The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions.
FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861-919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions.



FimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.
Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Diseases Research, McMaster University, Hamilton, Ontario, Canada.
Ryan N C
RN
Buensuceso
Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Diseases Research, McMaster University, Hamilton, Ontario, Canada.
Ylan
Y
Nguyen
Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Diseases Research, McMaster University, Hamilton, Ontario, Canada Department of Biochemistry, Western University, London, Ontario, Canada.
Kun
K
Zhang
Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Diseases Research, McMaster University, Hamilton, Ontario, Canada.
Martin
M
Daniel-Ivad
Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Diseases Research, McMaster University, Hamilton, Ontario, Canada.
Seiji N
SN
Sugiman-Marangos
Computer Science and Mathematics Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA, and Department of Microbiology, University of Tennessee, Knoxville, Tennessee, USA.
Aaron D
AD
Fleetwood
Computer Science and Mathematics Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA, and Department of Microbiology, University of Tennessee, Knoxville, Tennessee, USA.
Igor B
0000-0002-6708-5323
IB
Zhulin
Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Diseases Research, McMaster University, Hamilton, Ontario, Canada Department of Biochemistry, Western University, London, Ontario, Canada.
Murray S
MS
Junop
Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada howell@sickkids.ca burrowl@mcmaster.ca.
P Lynne
PL
Howell
Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Diseases Research, McMaster University, Hamilton, Ontario, Canada howell@sickkids.ca burrowl@mcmaster.ca.
Lori L
0000-0003-0838-5040
LL
Burrows
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence / Q000502:physiology*; D018360:Crystallography, X-Ray; D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008958:Models, Molecular; D010802:Phylogeny; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D000069178:Type II Secretion Systems
D001426:Bacterial Proteins; D000069178:Type II Secretion Systems; D000242:Cyclic AMP