title: Kinetics of insulin-like growth factor II (IGF-II) interaction with domain 11 of the human IGF-II/mannose 6-phosphate receptor: function of CD and AB loop solvent-exposed residues.
abstract: Ligands of the IGF-II/mannose 6-phosphate receptor (IGF2R) include IGF-II and mannose 6-phosphate modified proteins. Disruption of the negative regulatory effects of IGF2R on IGF-II-induced growth can lead to embryonic lethality and cancer promotion. Of the 15 IGF2R extracellular domains, domains 1-3 and 11 are known to have a conserved beta-barrel structure similar to that of avidin and the cation-dependent mannose 6-phosphate receptor, yet only domain 11 binds IGF-II with high specificity and affinity. In order to define the functional basis of this critical biological interaction, we performed alanine mutagenesis of structurally determined solvent-exposed loop residues of the IGF-II-binding site of human domain 11, expressed these mutant forms in Pichia pastoris, and determined binding kinetics with human IGF-II using isothermal calorimetry and surface plasmon resonance with transition state thermodynamics. Two hydrophobic residues in the CD loop (F1567 and I1572) were essential for binding, with a further non-hydrophobic residue (T1570) that slows the dissociation rate. Aside from alanine mutations of AB loop residues that decrease affinity by modifying dissociation rates (e.g. Y1542), a novel mutation (E1544A) of the AB loop enhanced affinity by threefold compared to wild-type. Conversion from an acidic to a basic residue at this site (E1544K) results in a sixfold enhancement of affinity via modification principally of the association rate, with enhanced salt-dependence, decreased entropic barrier and retained specificity. These data suggest that a functional hydrophobic binding site core is formed by I1572 and F1567 located in the CD loop, which initially anchors IGF-II. Within the AB loop, residues normally act to either stabilise or function as negative regulators of the interaction. These findings have implications for the molecular architecture and evolution of the domain 11 IGF-II-binding site, and the potential interactions with other domains of IGF2R.
authors.affiliation: Cancer Research UK Molecular Oncology and Growth Factor Research Group, Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK. o.j.zaccheo@bristol.ac.uk
authors.forename: Oliver J
authors.initials: OJ
authors.lastname: Zaccheo
authors.forename: Stuart N
authors.initials: SN
authors.lastname: Prince
authors.forename: David M
authors.initials: DM
authors.lastname: Miller
authors.forename: Christopher
authors.initials: C
authors.lastname: Williams
authors.forename: C Fred
authors.initials: CF
authors.lastname: Kemp
authors.forename: James
authors.initials: J
authors.lastname: Brown
authors.forename: E Yvonne
authors.initials: EY
authors.lastname: Jones
authors.forename: Lucy E
authors.initials: LE
authors.lastname: Catto
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Crump
authors.forename: A Bassim
authors.initials: AB
authors.lastname: Hassan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002151:Calorimetry; D002942:Circular Dichroism; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017527:Receptor, IGF Type 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D012997:Solvents / Q000737:chemistry*; D020349:Surface Plasmon Resonance
chemical_list: D017527:Receptor, IGF Type 2; D012997:Solvents; D007335:Insulin-Like Growth Factor II