The structure of recombinant plasminogen kringle 1 and the fibrin binding site

Wu, Tswei Ping; Padmanabhan, K.; Tulinsky, A.

doi:10.1097/00001721-199404000-00001

Cited by 45 publications

(51 citation statements)

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“…The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements. The structurally most similar Kringle domain is that of human plasminogen (PDB: 1PKR; Wu et al., 1994) with an root-mean-square deviation (RMSD) of 1.7 Å for 73 aligned C α (Figure 1B). The KRM1 structure reveals the fold of the WSC domain for the first time.…”

Section: Resultsmentioning

confidence: 99%

“…The structure was initially solved from crystal form III by molecular replacement (MR) with PHASER (McCoy et al., 2007), placing models for the CUB domain (PDB: 2WNO, CUB_C domain of Tsg-6 (Briggs et al., 2015), 37% sequence identity), and the KR domain (PDB: 1PKR, Kringle 1 of plasminogen; Wu et al., 1994; 39% sequence identity). Traceable density for the WSC domain became immediately evident.…”

Section: Methodsmentioning

confidence: 99%

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Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf

et al. 2016

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SummaryKremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling. We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains. The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor. We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD. DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC. Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf

et al. 2016

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“…8). NMR (19,29,30) and x-ray crystallographic (23,24,31,32) studies have demonstrated that the homology translates into a remarkable conformational uniformity. Among these structures, the lysine-binding K1 is the most potent inhibitory segment of endothelial cell proliferation.…”

Section: Discussionmentioning

confidence: 99%

Kringle Domains of Human Angiostatin

Cao

Davidson

et al. 1996

Journal of Biological Chemistry

347

118

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Recently we have identified angiostatin, an endogenous angiogenesis inhibitor of 38 kDa which specifically blocks the growth of endothelial cells (O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994) Cell 79, 315-328; Folkman, J. (1995) Nat. Med. 1, 27-31). Angiostatin was shown to represent an internal fragment of plasminogen containing the first four kringle structures. We now report on the inhibitory effects of individual or combined kringle structures of angiostatin on capillary endothelial cell proliferation. Recombinant kringle 1 and kringle 3 exhibit potent inhibitory activity with half-maximal concentrations (ED 50 ) of 320 nM and 460 nM, respectively. Also, recombinant kringle 2 displays a significant inhibition, although decreased compared with both kringle 1 and kringle 3. In contrast, kringle 4 is an ineffective inhibitor of basic fibroblast growth factor-stimulated endothelial cell proliferation. Among the tandem kringle arrays, the recombinant kringle 2-3 fragment exerts inhibitory activity similar to kringle 2 alone. However, relative to kringle 2-3, a marked enhancement in inhibition is observed when individual kringle 2 and kringle 3 are added together to endothelial cells. This implies that it is necessary to open the cystine bridge between kringle 2 and kringle 3 to obtain the maximal inhibitory effect of kringle 2-3. An increased (<2-fold) inhibitory activity is observed for the kringle 1-3 fragment (ED 50 ‫؍‬ 70 nM) compared with kringle 1-4 (ED 50 ‫؍‬ 135 nM). These data indicate that the anti-proliferative activity of angiostatin on endothelial cells is shared by kringle 1, kringle 2, and kringle 3, but probably not by kringle 4 and that more potent inhibition results when kringle 4 is removed from angiostatin. Thus, in view of the variable lysine affinity of the homologous domains, it would appear that lysine binding capability does not correlate with the relative inhibitory effects of the kringle-containing constructs. However, as we also demonstrate, appropriate folding of kringle structures is essential for angiostatin to maintain its full anti-endothelial activity.

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“…These Kringle domains are ϳ80 amino acids long; some of them possess the lysine binding capability (21)(22)(23). Thus, plasminogen binds abundantly to fibrin clots during clot lysis since more C-terminal lysine residues are generated through the action of plasmin, a trypsinlike protease that cuts after lysine and arginine residues.…”

mentioning

confidence: 99%

A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy

Wong

2003

Journal of Biological Chemistry

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To develop a fast-acting clot dissolving agent, a clottargeting domain derived from the Kringle-1 domain in human plasminogen was fused to the C-terminal end of staphylokinase with a linker sequence in between. Production of this fusion protein in Bacillus subtilis and Pichia pastoris was examined. The Kringle domain in the fusion protein produced from B. subtilis was improperly folded because of its complicated disulfidebond profile, whereas the staphylokinase domain produced from P. pastoris was only partially active because of an N-linked glycosylation. A change of the glycosylation residue, Thr-30, to alanine resulted in a non-glycosylated biologically active fusion. The resulting mutein, designated SAKM3-L-K1, was overproduced in P. pastoris. Each domain in SAKM3-L-K1 was functional, and this fusion showed fibrin binding ability by binding directly to plasmin-digested clots. In vitro fibrin clot lysis in a static environment and plasma clot lysis in a flowcell system demonstrated that the engineered fusion outperformed the non-fused staphylokinase. The time required for 50% clot lysis was reduced by 20 to 500% under different conditions. Faster clot lysis can potentially reduce the degree of damage to occluded heart tissues.

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The structure of recombinant plasminogen kringle 1 and the fibrin binding site

Cited by 45 publications

References 0 publications

Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf

Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf

Kringle Domains of Human Angiostatin

A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy

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