Structure–function analysis of full-length midkine reveals novel residues important for heparin binding and zebrafish embryogenesis

Abstract

Midkine is a heparin-binding di-domain growth factor, implicated in many biological processes as diverse as angiogenesis, neurogenesis and tumorigenesis. Elevated midkine levels reflect poor prognosis for many carcinomas, yet the molecular and cellular mechanisms orchestrating its activity remain unclear. At the present time, the individual structures of isolated half domains of human midkine are known and its functionally active C-terminal half domain remains a popular therapeutic target. In the present study, we determined the structure of full-length zebrafish midkine and show that it interacts with fondaparinux (a synthetic highly sulfated pentasaccharide) and natural heparin through a previously uncharacterized, but highly conserved, hinge region. Mutating six consecutive residues in the conserved hinge to glycine strongly abates heparin binding and midkine embryogenic activity. In contrast with previous in vitro studies, we found that the isolated C-terminal half domain is not active in vivo in embryos. Instead, we have demonstrated that the N-terminal half domain is needed to enhance heparin binding and mediate midkine embryogenic activity surprisingly in both heparin-dependent and -independent manners. Our findings provide new insights into the structural features of full-length midkine relevant for embryogenesis, and unravel additional therapeutic routes targeting the N-terminal half domain and conserved hinge.

Current structural biology of the heparin interactome

“…Lim and coworkers have studied the structural binding mechanism of fondaparinux and a natural heparin to midkine, a heparin-binding di-domain growth factor [52]. Results have indicated that residues W55, K56 and K57 in the hinge, which are spatially located between basic clusters 1 and 3 of both composing domains, are involved in the heparin binding.…”

Pharmacology of Heparin and Related Drugs

“…The interaction between the multifunctional growth factor midkine and heparin is unusual in that the hinge region between the two domains is important for heparin binding, as determined by an NMR study using the pentasaccharide fondaparinux (Lim et al, 2013). Pleiotrophin is associated with chondroitin sulfate-mediated neurite outgrowth and is important in bone repair (Lamprou et al, 2014).…”

Structure and function of midkine as the basis of its pharmacological effects

“…In particular, there is a flexible loop in the middle of the C‐domain (amino acids 86–93), and a pocket is created in the domain (Figure ). Recently, detailed analysis of the whole molecule of zebrafish Mdkb has been performed and has revealed the whole structure of MK, including the hinge region, which has an extended structure (Lim et al ., ).…”

“…N‐domain has functions different from those of C‐domain. Firstly, another heparin‐binding site has been identified in the N‐domain of Mdkb (Lim et al ., ). The key residue of the new binding site is R36, corresponding to R35 in human MK (Lim et al ., ).…”

“…The other site in the flexible loop called cluster 2 (K86, K87, R89) is conserved only partly (open circle, Figure ). However, in zebrafish Mdkb, which has only two basic amino acids in cluster 2, the cluster still functions as a heparin‐binding site (Lim et al ., ). Cluster 1 appears to be important not only to heparin binding but also to MK function because a mutation in cluster 1 reduces neurite‐promoting activity (Asai et al ., ) and binding to PTPζ (Maeda et al ., ).…”