Determination of the Disulphide Bonding Pattern in Proteins by Local and Global Analysis of Nuclear Magnetic Resonance Data

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...

Section: Resultsmentioning

confidence: 60%

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

Mayasundari¹,

Whittemore²,

Serpersu

et al. 2004

“…Assignment of Tpx-1 Crisp Domain Disulfide Bonds-The disulfide bond connectivity in the Tpx-1 Crisp domain was determined from analysis of the NOESY spectra, which were examined for the presence of C A H␣-C B H␤ and C A H␤-C B H␤ correlations (55,56 (57). This analysis was supported by the recently reported crystal structure of a native Crisp domain from Stecrisp (9).…”

Section: Journal Of Biological Chemistry 4157mentioning

confidence: 94%

The Cysteine-rich Secretory Protein Domain of Tpx-1 Is Related to Ion Channel Toxins and Regulates Ryanodine Receptor Ca2+ Signaling

Gibbs

Scanlon

Swarbrick

et al. 2006

133

126

The cysteine-rich secretory proteins (Crisp) are predominantly found in the mammalian male reproductive tract as well as in the venom of reptiles. Crisps are two domain proteins with a structurally similar yet evolutionary diverse N-terminal domain and a characteristic cysteine-rich C-terminal domain, which we refer to as the Crisp domain. We presented the NMR solution structure of the Crisp domain of mouse Tpx-1, and we showed that it contains two subdomains, one of which has a similar fold to the ion channel regulators BgK and ShK. Furthermore, we have demonstrated for the first time that the ion channel regulatory activity of Crisp proteins is attributed to the Crisp domain. Specifically, the Tpx-1 Crisp domain inhibited cardiac ryanodine receptor (RyR) 2 with an IC 50 between 0.5 and 1.0 M and activated the skeletal RyR1 with an AC 50 between 1 and 10 M when added to the cytoplasmic domain of the receptor. This activity was nonvoltage-dependent and weakly voltage-dependent, respectively. Furthermore, the Tpx-1 Crisp domain activated both RyR forms at negative bilayer potentials and showed no effect at positive bilayer potentials when added to the luminal domain of the receptor. These data show that the Tpx-1 Crisp domain on its own can regulate ion channel activity and provide compelling evidence for a role for Tpx-1 in the regulation of Ca 2؉ fluxes observed during sperm capacitation.Tpx-1 (testis specific protein-1) was originally identified in the mouse (1) and later found in the male reproductive tract of the human, guinea pig, rat, and horse (2-5). Tpx-1 is a member of the cysteine-rich secretory proteins (Crisp) 2 that are in turn a subgroup of the CAP protein superfamily (abbreviated from Crisp, Antigen 5, and Pr-1 (6)). The CAP proteins each contain a structurally related and unique domain, the CAP domain (7)(8)(9), that at present has no clearly defined biological function, although their spatial and temporal expression suggests a function related to the regulation of the innate immune system (10, 11) and male reproductive function. The Crisp proteins have a characteristic C-terminal sequence containing 10 absolutely conserved cysteines. The Crisp domain is unique and is only observed in association with the CAP domain. Previously, there has been no biological activity attributed specifically to this domain.In mammals, there are at least four Crisp proteins, Crisp-1, Tpx-1 (or Crisp-2), Crisp-3, and Crisp-4. Crisp-1 proteins are expressed predominantly in the epididymides where they coat the surface of sperm during epididymal maturation (12) and have been implicated in sperm oocyte binding and the regulation of capacitation (13-17). Tpx-1 is expressed only in the testis and localized to specific regions in the spermatozoa, notably the acrosome of the head, the outer dense fibers, and longitudinal columns of the fibrous sheath in the sperm tail and the connecting piece of the neck (3, 18). Transfection experiments have also suggested that Tpx-1 is involved in adhesion between germ cells and Sertoli...

“…NMR, Identification of Disulfide Bonds-In addition to the visual analysis of structures calculated without disulfide bonds, three established methods for the determination of disulfide bonds based on NMR data were used: (a) ambiguous intersulfur restraints (17), (b) probability of a certain disulfide bond ensemble (18), and (c) the averaged target function (19). (a) The cysteines were forced to form disulfide bonds by the CYANA algorithm using ambiguous distance restraints, for every cysteine, between one specific cysteine and all other cysteines (15).…”

Section: Methodsmentioning

confidence: 99%

“…The cells were washed twice with PBS and then immobilized on ␤ distance analysis as previously described (18). Averaged distances were extracted from the structure ensemble generated without any disulfide constraints.…”

Section: Methodsmentioning

confidence: 99%

Copsin, a Novel Peptide-based Fungal Antibiotic Interfering with the Peptidoglycan Synthesis

Essig

Hofmann

Münch

et al. 2014

139

124

Background: Secreted antibacterial substances of fungi provide a rich source for new antibiotics. Results: Copsin is a novel fungal antimicrobial peptide that binds in a unique manner to the cell wall precursor lipid II. Conclusion: As part of the defense strategy of a mushroom, copsin kills bacteria by inhibiting the cell wall synthesis. Significance: Copsin provides a novel highly stabilized scaffold for antibiotics.