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The disulfide-bridged chains in the kringle (K) and fibronectin type II (FN2) domains are characterized using a taxonomy that considers the regularities in both β-secondary structure and cystine cluster. The structural core of the kringle fold comprises an assembly of two β-hairpins (a “β meander”) accommodating two overlapping disulfides; one cystine is incorporated in adjacent β-strands, whereas the other is located just beyond the ends of non-adjacent β-strands. The dispositions of the (N, C) termini of the two overlapping disulfides in the kringle fold are given as (m, j+1) and (i−1, k+1), in which m, i, j, and k (m < i < j < k) are residues fulfilling the relations m ∼w j+3 and i ∼n j ∼w k, where the relationship ∼n/w associates residues belonging to a narrow/wide hydrogen-bonded pair of an antiparallel β-sheet. This pattern is the structural signature of the kringle fold and is referred to as the “disulfide kringle-cross”. The metrics of this motif are quantified, revealing structural differences between the two families of the kringle fold. The conformations of disulfides in the kringle fold are poorly accommodated by existing classification schemes. To elucidate the nature of these rotamers we have performed density functional theory (DFT) calculations for diethyl disulfide. A new classification for the disulfide conformations in proteins is proposed, consisting of six rotamer types: spiral, trans–spiral, corner, trans, hook, and staple. Its relation with previous classification schemes is specified. A survey of high-resolution X-ray structures reveals that the disulfide conformations are clustered around the averaged conformations for the six classes. Average conformation dihedral and distance values are in excellent agreement with the DFT values. The two overlapping disulfides in kringle domains adopt the trans-spiral conformation that appears to be ubiquitous (∼ 17%) in proteins. One of the disulfides stretches across the β-meander, invoking “strain” in the disulfide conformational state. The relevance of the new classification and the concept of strain are briefly discussed in the context of disulfide bond cleavage in proteins.

Analysis of complete genome sequences has made it clear that fibronectin type II (FN2) modules are present only in the vertebrate lineage, raising intriguing questions about the origin of this module type. Kringle domains display many similarities to FN2 domains; therefore it was suggested previously that they are highly divergent descendants of the same ancestral protein-fold. Since kringles are present in arthropodes, nematodes, and invertebrate chordates as well as in vertebrates, it is suggested that the FN2 domain arose in the vertebrate lineage through major structural modification of the more ancestral kringle fold. To explore this structural transition, in the present work we compare key structural features of two highly divergent kringle domains (the kringle of Caenorhabditis elegans Ror receptor tyrosine kinase and the kringle of rat neurotrypsin) with those of plasminogen kringles and FN2 domains. Our NMR conformation fingerprinting analysis indicates that characteristic 1 H-NMR markers of kringle or FN2 native folding, such as the dispersion of Trp aromatic connectivities and shifts of the Leu 46 /Thr 16 methyl signals, both decrease in the order kringles > neurotrypsin kringle > FN2 domains. These results suggest that the neurotrypsin kringle may represent an intermediate form between typical kringles and FN2 domains.Keywords: Fibronectin type II domain; kringle domain; neurotrypsin; NMR spectroscopy; evolution of protein folds Fibronectin type II modules (FN2 modules) are small, compact two-disulphide-bond domains of about 40 amino acid residues first identified in the extracellular matrix protein, fibronectin, and in some seminal fluid proteins (Esch et al. 1983;Skorstengaard et al. 1986;Seidah et al. 1987;). Related domains are found in the extracytoplasmic parts of membrane-associated proteins, such as members of the mannose receptor-phospholipase A2 receptor family (Taylor et al. 1990;Ishizaki et al. 1994;Jiang and Nussenzweig 1995), mannose-6-phosphate receptors (Morgan et al. 1987), and the pancreas-specific sel-1 proteins of vertebrates (Harada et al. 1999;Biunno et al. 2000). FN2 modules are also present in matrix metalloproteinases MMP-2 and MMP-9 (Collier et al. 1988;Wilhelm et al. 1989) as well as in the serine proteases, factor XII, and hepatocyte growth factor activator (McMullen and Fujikawa 1985;Miyazawa et al. 1993). Abbreviations: CD, circular dichroism; COSY, two-dimensional NMR chemical shift correlated spectroscopy; CRor, Ror-type receptor tyrosine kinase of C. elegans; CRor/K, the kringle domain of the Ror receptor tyrosine kinase of C. elegans; FN2, fibronectin type II domain; IPTG, isopropyl-␤-D-thiogalactopyranoside; K, kringle domain; NMR, nuclear magnetic resonance; NOESY, two-dimensional NMR nuclear Overhauser effect correlated spectroscopy; NT/K, the kringle domain of neurotrypsin; Pgn/K4, human plasminogen kringle 4; PDC-109/b, second fibronectin type II domain of bovine PDC-109; ppm, parts-per-million; PMSF, phenylmethyl sulfonyl fluride; SDS-PAGE, sodium dode...

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Neurotrypsin is a multidomain protein that serves as a brain-specific serine protease. Here we report the NMR structure of its kringle domain, NT/K. The data analysis was performed with the BACUS (Bayesian analysis of coupled unassigned spins) algorithm. This study presents the first application of BACUS to the structure determination of a 13 C unenriched protein for which no prior experimental 3D structure was available. NT/K adopts the kringle fold, consisting of an antiparallel β-sheet bridged by an overlapping pair of disulfides. The structure reveals the presence of a surface-exposed lefthanded polyproline II helix that is closely packed to the core β-structure. This feature distinguishes NT/K from other members of the kringle fold and points toward a novel functional role for a kringle domain. Functional divergence among kringle domains is discussed on the basis of their surface and electrostatic characteristics.The kringle fold occurs in combination with other folds in multidomain proteins, often as a part of tandem domains (1). The most frequent number of homologous repeats is 2, and the maximum number of consecutive kringle domains is 37 in apolipoprotein A (2). The kringle family has a broad repertoire of domain partners. For example, the hyaluronan-binding protein is the combination of a kringle domain with three epidermal growth factor domains and a serine protease. These proteins perform a variety of functions and can act as proteases (plasminogen and prothrombin) (3), protease activators (urokinase, tissue plasminogen activator, and hepatocyte growth factor activator) (3,4), growth factors (hepatocyte growth factor and macrophage-stimulating protein) (5), and lipid transporters (apolipoprotein A) (2).Human neurotrypsin is a multidomain protein of 875 amino acids, composed of a proline-rich basic segment at the N-terminus, followed by a kringle domain, four scavenger receptor † This work was sponsored by NIH Grant HL-29409 to M.L., and E.L.B. acknowledges financial support from NSF Grant MCB-0424494. ‡ Coordinates for the ensemble of 29 NT/K structures (accession code 2k4r) and a representative conformer (accession code 2k51) have been deposited in the RCSB Protein Data Bank. Experimental NMR restraints and 1 H and 15 N chemical shift assignments (accession code 15806) have been deposited in the Biological Magnetic Resonance Bank. * Corresponding authors. O.A.O.: e-mail, oao.sputnik@gmail.com; telephone, 412-268-5671. M.L.: e-mail: llinas@andrew.cmu.edu; telephone, 412-268-3140; fax, 412-268-1061. Table S1 listing the numbers of residues in the loops of proteins from the kringle family, Tables S2 and S3 listing RMSDs obtained from pairwise alignments of inner disulfides and of core residues of proteins in the K family, Figure S1 giving the sequence alignment of Figure 3, but now using the plasminogen kringle 5 domain residue numbering as a reference, Figure S2 showing the overlay of fluorescence spectra obtained at different POPC concentrations after normalization, and Figure S3 showing the...