Cystine Knot of the Gonadotropin α Subunit Is Critical for Intracellular Behavior but Not for in Vitro Biological Activity

100

118

Norrin and Frizzled4 (Fz4) function as a ligand-receptor pair to control vascular development in the retina and inner ear. In mice and humans, mutations in either of the corresponding genes lead to defects in vascular development. The present work is aimed at defining the sequence determinants of binding specificity between Norrin and the Fz4 amino-terminal ligand-binding domain (the "cysteine-rich domain" (CRD)). The principal conclusions are as follows: 1) Norrin binds to the Fz4 CRD and does not detectably bind to the 14 other mammalian Frizzled and secreted Frizzled-related protein CRDs; 2) Norrin and Xenopus Wnt8 recognize largely overlapping regions of the Fz4 CRD; 3) surface determinants on the Fz4 and Fz8 CRDs that allow Norrin to distinguish between these two CRDs reside within several small regions on one face of the CRD; 4) Norrin function depends critically on three pairs of cysteines that form the highly conserved trio of disulfide bonds shared among all cystine knot proteins, but the remaining two putative disulfide bonds are less important; 5) Norrin-CRD binding depends on a largely contiguous group of amino acids in the extended ␤-sheet domain of Norrin that are predicted to face away from the interface between the two monomers in the Norrin homodimer; 6) Norrin-CRD binding is strongly modulated by interactions involving charged amino acid side chains; and 7) Norrin-CRD binding is enhanced ϳ10-fold by the addition of heparin. These observations are discussed in the context of Frizzled signaling and the structure and function of other cystine knot proteins.

Section: Discussionmentioning

confidence: 59%

Mutational Analysis of Norrin-Frizzled4 Recognition

Smallwood

Williams

et al. 2007

100

118

“…In the large family of CSH domains, it is observed that a variety of disulfide-bonding patterns can be tolerated within the core of the domain, providing a structural scaffold that orients the ␣-helix on the surface (41). Thus, the curious retention of PAI-1 binding among this set of clearly different folds for the SMB domain is consistent with the demonstrated malleability of cystine knot structures to accommodate cysteine substitutions, yet retain activity (41)(42)(43)(44)(45). Even though the recombinant forms of the SMB domain do not have the correct cystine bonds found in circulating vitronectin, PAI-1 binding appears to be maintained because the various disulfide cross-linked frameworks support the formation of the sole ␣-helix in this domain.…”

Section: Figmentioning

confidence: 63%

The Solution Structure of the N-terminal Domain of Human Vitronectin

Mayasundari¹,

Whittemore²,

Serpersu

et al. 2004

The three-dimensional structure of an N-terminal fragment comprising the first 51 amino acids from human plasma vitronectin, the somatomedin B (SMB) domain, has been determined by two-dimensional NMR approaches. An average structure was calculated, representing the overall fold from a set of 20 minimized structures. The core residues (18 -41) overlay with a root mean square deviation of 2.29 ؎ 0.62 Å. The N-and Cterminal segments exhibit higher root mean square deviations, reflecting more flexibility in solution and/or fewer long-range NOEs for these regions. Residues 26 -30 form a unique single-turn ␣-helix, the locus where plasminogen activator inhibitor type-1 (PAI-1) is bound. This structure of this helix is highly homologous with that of a recombinant SMB domain solved in a co-crystal with PAI-1 (Zhou, A., Huntington, J. A., Pannu, N. S., Carrell, R. W., and Read, R. J. (2003) Nat. Struct. Biol. 10, 541-544), although the remainder of the structure differs. Significantly, the pattern of disulfide cross-links observed in this material isolated from human plasma is altogether different from the disulfides proposed for recombinant forms. The NMR structure reveals the relative orientation of binding sites for cell surface receptors, including an integrin-binding site at residues 45-47, which was disordered and did not diffract in the co-crystal, and a site for the urokinase receptor, which overlaps with the PAI-1-binding site.Human vitronectin is a glycoprotein found in the circulation, where it contributes to hemostasis by regulating blood coagulation and fibrinolysis (1, 2). Vitronectin is also found in the ECM, 1 where it plays important roles in cell adhesion, pericellular proteolysis, tissue invasion, angiogenesis, and metastasis (3-7). The variety of functions of vitronectin in the two microenvironments is a manifestation of its ability to interact with numerous humoral and cellular proteins. An important binding partner for vitronectin is the serine protease inhibitor PAI-1, which also is found both in circulation and the ECM. Furthermore, it has different activities depending upon this localization; the anti-protease activity of PAI-1 that regulates thrombolysis in the circulation is targeted instead toward pericellular proteolysis when localized to the ECM or cell/matrix boundary. Vitronectin binds to PAI-1 with high affinity and stabilizes the inhibitor in its active conformation (8, 9). Vitronectin can also associate with PAI-1 and assemble to form higher order complexes (10) that exhibit altered adhesive functions (11). Key to the adhesive functions of vitronectin are its interactions with cell-surface receptors including integrins and uPAR.A widely accepted model suggests that vitronectin is organized into functional domains that provide the broad repertoire necessary for binding to target ligands (12)(13)(14). We recently used computational methods to predict the structure of the three domains comprising vitronectin (15). A threading algorithm gave high confidence predictions for the central domai...

“…Furthermore, site-directed mutagenesis of hCG-␤ has shown that the removal of disulfide bonds involved in discrete steps of the hCG-␤ folding pathway either disrupts the folding and assembly of hCG-␤ or slows progression to subsequent folding intermediates (10). Previous studies have shown that the three cystine knot disulfide bonds of GPH-␣ are necessary for efficient secretion and heterodimerization with hCG-␤ (13,14). However, little is known about the role of individual disulfide bonds as GPH-␣ folds to its native conformation and how this folding process relates to GPH-␣ secretion and heterodimer formation.…”

mentioning

confidence: 99%

Cystine Knot Mutations Affect the Folding of the Glycoprotein Hormone α-Subunit

Darling

Ruddon

Perini

et al. 2000

The common glycoprotein hormone ␣-subunit (GPH-␣) contains five intramolecular disulfide bonds, three of which form a cystine knot motif (10 -60, 28 -82, and 32-84). By converting each pair of cysteine residues of a given disulfide bond to alanine, we have studied the role of individual disulfide bonds in GPH-␣ folding and have related folding ability to secretion and assembly with the human chorionic gonadotropin ␤-subunit (hCG-␤). Mutation of non-cystine knot disulfide bond 7-31, bond 59 -87, or both (leaving only the cystine knot) resulted in an efficiently secreted folding form that was indistinguishable from wild type. Conversely, the cystine knot mutants were inefficiently secreted (<25%). Furthermore, mutation of the cystine knot disulfide bonds resulted in multiple folding intermediates containing 1, 2, or 4 disulfide bonds. High performance liquid chromatographic separation of intracellular and secreted forms of the folding intermediates demonstrated that the most folded forms were preferentially secreted and combined with hCG-␤. From these studies we conclude that: (i) the cystine knot of GPH-␣ is necessary and sufficient for folding and (ii) there is a direct correlation between the extent of GPH-␣ folding, its ability to be secreted, and its ability to heterodimerize with hCG-␤.