Crystal structure of ADAMTS13 CUB domains reveals their role in global latency.
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ABSTRACT:
SUBMITTER: Kim HJ
PROVIDER: S-EPMC8051872 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Hereditary thrombotic thrombocytopenic purpura (TTP) is an autosomal recessive thrombosis disorder, caused by loss-of-function mutations in ADAMTS13. Mutations in the CUB domains of ADAMTS13 are rare, and the exact mechanisms through which these mutations result in the development of TTP have not yet been fully elucidated. In this study, we identified two novel mutations in the CUB domains in a TTP family with an acceptor splice-site mutation (c.3569-1, G>A, intron 25) and a point missense mutation (c.3923, G>A, exon 28), resulting in a glycine to aspartic acid substitution (p.G1308D). In vitro splicing analysis revealed that the intronic mutation resulted in abnormal pre-mRNA splicing, and an in vitro expression assay revealed that the missense mutation significantly impaired ADAMTS13 secretion. Although both the patient and her brother displayed significantly reduced ADAMTS13 activity and increased levels of ultra-large VWF (ULVWF) multimers in plasma, only the female developed acute episodes of TTP. Our findings indicate the importance of the CUB domains for the protein stability and extracellular secretion of ADAMTS13.
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Project description:ADAMTS13 specifically cleaves plasma von Willebrand factor (VWF) and thereby controls VWF-mediated platelet thrombus formation. Severe deficiencies in ADAMTS13 can cause life-threatening thrombotic thrombocytopenic purpura. Here, we determined 2 crystal structures of ADAMTS13-DTCS (residues 287-685), an exosite-containing human ADAMTS13 fragment, at 2.6-A and 2.8-A resolution. The structures revealed folding similarities between the disintegrin-like (D) domain and the N-terminal portion of the cysteine-rich domain (designated the C(A) domain). The spacer (S) domain forms a globular functional unit with a 10-stranded beta-sandwich fold that has multiple interaction sites with the C(A) domain. We expressed 25 structure-based mutants of ADAMTS13-MDTCS (residues 75-685) and measured their enzymatic activity. We identified 3 VWF-binding exosites on the linearly aligned discontinuous surfaces of the D, C(A), and S domains traversing the W-shaped molecule. Since the MDTCS domains are conserved among ADAMTS family proteins, the structural framework of the multiple enzyme-substrate interactions identified in the ADAMTS13-VWF system provides the basis for a common substrate recognition mode in this class of proteinases.
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2016-12-20 | GSE87157 | GEO
Project description:Developmentally Regulated GTP-binding (DRG) proteins are highly conserved GTPases that associate with DRG Family Regulatory Proteins (DFRP). The resulting complexes have recently been shown to participate in eukaryotic translation. The structure of the Rbg1 GTPase, a yeast DRG protein, in complex with the C-terminal region of its DFRP partner, Tma46, was solved by X-ray diffraction. These data reveal that DRG proteins are multimodular factors with three additional domains, helix-turn-helix (HTH), S5D2L and TGS, packing against the GTPase platform. Surprisingly, the S5D2L domain is inserted in the middle of the GTPase sequence. In contrast, the region of Tma46 interacting with Rbg1 adopts an extended conformation typical of intrinsically unstructured proteins and contacts the GTPase and TGS domains. Functional analyses demonstrate that the various domains of Rbg1, as well as Tma46, modulate the GTPase activity of Rbg1 and contribute to the function of these proteins in vivo. Dissecting the role of the different domains revealed that the Rbg1 TGS domain is essential for the recruitment of this factor in polysomes, supporting further the implication of these conserved factors in translation.
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Project description:SSRP1 is a subunit of the FACT complex, an important histone chaperone required for transcriptional regulation, DNA replication and damage repair. SSRP1 also plays important roles in transcriptional regulation independent of Spt16 and interacts with other proteins. Here, we report the crystal structure of the middle domain of SSRP1. It consists of tandem pleckstrin homology (PH) domains. These domains differ from the typical PH domain in that PH1 domain has an extra conserved ??? topology. SSRP1 contains the well-characterized DNA-binding HMG-1 domain. Our studies revealed that SSRP1-M can also participate in DNA binding, and that this binding involves one positively charged patch on the surface of the structure. In addition, SSRP1-M did not bind to histones, which was assessed through pull-down assays. This aspect makes the protein different from other related proteins adopting the double PH domain structure. Our studies facilitate the understanding of SSRP1 and provide insights into the molecular mechanisms of interaction with DNA and histones of the FACT complex.
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Project description:BackgroundType G immunoglobulins against ADAMTS13 are the primary cause of acquired (idiopathic) thrombotic thrombocytopenic purpura. However, the domains of ADAMTS13 which the type G anti-ADAMT13 immunoglobulins target have not been investigated in a large cohort of patients with thrombotic thrombocytopenic purpura.Design and methodsSixty-seven patients with acquired idiopathic thrombotic thrombocytopenic purpura were prospectively collected from three major U.S. centers. An enzyme-linked immunosorbent assay determined plasma concentrations of anti-ADAMTS13 type G immunoglobulins, whereas immunoprecipitation plus western blotting determined the binding domains of these type G immunoglobulins.ResultsPlasma anti-ADAMTS13 type G immunoglobulins from 67 patients all bound full-length ADAMTS13 and a variant truncated after the eighth TSP1 repeat (delCUB). Approximately 97% (65/67) of patients harbored type G immunoglobulins targeted against a variant truncated after the spacer domain (MDTCS). However, only 12% of patients' samples reacted with a variant lacking the Cys-rich and spacer domains (MDT). In addition, approximately 37%, 31%, and 46% of patients' type G immunoglobulins interacted with the ADAMTS13 fragment containing TSP1 2-8 repeats (T2-8), CUB domains, and TSP1 5-8 repeats plus CUB domains (T5-8CUB), respectively. The presence of type G immunoglobulins targeted against the T2-8 and/or CUB domains was inversely correlated with the patients' platelet counts on admission.ConclusionsThis multicenter study further demonstrated that the multiple domains of ADAMTS13, particularly the Cys-rich and spacer domains, are frequently targeted by anti-ADAMTS13 type G immunoglobulins in patients with acquired (idiopathic) thrombotic thrombocytopenic purpura. Our data shed more light on the pathogenesis of acquired thrombotic thrombocytopenic purpura and provide further rationales for adjunctive immunotherapy.
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