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Plenary Paper Plenary Paper THROMBOSIS AND HEMOSTASIS Highly reinforced structure of a C-terminal dimerization domain in von Willebrand factor Yan-Feng Zhou and Timothy A. Springer Program in Cellular and Molecular Medicine, Children’s Hospital Boston and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA Key Points The C-terminal cystine knot (CK) (CTCK) domain in von Willebrand factor (VWF) mediates dimerization of proVWF in the endoplasmic reticulum and is essential for long multimers • C-terminal cystine knot required for hemostatic function. The CTCK dimer crystal structure reveals highly monomers in VWF are highly elongated monomers with 2 b-ribbons and 4 intra-chain disulfides, including 3 in the CK. elongated and form Dimerization buries an extensive interface of 1500 A˚ 2 corresponding to 32% of the surface antiparallel dimers. of each monomer and forms a super b-sheet and 3 inter-chain disulfides. The shape, • Three disulfides across dimensions, and N-terminal connections of the crystal structure agree perfectly with the dimer interface flanked by previous electron microscopic images of VWF dimeric bouquets with the CTCK dimer forming a down-curved base. The dimer interface is suited to resist hydrodynamic force the cystine knots in each and disulfide reduction. CKs in each monomer flank the 3 inter-chain disulfides, and their monomer form a highly force- presence in b-structures with dense backbone hydrogen bonds creates a rigid, highly resistant structure. crosslinked interface. The structure reveals the basis for von Willebrand disease phenotypes and the fold and disulfide linkages for CTCK domains in diverse protein families involved in barrier function, eye and inner ear development, insect coagulation and innate immunity, axon guidance, and signaling in extracellular matrices. (Blood. 2014;123(12):1785-1793) Introduction Von Willebrand factor (VWF) is a mosaic of domains with many sequence homology to domains at the C termini of VWF, some gel- types of binding sites that crosslinks platelets to one another and to forming mucins, hemolectin, and norrie disease protein (norrin).4 the vessel wall in hemostasis and thrombosis.1,2 Long length is key to Eight of 10 or 11 cysteines were invariant in CT domains. Finally, VWF’s function as a sensor for flow changes at sites of hemostasis. Bork4,5 found still weaker homology, but conservation of the Polymerization is mediated by specialized N- and C-terminal do- spacing of 6 of the cysteines, in CK cytokines such as trans- mains. Dimerization through a C-terminal cystine knot (CK) (CTCK) forming growth factor b (TGFb). domain3-6 occurs first in the endoplasmic reticulum and is followed Twenty years has passed since the CTCK domain family was later in biosynthesis by N-terminal linkage through the D3 domain as identified4 and suggested to be structurally related to TGFb.5 How- VWF dimers assemble into helical tubules in the Golgi and Weibel- ever, despite presence in a diverse group of functionally impor- Palade bodies.7 tant proteins mutated in disease, the CTCK domain has been Domains with CKs contain a unique motif of 3 disulfide recalcitrant to structural elucidation. Early work on VWF showed bonds.8 Two closely spaced cysteines on each of 2 polypeptide that at least one inter-chain disulfide was present in the C-terminal segments disulfide link to form a ring through which a third region.3 Later chemical and mutational studies characterized the disulfide linking other polypeptide segments crosses. The CK is 11 cysteines present in the VWF CTCK domain.12 Intra-chain actually a motif and not a domain; CKs are found in domains connections were defined for 8 cysteines, and among the 3 re- including knottins and cytokines that are otherwise completely maining cysteines, either 1 or 3 were proposed to mediate inter- unrelated.9 chain dimerization. Here, we report the crystal structure of the CTCK domains were called C-terminal or CT domains by Bork,10 VWF CTCK dimer. Our structure defines the principles for assembly who found them at the C termini of the CCN (cysteine-rich 61, con- of proteins with functions as diverse as hemostasis (VWF), barrier nective tissue growth factor, nephroblastoma overexpressed) function (mucins), axonal guidance (Slit), and regulation of growth, family of regulatory matricellular proteins and the Slit family of migration, and differentiation (the CCN family and Norrie disease proteins that regulate axon guidance.4 Bork11 recognized lesser protein). Submitted November 12, 2013; accepted December 16, 2013. Prepublished The publication costs of this article were defrayed in part by page charge online as Blood First Edition paper, January 6, 2014; DOI 10.1182/blood-2013- payment. Therefore, and solely to indicate this fact, this article is hereby 11-523639. marked “advertisement” in accordance