title: Structural and functional analysis of cyclophilin PpiB mutants supports an in vivo function not limited to prolyl isomerization activity.
abstract: Escherichia coli cyclophilin PpiB is a peptidyl-prolyl cis/trans isomerase (PPIase, EC: 5.2.1.8), involved in the negative modulation of various bacterial processes, such as swimming and swarming motility and biofilm formation ability. In this study, we show that PpiB possesses also a chaperone function as it can prevent the thermal denaturation of citrate synthase even with essentially eliminated PPIase activity. We demonstrate, using active site mutations, that the PPIase activity of PpiB is required in all processes, except for the negative effect on swimming, indicating a possible isomerase-independent function. Additionally, we show that the reduced PPIase activity of PpiB does not prevent the association with all prey proteins tested and that the PPIase active site is not involved necessarily in each association. We also used a random mutagenesis approach, to identify amino acid residues apart from the catalytic site, which are necessary for PpiB function. The combination of enzymatic studies concerning the PPIase and chaperone activities of each mutant protein, with structural analyses based on 3D models, provided further insights into the effects of the mutations on the function of PpiB and showed the importance of structural features in addition to the catalytic site, for its in vivo role.
authors.affiliation: Laboratory of General and Agricultural Microbiology, Faculty of Crop Science, Agricultural University of Athens, Iera Odos 75, Votanikos, Athens, 11855, Greece.
authors.forename: Aggeliki
authors.initials: A
authors.lastname: Skagia
authors.affiliation: Laboratory of General and Agricultural Microbiology, Faculty of Crop Science, Agricultural University of Athens, Iera Odos 75, Votanikos, Athens, 11855, Greece.
authors.forename: Eleni
authors.initials: E
authors.lastname: Vezyri
authors.affiliation: Laboratory of General and Agricultural Microbiology, Faculty of Crop Science, Agricultural University of Athens, Iera Odos 75, Votanikos, Athens, 11855, Greece.
authors.forename: Markezina
authors.initials: M
authors.lastname: Sigala
authors.affiliation: Laboratory of Physics, Department of Biotechnology, Agricultural University of Athens, Iera Odos 75, Votanikos, Athens, 11855, Greece.
authors.forename: Areti
authors.initials: A
authors.lastname: Kokkinou
authors.affiliation: Laboratory of Physics, Department of Biotechnology, Agricultural University of Athens, Iera Odos 75, Votanikos, Athens, 11855, Greece.
authors.forename: Michael
authors.initials: M
authors.lastname: Karpusas
authors.affiliation: Laboratory of General and Agricultural Microbiology, Faculty of Crop Science, Agricultural University of Athens, Iera Odos 75, Votanikos, Athens, 11855, Greece.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Venieraki
authors.affiliation: Laboratory of General and Agricultural Microbiology, Faculty of Crop Science, Agricultural University of Athens, Iera Odos 75, Votanikos, Athens, 11855, Greece.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Katinakis
authors.affiliation: Laboratory of General and Agricultural Microbiology, Faculty of Crop Science, Agricultural University of Athens, Iera Odos 75, Votanikos, Athens, 11855, Greece.
authors.forename: Maria
authors.initials: M
authors.lastname: Dimou
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D020134:Catalytic Domain; D021983:Cyclophilins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D018832:Molecular Chaperones / Q000235:genetics; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D013329:Structure-Activity Relationship
chemical_list: D018832:Molecular Chaperones; D050505:Mutant Proteins; C081786:cyclophilin B; D021983:Cyclophilins