Crystal Structure of the Cysteine-rich Secretory Protein Stecrisp Reveals That the Cysteine-rich Domain Has a K+ Channel Inhibitor-like Fold

Cornell University, Итак, New York, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2005; 280(13):12405-12. DOI: 10.1074/jbc.M413566200
Source: PubMed


Stecrisp from Trimeresurus stejnegeri snake venom belongs to a family of cysteine-rich secretory proteins (CRISP) that have various functions related to sperm-egg fusion, innate host defense, and the blockage of ion channels. Here we present the crystal structure of stecrisp refined to 1.6-angstrom resolution. It shows that stecrisp contains three regions, namely a PR-1 (pathogenesis-related proteins of group1) domain, a hinge, and a cysteine-rich domain (CRD). A conformation of solvent-exposed and -conserved residues (His60, Glu75, Glu96, and His115) in the PR-1 domain similar to that of their counterparts in homologous structures suggests they may share some molecular mechanism. Three flexible loops of hypervariable sequence surrounding the possible substrate binding site in the PR-1 domain show an evident difference in homologous structures, implying that a great diversity of species- and substrate-specific interactions may be involved in recognition and catalysis. The hinge is fixed by two crossed disulfide bonds formed by four of ten characteristic cysteines in the carboxyl-terminal region and is important for stabilizing the N-terminal PR-1 domain. Spatially separated from the PR-1 domain, CRD possesses a similar fold with two K+ channel inhibitors (Bgk and Shk). Several candidates for the possible functional sites of ion channel blocking are located in a solvent-exposed loop in the CRD. The structure of stecrisp will provide a prototypic architecture for a structural and functional exploration of the diverse members of the CRISP family.

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    • "Annotation and fold appraisal was guided by results from automated model predictions performed with I-TASSER (Yang et al., 2015). Using PSIPRED (Bryson et al., 2005) secondary structure predictions for inferred H. contortus CAP proteins, structure-based amino acid sequence alignments were made using SBAL (Wang et al., 2012; Wang and Hofmann, 2015), and the amino acid sequence of SteCRISP was added using the crystal structure deposited in PDB entry 1rc9 (Guo et al., 2005). In preparation for comparative modelling of Hc-CAP-15, SteCRISP was identified as the most suitable template (P-value: 2x10 -6 ) by surveying the PDB with pGenThreader (Bryson et al., 2005) for the protein with highest homology. "
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    ABSTRACT: Parasitic worm proteins that belong to the cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 (CAP) superfamily are proposed to play key roles in the infection process and the modulation of immune responses in host animals. However, there is limited information on these proteins for most socio-economically important worms. Here, we review the CAP protein superfamily of Haemonchus contortus (barber's pole worm), a highly significant parasitic roundworm (order Strongylida) of small ruminants. To do this, we mined genome and transcriptomic datasets, predicted and curated full-length amino acid sequences (n=45), undertook systematic phylogenetic analyses of these data and investigated transcription throughout the life cycle of H. contortus. We inferred functions for selected C. elegans orthologs (including vap-1, vap-2, scl-5 and lon-1) based on genetic networking and by integrating data and published information, and were able to infer that a subset of orthologs and their interaction partners play pivotal roles in growth and development via the insulin-like and/or the TGF-beta signaling pathways. The identification of the important and conserved growth regulator LON-1 led us to appraise the three-dimensional structure of this CAP protein by comparative modelling. This model revealed the presence of different topological moieties on the canonical fold of the CAP domain, which coincide with an overall charge separation as indicated by the electrostatic surface potential map. These observations suggest the existence of separate sites for effector binding and receptor interactions, and thus support the proposal that these worm molecules act in similar ways as venoms act as ligands for chemokine receptors or G protein-coupled receptor effectors. In conclusion, this review should guide future molecular studies of these molecules, and could support the development of novel interventions against haemonchosis. Copyright © 2015. Published by Elsevier Inc.
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    • "ShK domains are common in the astacin family of metalloendopeptidases, which includes BMP1/Tolloid, meprins, and MMP23 as examples. The ShK motif in astacins is thought to mediate protein-protein interactions [65]. "
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    ABSTRACT: The microfibril-associated glycoproteins MAGP-1 and MAGP-2 are extracellular matrix proteins that interact with fibrillin to influence microfibril function. The two proteins are related through a 60 amino acid matrix-binding domain but their sequences differ outside of this region. A distinguishing feature of both proteins is their ability to interact with TGFβ family growth factors, Notch and Notch ligands, and multiple elastic fiber proteins. MAGP-2 can also interact with αvβ3 integrins via an RGD sequence that is not found in MAGP-1. Morpholino knockdown of MAGP-1 expression in zebrafish results in abnormal vessel wall architecture and altered vascular network formation. In the mouse, MAGP-1 deficiency had little effect on elastic fibers in blood vessels and lung but resulted in numerous unexpected phenotypes including bone abnormalities, hematopoietic changes, increased fat deposition, diabetes, impaired wound repair, and a bleeding diathesis. Inactivation of the gene for MAGP-2 in mice produced a neutropenia yet had minimal effects on bone or adipose homeostasis. Double knockouts had phenotypes characteristic of each individual knockout as well as several additional traits only seen when both genes are inactivated. A common mechanism underlying all of the traits associated with the knockout phenotypes is altered TGFβ signaling. This review summarizes our current understanding of the function of the MAGPs and discusses ideas related to their role in growth factor regulation. Copyright © 2015. Published by Elsevier B.V.
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    • "Sequence variability in these regions is therefore likely to confer selectivity and affinity for binding partners, accounting for the diverse range of CRISP ion channel targets. A comparison of CRISP and CAP proteins from multiple species similarly identified three hypervariable regions in the CAP domain which were postulated to confer substrate specificity30. The mouse CRISP3 glycosylation site N113 lies within one of these hypervariable regions, corresponding to G109-V117, in the CAP domain. "
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    ABSTRACT: While the Cysteine-Rich Secretory Proteins (CRISPs) have been broadly proposed as regulators of reproduction and immunity, physiological roles have yet to be established for individual members of this family. Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family. Here we report the expression and purification of native, glycosylated CRISP3 from human and mouse, expressed in HEK 293 cells and isolated using ion exchange and size exclusion chromatography. Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution). Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences.
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