title: N-Terminal domain linkage modulates the folding properties of protein S epidermal growth factor-like modules.
abstract: Protein S interacts with activated protein C to play a crucial role in blood anticoagulation, and protein S deficiency is associated with increased risk of thrombosis. Despite the large volume of functional data available for this protein, no atomic resolution structure data have yet been reported. This is due at least in part to difficulties encountered when trying to produce fragments dissected from the intact protein; however, a few successful strategies have been described. In this research we have expressed a number of constructs containing protein S epidermal growth factor-like (EGF) domains 1 and 2 in Escherichia coli and Pichia pastoris. None of the proteins produced was stably folded as assayed by solution nuclear magnetic resonance spectroscopy. We therefore constructed a series of non-native protein S EGF concatemers to investigate the role of pairwise domain linkage in domain folding. Our results demonstrate that N-terminal domain linkage can either positively or negatively impact on the refolding of an adjacent domain. Furthermore, analysis of the NMR data for EGF3-4 reveals the expected interdomain NOEs that are characteristic of an extended arrangement of calcium-binding EGF domains and a similar average [(1)H]-(15)N heteronuclear NOE value for each of the two domains. These results provide the first data in support of protein S EGF3-4 adopting the same extended domain orientation as observed for the functionally distinct proteins fibrillin-1 and the low-density lipoprotein receptor. The results also have important implications for future studies, particularly when a dissection approach is used, of tandem EGF domains from protein S and other proteins.
authors.affiliation: Division of Structural Biology, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors.forename: Nyoman D
authors.initials: ND
authors.lastname: Kurniawan
authors.forename: Joanne M
authors.initials: JM
authors.lastname: O'Leary
authors.forename: Ann-Marie
authors.initials: AM
authors.lastname: Thamlitz
authors.forename: Raphael
authors.initials: R
authors.lastname: Sofair
authors.forename: Jörn M
authors.initials: JM
authors.lastname: Werner
authors.forename: Johan
authors.initials: J
authors.lastname: Stenflo
authors.forename: A Kristina
authors.initials: AK
authors.lastname: Downing
mesh_terms: D000595:Amino Acid Sequence; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D017510:Protein Folding; D017293:Protein S / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D017293:Protein S; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor