Origin of fibronectin type II (FN2) modules: Structural analyses of distantly‐related members of the kringle family idey the kringle domain of neurotrypsin as a potential link between FN2 domains and kringles

Abstract

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...

A Ligand-induced Conformational Change in Apolipoprotein(a) Enhances Covalent Lp(a) Formation

“…tor/scatter factor (33,34), and fibronectin (35,36). The closed conformation in these functionally diverse proteins provides a mechanism for functional regulation by masking important binding sites that are required for the functions of the respective proteins.…”

Differential Mitogenic Effects of Single Chain Hepatocyte Growth Factor (HGF)/Scatter Factor and HGF/NK1 following Cleavage by Factor Xa

“…Our experiments clearly show that digestion of scHGF/SF by fXa does not alter the associated mitogenic activity. It is possible that subdomains within the first kringle exist which are sufficient to allow high affinity binding of HGF/SF to c-Met, such as fibronectin type II sub-domains contained with in the kringle (47).…”

Characterization of the kringle fold and identification of a ubiquitous new class of disulfide rotamers

“…By reason of these criteria, the neurotrypsin kringle domain (i) belongs to the kringle fold, (ii) has the conserved arrangement of cystine clusters and the secondary structure of the K family, and (iii) lacks the distinct conserved β-structure of the FN2 family. Hence, NT/K is an unambiguous member of the K family and not a structural intermediate between the K and FN2 families as it was previously suggested [5]. …”

“…The K domains are ∼ 80 amino acid residue-long proteins with three disulfides. The FN2 domains comprise ∼ 40 amino acids residues with two disulfides and have been proposed to have evolved from the K domains through partial deletion of residues from the terminal strands [4, 5]. Since the N- and C-termini of the neurotrypsin kringle (NT/K) are shorter than in other K domains, it seemed attractive to hypothesize that NT/K represents a structural intermediate in the evolutionary transition from K to FN2 domains [5].…”

“…The FN2 domains comprise ∼ 40 amino acids residues with two disulfides and have been proposed to have evolved from the K domains through partial deletion of residues from the terminal strands [4, 5]. Since the N- and C-termini of the neurotrypsin kringle (NT/K) are shorter than in other K domains, it seemed attractive to hypothesize that NT/K represents a structural intermediate in the evolutionary transition from K to FN2 domains [5]. This example illustrates the dilemma of what constitutes a meaningful definition of “fold” in the evolutionary context: either an exact recapitulation of the chain path or just a common structural core of the chain?…”