interactions can have profound effects on function and pathogenesis, and suggest that T4P systems may be disrupted by inhibitors that do not preclude component assembly.
KW - GspE ATPase
KW - crystal structure
KW - protein-protein interaction
KW - type II secretion
KW - type IV pili
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U2 - 10.1016/j.jmb.2012.02.041
DO - 10.1016/j.jmb.2012.02.041
M3 - Article
C2 - 22387466
AN - SCOPUS:84860303391
SN - 0022-2836
VL - 419
SP - 110
EP - 124
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1-2
ER -