The two cysteine-rich head domains of minicollagen from Hydra nematocysts differ in their cystine framework and overall fold despite an identical cysteine sequence pattern.

Synthetic replicates of naturally occurring cysteine-rich peptides such as hormones, neurotransmitters, growth factors, enzyme inhibitors, defensins and toxins often can be oxidatively folded in high yields to their native structure in simple redox buffers. Thereby, identical cysteine patterns in the sequence were found to generate identical disulfide connectivities and homologous spatial structures despite significant variability in the non-cysteine positions. Minicollagen-1 from the nematocysts of Hydra is a trimeric protein that contains cysteine-rich domains at the N and C termini, which are involved in the assembly of an intermolecular disulfide network. Determination of the three-dimensional structures of peptides corresponding to the N-terminal and C-terminal domains by NMR spectroscopy revealed a remarkable exception from the general rule. Despite an identical cysteine pattern, the two domains of minicollagen-1 form different disulfide bridges and exhibit distinctly different folds, both of which are not found in the current structural databases. To our knowledge, this is the first case where two relatively short peptides with the abundant cysteine residues in identical sequence positions fold uniquely and with high yields into defined, but differing, structures. Therefore, the cysteine-rich domains of minicollagen constitute ideal model systems for studies of the interplay between folding and oxidation in proteins.

[1]  Folding of omega-conotoxins. 1. Efficient disulfide-coupled folding of mature sequences in vitro. , 1996, Biochemistry.

[2]  Luis Moroder,et al.  The Structure of the Cys-rich Terminal Domain of Hydra Minicollagen, Which Is Involved in Disulfide Networks of the Nematocyst Wall* , 2004, Journal of Biological Chemistry.

[3]  M. Uhlén,et al.  Scaffolds for engineering novel binding sites in proteins. , 1997, Current opinion in structural biology.

[4]  J. Thornton.  Disulphide bridges in globular proteins. , 1981, Journal of molecular biology.

[5]  Y. Shimonishi,et al.  Dual Function of the Propeptide of Prouroguanylin in the Folding of the Mature Peptide , 2000, The Journal of Biological Chemistry.

[6]  G H Snyder,et al.  Dependence of formation of small disulfide loops in two-cysteine peptides on the number and types of intervening amino acids. , 1989, The Journal of biological chemistry.

[7]  R. Nussinov,et al.  A disulphide-reinforced structural scaffold shared by small proteins with diverse functions , 1995, Nature Structural Biology.

[8]  S. Grzesiek,et al.  Determination of a high‐precision NMR structure of the minicollagen cysteine rich domain from Hydra and characterization of its disulfide bond formation , 2004, FEBS letters.

[9]  D Thirumalai,et al.  Theoretical predictions of folding pathways by using the proximity rule, with applications to bovine pancreatic trypsin inhibitor. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Craig J. Benham,et al.  Disulfide bonding patterns and protein topologies , 1993, Protein science : a publication of the Protein Society.

[11]  D. Goldenberg,et al.  Folding of omega-conotoxins. 2. Influence of precursor sequences and protein disulfide isomerase. , 1996, Biochemistry.

[12]  D. Craik,et al.  A common structural motif incorporating a cystine knot and a triple‐stranded β‐sheet in toxic and inhibitory polypeptides , 1994, Protein science : a publication of the Protein Society.

[13]  C. Roumestand,et al.  Scorpion toxins as natural scaffolds for protein engineering. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[14]  T. Blundell,et al.  Snail and spider toxins share a similar tertiary structure and ‘cystine motif’ , 1994, Nature Structural Biology.

[15]  E Schwarz,et al.  Pro-sequence assisted folding and disulfide bond formation of human nerve growth factor. , 2001, Journal of molecular biology.

[16]  H. Kuroda,et al.  Structure-activity relationship of endothelin: importance of charged groups. , 1989, Biochemical and biophysical research communications.

[17]  K. Wüthrich.  NMR of proteins and nucleic acids , 1988 .

[18]  M. Sternberg,et al.  The disulphide beta-cross: from cystine geometry and clustering to classification of small disulphide-rich protein folds. , 1996, Journal of molecular biology.

[19]  G. Bulaj,et al.  Propeptide does not act as an intramolecular chaperone but facilitates protein disulfide isomerase-assisted folding of a conotoxin precursor. , 2004, Biochemistry.

[20]  J. Engel,et al.  Mini-collagens in hydra nematocytes , 1991, The Journal of cell biology.

[21]  Juswinder Singh,et al.  A classification of disulfide patterns and its relationship to protein structure and function , 2004, Protein science : a publication of the Protein Society.

[22]  H. A. Sober,et al.  Handbook of Biochemistry: Selected Data for Molecular Biology , 1971 .

[23]  P. S. Kim,et al.  The pro region of BPTI facilitates folding , 1992, Cell.

[24]  P E Bourne,et al.  The Protein Data Bank. , 2002, Nucleic acids research.

[25]  U. Marx,et al.  Role of the prosequence of guanylin , 1999, Protein science : a publication of the Protein Society.

[26]  W. D. Fairlie,et al.  The Propeptide of the Transforming Growth Factor-β Superfamily Member, Macrophage Inhibitory Cytokine-1 (MIC-1), Is a Multifunctional Domain That Can Facilitate Protein Folding and Secretion* , 2001, The Journal of Biological Chemistry.

[27]  Y. Shimonishi,et al.  In vitro disulfide-coupled folding of guanylyl cyclase-activating peptide and its precursor protein. , 1998, Biochemistry.

[28]  P. Lyu,et al.  Relationship between protein structures and disulfide‐bonding patterns , 2003, Proteins.

[29]  A. Molina,et al.  Plant defense peptides. , 1998, Biopolymers.

[30]  Richard R. Ernst,et al.  Investigation of exchange processes by two‐dimensional NMR spectroscopy , 1979 .

[31]  H A Scheraga,et al.  Coupling of conformational folding and disulfide-bond reactions in oxidative folding of proteins. , 2001, Biochemistry.

[32]  R. Norton,et al.  The cystine knot structure of ion channel toxins and related polypeptides. , 1998, Toxicon : official journal of the International Society on Toxinology.

[33]  Mark W. Maciejewski,et al.  Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge. , 2000 .

[34]  Vladimir Sklenar,et al.  Gradient-Tailored Water Suppression for 1H-15N HSQC Experiments Optimized to Retain Full Sensitivity , 1993 .

[35]  J. Pieters.  Protein folding coupled to disulphide bond formation. , 1997 .

[36]  G. Bulaj,et al.  Efficient oxidative folding of conotoxins and the radiation of venomous cone snails , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Friedrich Lottspeich,et al.  Nowa, a novel protein with minicollagen Cys-rich domains, is involved in nematocyst formation in Hydra , 2002, Journal of Cell Science.

[38]  Y. Kyōgoku,et al.  Folding motifs induced and stabilized by distinct cystine frameworks. , 1998, Protein engineering.

[39]  Ad Bax,et al.  MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy , 1985 .

[40]  H. Scheraga,et al.  Conformational propensities of protein folding intermediates: distribution of species in the 1S, 2S, and 3S ensembles of the [C40A,C95A] mutant of bovine pancreatic ribonuclease A. , 2002, Biochemistry.

[41]  C. David,et al.  A switch in disulfide linkage during minicollagen assembly in Hydra nematocysts , 2001, The EMBO journal.