Structure of the calcium-rich signature domain of human thrombospondin-2

Abstract

Thrombospondins (TSPs) are secreted glycoproteins that play key roles in interactions between cells and the extracellular matrix. Here, we describe the 2.6 Å resolution crystal structure of the glycosylated signature domain of human TSP-2, which includes three epidermal growth factor-like (EGF-like) modules, 13 aspartate-rich repeats, and a lectin-like module. These elements interact extensively to form three striking structural regions termed the stalk, wire, and globe. The TSP-2 signature domain is stabilized by these interactions and by a network of 30 bound Ca 2+ ions and 18 disulfide bonds. The structure suggests how genetic alterations of TSPs result in disease.Keywords calcium binding; extracellular matrix; lectin-like module; skeletal dysplasia; epidermal growth factor-like module; protein crystallography Thrombospondins (TSPs) are a family of five secreted multimodular metallo/glycoproteins that have diverse roles involving interactions between cells and the extracellular matrix {Adams, 2004 #3568}. For TSP-2, these functions are critical for such processes as synaptogenesis{Christopherson, 2005 #3578}, megakaryocytopoiesis{Kyriakides, 2003, and the foreign body reaction{Kyriakides, 2001 #3588}. All TSPs contain a highly conserved "signature domain" consisting of tandem epidermal growth factor-like (EGF-like) modules, aspartate-rich repeats, and a lectin-like module at their C-termini ( Fig. 1a). These modules are conserved with remarkable fidelity as is evidenced by a 458-residue stretch of fly TSP and human TSP-2 in the signature domain that are 60% identical without an insertion or deletion{Adams, 2003 #3571;LaBell, 1993 #340}. The signature domain contains the sites of polymorphisms and mutations linked to familial coronary artery disease and two skeletal disorders, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1){Topol, 2001 #3537; Kennedy, 2005 #3585;Posey, 2004 #3579}. Here, we describe the 2.6 Å resolution crystal structure of the complete glycosylated signature domain of human TSP-2, which includes three epidermal growth factor-like (EGF-like) modules, 13 aspartate-rich repeats, and a lectin-like module, along with 30 bound Ca 2+ ions. The structure reveals the highly intertwined nature of the signature domain that includes major interactions among the different parts of the molecule. The structure furthermore establishes a molecular explanation of how known genetic alterations of TSPs result in disease.Correspondence and requests for materials should be addressed to D. NIH Public Access Results Structure of the human TSP-2 signature domainThe crystal structure of the signature domain of human TSP-2 was solved using a combination of anomalous scattering and molecular replacement approaches. Electron-density maps, particularly those derived from molecular replacement using the structure of a fragment of the signature domain from TSP-1{Kvansakul, 2004 #3561}, were of high quality ( Fig. 1b), and allowed modelling of the entire domain structure.The overall structu...

Thrombospondins: from structure to therapeutics

“…EGF1 and EGF2 form an elongated stalk in the crystal structure of the signature domain of THBS-2 [11]. Each has the expected 1-3, 2-4, 5-6 disulfide connectivity of modules in the cEGF subtype 1 [32], despite the unusual two-cysteine separation of the fourth and fifth cysteines.…”

“…The EGF-like repeats from THBS have been solved within the context of the signature domain (2.6 Å resolution with R work /R free = 21.8/28.4) (Fig. 2E,F) [11]. THBSs have a mixture of calcium-binding and non-calcium-binding EGFlike repeats.…”

“…The consensus sequence for calcium-binding EGF-like repeats is ZXZZ-C-X variable -C-X variable -C-X-D/N-X 4 -Y/F-X-C, where Z is an aspartate, glutamate, asparagine, or glutamine [30]. THBS-2 has been shown to have one calcium-binding EGF [11]. There is one EGF-like repeat with the calcium-binding consensus sequence in THBS-1, two in vertebrate group B THBSs, and two in Drosophilia THBS.…”

“…Group B THBSs lack the 3-residue NAT glycosylation recognition sequence found in insert A. In the structure of the THBS-2 signature domain, the insertion does not perturb the folding of repeat 1C, which overlays the other Ctype repeats nearly exactly [11]. The second insert, designated insert B, is found only in THBS-3 and THBS-4, is located in repeat 11C of wire, and consists of four amino acids.…”

“…In many cases, the distinct structural domains have been shown to have unique functional properties. Structures of each of the modules found in THBSs have been solved for THBS or a related protein [5][6][7][8][9][10][11]. These structures provide a working model of group A THBSs in high calcium (Fig.…”