Characterization of an Extensive Interface on Vitronectin for Binding to Plasminogen Activator Inhibitor-1: Adoption of Structure in an Intrinsically Disordered Region

Abstract

Small-angle neutron scattering (SANS) measurements were pursued to study human vitronectin, a protein found in tissues and the circulation that regulates cell adhesion/ migration and proteolytic cascades that govern hemostasis and pericellular proteolysis. Many of these functions occur via interactions with its binding partner, plasminogen activator inhibitor-1 (PAI-1), the chief inhibitor of proteases that lyse and activate plasminogen. We focused on a region of vitronectin that remains uncharacterized from previous X-ray scattering, nuclear magnetic resonance, and computational modeling approaches and which we propose is involved in binding to PAI-1. This region, which bridges the N-terminal somatomedin B (SMB) domain with a large central β-propeller domain of vitronectin, appears unstructured and has characteristics of an intrinsically disordered domain (IDD). The effect of osmolytes was evaluated using circular dichroism and SANS to explore the potential of the IDD to undergo a disorder-to-order transition. The results suggest that the IDD favors a more ordered structure under osmotic pressure; SANS shows a smaller radius of gyration (R g ) and a more compact fold of the IDD upon addition of osmolytes. To test whether PAI-1 binding is also coupled to folding within the IDD structure, a set of SANS experiments with contrast variation were performed on the complex of PAI-1 with a vitronectin fragment corresponding to the N-terminal 130 amino acids (denoted the SMB-IDD because it contains the SMB domain and IDD in linear sequence). Analysis of the SANS data using the Ensemble Optimization Method confirms that the SMB-IDD adopts a more compact configuration when bound to PAI-1. Calculated structures for the PAI-1:SMB-IDD complex suggest that the IDD provides an interaction surface outside of the primary PAI-1-binding site located within the SMB domain; this binding is proposed to lead to the assembly of higher-order structures of vitronectin and PAI-1 commonly found in tissues.

Identification of a PAI‐1‐binding site within an intrinsically disordered region of vitronectin

“…61 Recent studies with the isolated SMB-IDD fragment from vitronectin demonstrated the adoption of structure within the IDD upon binding to PAI-1. 57 These previous results laid the groundwork and provided the rationale for this study. Our goals here were to determine whether the SMB and IDD regions account for the biphasic binding interactions, and, if so, to localize the PAI-1-binding site within the IDD.…”

“…49 Our initial computational analysis of the vitronectin domain structure suggested that the ~80 amino acid linker between the SMB and central domains is prone to disorder, 49 and further studies have demonstrated that this region has hallmark features of an intrinsically disordered domain (IDD). 47,57 We have sustained interest in this region and its role in the functional properties of vitronectin.…”

“…The location of an additional PAI-1 binding site within vitronectin has been hotly debated over the years. 36,57,58,[61][62][63][64][65][66] The picture that has emerged from mutagenesis and kinetic studies shows that PAI-1 and vitronectin interact over extensive binding surfaces. 58,61,62 A binding site located outside the SMB domain of vitronectin was demonstrated by engineering a vitronectin variant lacking the SMB domain and showing that it still bound to PAI-1, albeit with a lower affinity.…”

“…72 We have recently investigated the role of the vitronectin IDD with regards to PAI-1 binding by utilizing small-angle neutron scattering (SANS) approaches, which indicated that the IDD gains structure and becomes more compact upon PAI-1 binding. 57 The highly positively charged nature of residues in PAI-1 that comprise the more extensive binding site 61 suggests that the complementary binding site on vitronectin may include several negatively charged residues. Recognizing the density of negatively charged residues within the linker as one hallmark feature of IDD regions, we hypothesized that residues in this unstructured linker region of vitronectin comprise the PAI-1 binding site that exists external to the SMB domain.…”

“…Within the IDD region, the sequence "VFTMPEDEYTVYDDGEEKNNATVH" contains many negatively charged residues (in bold), including Glu-53, Asp-54, Glu-55, Asp-60, Asp-61, Glu63, and Glu-64. Steadystate and stopped-flow fluorescence approaches were utilized to study the binding of PAI-1 with several vitronectin variants, including the isolated SMB domain, a 130 amino acid variant harboring the SMB and IDD regions (SMB-IDD), 57 and the truncated SMB-IDDΔ71 form. This work demonstrates that the SMB-IDDΔ71 protein exhibits similar binding properties with the SMB-IDD and full-length vitronectin, thus localizing the additional PAI-1-binding site to the highly charged region spanning residues 48-71 in vitronectin.…”