Crystallization and preliminary crystallographic studies of human TGF-β type II receptor ligand-binding domain

Abstract

Three constructs (residues 15-136, 22-136 and 27-136) of the truncated extracellular domain of human transforming growth factor beta type II receptor (TBRII) were overexpressed in Escherichia coli. The constructs are referred to as TBRII(15-136), TBRII(22-136) and TBRII(27-136). The refolded receptors were purified using a combination of ion-exchange and size-exclusion chromatography. The purified receptors have an apparent molecular weight of 14 kDa as judged by size-exclusion chromatography. In the crystallization trials, TBRII(15-136) and TBRII(22-136) formed mostly crystal-like spheres but failed to produce data-quality crystals. TBRII(27-136) yielded large single crystals from hanging drops using the vapor-diffusion procedure with PEG 2000 or 4000 at pH 5.0. The crystals diffracted to 1.05 A [using the X9B beamline operated at lambda = 1.0092 A of the National Synchrotron Light Source (NSLS) at the Brookhaven National Laboratory] and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 35.5, b = 40.7, c = 76.2 A. There was one molecule in the asymmetric unit, which corresponds to a solvent content of 42.1%.

Cooperative Assembly of TGF-β Superfamily Signaling Complexes Is Mediated by Two Disparate Mechanisms and Distinct Modes of Receptor Binding

“…To test the role of the tether in recruitment of TbRI, the activities of N-terminal variants of TbRII (Figure 5A) were analyzed by in vitro binding assays. In native gels, binary complexes of a series of TbRII truncation variants (D14, D20, and D25) and TGF-b3 stained progressively less than binary complex with full-length TbRII and yet were clearly formed (Figure 5B), consistent with SPR interaction analyses (see below) and the dispensable role of the N-terminal segment in TGF-b binding established previously (Boesen et al, 2002a(Boesen et al, , 2002bHart et al, 2002). However, unlike the others, binary complex with fully truncated (D25) TbRII recruited little if any TbRI (right two lanes) into a stable ternary complex.…”

“…Furthermore, TGF-bs interact with their type II receptor in a manner distinct from BMPs, the three-finger toxin scaffold binding with its ''knuckles'' rather than ''fingers'' at a separate site (De Crescenzo et al, 2006;Hart et al, 2002). TbRII also has a unique N-terminal extension of 25 disordered residues not required for ligand binding (Boesen et al, 2002a(Boesen et al, , 2002bHart et al, 2002). TbRI is predicted to be structurally similar to BMPRIA with the exception of a small loop, the prehelix extension (Harrison et al, 2003), peculiar to TbRI and ActRIB.…”

The non-detergent sulfobetaine-201 acts as a pharmacological chaperone to promote folding and crystallization of the type II TGF-β receptor extracellular domain

“…Crystals appeared and grew to full size in 2–3 days. This time scale is accelerated compared with that reported previously (and consistent with our experience) of 7–14 days for conditions in which NDSB-201 is absent [21]. The crystals were cryoprotected using paratone, then vitrified and stored in liquid nitrogen.…”

“…Purified TBRII-ECD-PR could be crystallized using previously reported conditions [21]. When NDSB-201 was added, protein crystals grew and reached full size in 2–3 days as opposed to 1–2 weeks.…”

“…The protein concentration of the final solution was determined by UV absorbance at 280 nm using an extinction coefficient of 9230 M −1 cm −1 [20]. The resulting sample was subjected to previously reported crystallization conditions (100 mM sodium citrate pH 5.0, 30% PEG 2000) supplemented with 50 mM NDSB-201 [21]. Crystals appeared and grew to full size in 2–3 days.…”