with 18 USC section 1734. The online version of this article contains a data supplement. There is an Inside Blood commentary on this article in this issue. © 2014 by The American Society of Hematology BLOOD, 20 MARCH 2014 x VOLUME 123, NUMBER 12 1785 1786 ZHOU and SPRINGER BLOOD, 20 MARCH 2014 x VOLUME 123, NUMBER 12 Table 1. Crystal diffraction data and refinement statistics Methods Data collection Wavelength (A)˚ 1.28237 Constructs and cell culture Resolution range (A)˚ 47-3.28 (3.37-3.28)* Human VWF CTCK (residues 2720-2813 of pre-proVWF) was expressed Space group P4332 5 5 5 ˚ 5 5 5 ° in vector ET8.13 VWF sequence was fused C-terminal to the N-terminal Unit cell a b c 135.34 A, a b g 90 Solvent content (%) 88 signal peptide, His6 tag, and 3C protease site (LEVLFQGP). HEK293S N-acetylglucosaminyl transferase I-deficient cells14 were transfected using Total unique reflections† 12294 polyethyleneimine.15 Single colonies were selected in Dulbecco’s modified Redundancy‡ 13.7 (10.7) Eagle’s medium and 5% fetal bovine serum with 1 mg/mL G418.16 This study Completeness (%) 99.99 (99.85) was conducted in accordance with the Declaration of Helsinki. I/ s(I) 10.87 (0.43) Rsym‡ 0.228 (6.097) Characterization of the CTCK domain CC1/2 (%)§ 99.8 (12.0) Phasing and refinement fi CTCK was secreted as a disul de-linked dimer (supplemental Figure 1, Phasing figure of merit 0.245 available on the Blood Web site). Secretion was greatly decreased if the Rwork /Rfree|| 0.224 / 0.248 His6 tag and 3C protease site were not included in the expression construct. Monomer / asymmetric unit 1 Expression in supernatant was semiquantitatively measured by western Nonhydrogen atoms blotting with anti-VWF polyclonal antibody (Delta Biolabs, Gilroy, CA). Protein/ N-acetyl glucosamine /Zn/SO /water 721/14/0.5/25/6 N fi 4 One -glycosylation site in CTCK sequence was con rmed by endogly- RMSD bonds (A)˚ 0.003 cosidase H (Roche) digestion (supplemental Figure 1). RMSD angles (°) 0.46 Ramachandran plot{ Purification and crystallization (% favored/allowed/outliers) 94.6/5.4/0.0 CTCK in square bottle suspension culture Ex-CELL 293 serum-free medium Geometry and clash percentiles{ 100% / 100% supernatant was purified using Ni-NTA (Qiagen) and Hitrap Q (GE Health- Protein data bank ID 4NT5 care) chromatography as described for other C-terminal fragments of VWF, 13 *Statistics for the highest-resolution shell are shown in parentheses. including buffers, pH, and salt gradients, except that Hitrap Q was sub- †Friedel pairs are treated as separate reflections. stituted for mono Q. The ion exchange eluate was concentrated and ‡Rsym 5 ShklSi|Ii2,I.|/ShklSiIi, where Ii and ,I . are the i th and mean exchanged into 20 mM BisTris, pH 6.2, 0.15 M NaCl, and 3 mM EDTA and measurement of the intensity of reflection hkl. digested with a mass ratio of 1:400 of endoglycosidase H: CTCK for 48 hours §Pearson’s correlation coefficient between average intensities of random half- 38 at 22°C. The digest was concentrated and loaded onto a Superdex 200 10/300 data sets of the measurements for each unique reflection. 5 F 2 F F F F GL column (GE Healthcare) equilibrated with 20 mM sodium acetate, pH 5.5, ||Rwork Shkl|| obs| | calc||/| obs|, where obs and calc are the observed and calculated structure factors, respectively. Rfree is the crossvalidation R factor 0.15 M NaCl. Fractions containing CTCK dimer (supplemental Figure 1B) computed for the 8% (929) test set of unique reflections. were concentrated to 20 mg/mL. Material was crystallized with the N-terminal {Ramachandran, geometry, and clash values were reported by MOLPROBITY.25 His6 tag intact; material with the tag removed did not yield crystals. Single crystals were obtained with a 1:1 volume to volume ratio well versions of PHENIX (we used dev-1426) include the overall B factor in the solutionof0.1Mbicine,pH8.2to8.3,and1.3to1.4MZnSO4 at 20°C in individual atomic B factors, which increases the B factors reported in the hanging drops. Crystallization trays were moved to 4°C for cryoprotection. coordinate file by about 50. MolProbity was used for model validation.25 Saturated LiSO4 was mixed with 80, 60, 40, 20, and 0% volume to volume ratio well solution, and individual crystals in cryo loops were passed through drops of Electron microscopy (EM) image processing these solutions in the same order and then plunge frozen in liquid N . Anomalous 2 Class averages of A3-CK13 were recalculated with centering on the CTCK diffraction data at zinc peak (1.28237 A)˚ were collected at General Medical region using a previously described auto-centering script (supplemental Sciences/Cancer ID23-B beamline (Advanced Photon Source, Argonne Figure 4).6 National Laboratory, Argonne, IL). To minimize radiation damage, the beam position was vectorially scanned along the crystal during data collection.
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