Calcium binding properties of an epidermal growth factor-like domain from human thrombomodulin.
Two different disulfide-paired isomers of the peptide T422DIDECENG430GFCSGVCHNL440PGTFECISG449, spanning the junction between the fifth and sixth EGF-like domains plus the N-terminal part of the sixth EGF-like domain from human thrombomodulin (TM), and containing a consensus calcium binding sequence, were synthesized and studied by two-dimensional proton NMR spectroscopy. In the course of air oxidation of the fully reduced form of the peptide, only uncrossed non EGF-like [1-2, 3-4] disulfide-bonded isomer was produced, regardless of the presence of redox buffer and/or calcium. The crossed [1-3, 2-4] isomer was prepared from a peptide with acetamidomethyl-protected second and fourth cysteines. The isomer with the crossed disulfide pairing was a better thrombin inhibitor and was more strongly affected by calcium binding than the uncrossed [1-2, 3-4] isomer. Calcium-induced NMR resonance shifts observed for the [1-3, 2-4] isomer provide evidence for the presence of a specific calcium-binding site in the corresponding TM region. There was a limited dispersion of the proton chemical shifts and a general lack of nonsequential NOE's for both peptide isomers in the presence or absence of calcium. Therefore, neither the apo nor the calcium-bound forms of the peptides adopted a completely folded conformation, despite the fact that the [1-3, 2-4] isomer contains a potential folding nucleus existing in a number of disulfide-rich proteins. Apparently, other interactions have to be involved to determine the three-dimensional structure of the criss-cross fold in this peptide, most likely the interaction with the C-terminal parts of the fifth and/or sixth EGF-like domains.
Tolkatchev
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Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
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D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012996:Solutions; D013237:Stereoisomerism; D013917:Thrombin / Q000037:antagonists & inhibitors; D018180:Thrombomodulin / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology
D004220:Disulfides; D010446:Peptide Fragments; D012996:Solutions; D018180:Thrombomodulin; D004815:Epidermal Growth Factor; D013917:Thrombin; D008274:Magnesium; D002118:Calcium