title: Consequences of inducing intrinsic disorder in a high-affinity protein-protein interaction.
abstract: The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (K(d) ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost.
authors: †Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors: Grigorios
authors: G
authors: Papadakos
authors: ‡Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors: Amit
authors: A
authors: Sharma
authors: §School of Life Sciences, University of Lincoln, Brayford Pool, Lincoln LN6 7TS, United Kingdom.
authors: Lorna E
authors: LE
authors: Lancaster
authors: ∥Department of Biology, University of York, Heslington Road, York YO10 5DD, United Kingdom.
authors: Rebecca
authors: R
authors: Bowen
authors: †Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors: Renata
authors: R
authors: Kaminska
authors: ∥Department of Biology, University of York, Heslington Road, York YO10 5DD, United Kingdom.
authors: Andrew P
authors: AP
authors: Leech
authors: ⊥Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8AT, United Kingdom.
authors: Daniel
authors: D
authors: Walker
authors: †Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors: Christina
authors: C
authors: Redfield
authors: †Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors: Colin
authors: C
authors: Kleanthous
mesh_terms: D003087:Colicins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064267:Intrinsically Disordered Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D017434:Protein Structure, Tertiary; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D003087:Colicins; D064267:Intrinsically Disordered Proteins; D012269:Ribosomal Proteins; C488855:ceiC protein, E coli; D012260:Ribonucleases