Identification of the gate regions in the primary structure of the secretin pIV.
Secretins are a family of large bacterial outer membrane channels that serve as exit ports for folded proteins, filamentous phage and surface structures. Despite the large size of their substrates, secretins do not compromise the barrier function of the outer membrane, implying a gating mechanism. The region in the primary structure that forms the putative gate has not previously been determined for any secretin. To identify residues involved in gating the pIV secretin of filamentous bacteriophage f1, we used random mutagenesis of the gene followed by positive selection for mutants with compromised barrier function ('leaky' mutants). We identified mutations in 34 residues, 30 of which were clustered into two regions located in the centre of the conserved C-terminal secretin family domain: GATE1 (that spanned 39 residues) and GATE2 (that spanned 14 residues). An internal deletion constructed in the GATE2 region resulted in a severely leaky phenotype. Three of the four remaining mutations are located in the region that encodes the N-terminal, periplasmic portion of pIV and could be involved in triggering gate opening. Two missense mutations in the 24-residue region that separates GATE1 and GATE2 were also constructed. These mutant proteins were unstable, defective in multimerization and non-functional.
Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.
Julian
J
Spagnuolo
Natacha
N
Opalka
Wesley X
WX
Wen
Dragana
D
Gagic
Elodie
E
Chabaud
Pierdomenico
P
Bellini
Matthew D
MD
Bennett
Gillian E
GE
Norris
Seth A
SA
Darst
Marjorie
M
Russel
Jasna
J
Rakonjac
D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D048168:Escherichia coli K12 / Q000821:virology; D017912:Inovirus / Q000201:enzymology* / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D012633:Secretin / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism*
D017361:Viral Nonstructural Proteins; C119520:pIV protein, Enterobacteria phage f1; D012633:Secretin