title: Ion-specific modulation of protein interactions: anion-induced, reversible oligomerization of a fusion protein.
abstract: Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt-dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion-specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E. coli expressed, basic (pI = 8.8), homodimer (65.2 kDa), consisted of an IgG1-Fc with two, C-terminal peptide arms linked via penta-glycine linkers. Each peptide arm was composed of two, tandem, active sequences (SEYQGLPPQGWK) separated by a spacer (GSGSATGGSGGGASSGSGSATG). PbA was monomeric in 10 mM acetate, pH 5.0 but exhibited reversible self-association upon salt addition. The sedimentation coefficient (s(w)) and hydrodynamic diameter (D(H)) versus PbA concentration isotherms in the presence of 140 mM NaCl (A5N) displayed sharp increases in s(w) and D(H), reaching plateau values of 9 s and 16 nm by 10 mg/mL PbA. The D(H) and sedimentation equilibrium data in the plateau region (>12 mg/mL) indicated the oligomeric ensemble to be monodisperse (PdI = 0.05) with a z-average molecular weight (M(z)) of 433 kDa (stoichiometry = 7). There was no evidence of reversible self-association for an IgG1-Fc molecule in A5N by itself or in a mixture containing fluorescently labeled IgG1-Fc and PbA, indicative of PbA self-assembly being mediated through its peptide arms. Self-association increased with pH, NaCl concentration, and anion size (I(-) > Br(-) > Cl(-) > F(-)) but could be inhibited using soluble Trp-, Phe-, and Leu-amide salts (Trp > Phe > Leu). We propose that in the presence of salt (i) anion binding renders PbA self-association competent by neutralizing the peptidyl arginyl and lysyl amines, (ii) self-association occurs via aromatic and hydrophobic interactions between the ..xxCTRWPWMC..xxxCTRWPWMCxx.. motifs, and (iii) at >10 mg/mL, PbA predominantly exists as heptameric clusters.
authors.affiliation: Process and Product Development, Amgen Inc., Seattle, Washington 98119, USA. yating@gene.com
authors.forename: Yatin R
authors.initials: YR
authors.lastname: Gokarn
authors.forename: R Matthew
authors.initials: RM
authors.lastname: Fesinmeyer
authors.forename: Atul
authors.initials: A
authors.lastname: Saluja
authors.forename: Shawn
authors.initials: S
authors.lastname: Cao
authors.forename: Jane
authors.initials: J
authors.lastname: Dankberg
authors.forename: Andrew
authors.initials: A
authors.lastname: Goetze
authors.forename: Richard L
authors.initials: RL
authors.lastname: Remmele
authors.forename: Linda O
authors.initials: LO
authors.lastname: Narhi
authors.forename: David N
authors.initials: DN
authors.lastname: Brems
mesh_terms: D000838:Anions / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008027:Light; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D055503:Protein Multimerization / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012542:Scattering, Radiation; D014461:Ultracentrifugation
chemical_list: D000838:Anions; D015815:Cell Adhesion Molecules; D029968:Escherichia coli Proteins; C106607:FCGBP protein, human; D010455:Peptides; D011993:Recombinant Fusion Proteins