title: The periplasmic domains of Escherichia coli HflKC oligomerize through right-handed coiled-coil interactions.
abstract: The periplasmic domains of the Escherichia coli HflK and HflC were coexpressed and purified. The two polypeptides copurified in a 1:1 ratio, as determined by quantitative amino acid analysis. Circular dichroism studies showed the complex to have substantial helical/coiled-coil content that melted with midpoints in the range of 26-29 degrees C depending upon the concentration, implying a reversible oligomerization. The average molecular weight of the soluble HflKC determined by sedimentation equilibrium ultracentrifugation using a single-species model varied with rotor speed, providing further evidence of concentration-dependent oligomerization. The data were well-fit by models that specified a protomer to n-mer oligomerization, with the heterodimeric HflKC as the protomer and values of n between 7 and 10. Multiple-sequence alignments of both HflK and HflC revealed regions near the C termini to contain 11-residue hendecad repeats, indicative of right-handed coiled coils, with characteristic small residues in the a, f, h, and j positions. To test the importance of the small size of these positions, two residues in the HflC domain, Ala-262 in a f position and Gly-268 in an a position, were mutated to isoleucine. The HflKC:A262I mutant complex showed lower helicity than the wild type, and its melting was less concentration-dependent. During purification of HflKC:G268I, the mutated HflC subunit precipitated, leaving a preparation of the pure peripheral HflK domain. This polypeptide behaved as a monomer in sedimentation equilibrium experiments and showed low helicity, implying that the protein conformation is largely dependent upon heteromeric subunit interactions. These results demonstrate the importance of right-handed coiled-coil interactions in the oligomerization of HflKC, and a model entailing the formation of a right-handed helical barrel is proposed.
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
authors.forename: Lee-Ann K
authors.initials: LK
authors.lastname: Briere
authors.forename: Stanley D
authors.initials: SD
authors.lastname: Dunn
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D016415:Sequence Alignment; D014461:Ultracentrifugation
chemical_list: D029968:Escherichia coli Proteins; C512893:HflC protein, E coli; C512891:HflK protein, E coli; D010455:Peptides; D000409:Alanine; D005998:Glycine