Pro-metastatic collagen lysyl hydroxylase dimer assemblies stabilized by Fe2+-binding

Hou-Fu Guo,Sarah Alvarado,Mitchell D Miller,Mitsuo Yamauchi,Yulong Chen,Masahiko Terajima,Kevin N Dalby,Neus Bota-Rabassedas,Priyam Banerjee,Jonathan M Kurie,Xiaochao Tan,Tamer S Kaoud,George N Phillips,Jiang Yu,Chi-Lin Tsai,Jovita Byemerwa,John A Tainer,Xin Liu

doi:10.1038/s41467-018-02859-z

Abstract

Collagen lysyl hydroxylases (LH1-3) are Fe2+- and 2-oxoglutarate (2-OG)-dependent oxygenases that maintain extracellular matrix homeostasis. High LH2 levels cause stable collagen cross-link accumulations that promote fibrosis and cancer progression. However, developing LH antagonists will require structural insights. Here, we report a 2 Å crystal structure and X-ray scattering on dimer assemblies for the LH domain of L230 in Acanthamoeba polyphaga mimivirus. Loop residues in the double-stranded β-helix core generate a tail-to-tail dimer. A stabilizing hydrophobic leucine locks into an aromatic tyrosine-pocket on the opposite subunit. An active site triad coordinates Fe2+. The two active sites flank a deep surface cleft that suggest dimerization creates a collagen-binding site. Loss of Fe2+-binding disrupts the dimer. Dimer disruption and charge reversal in the cleft increase Km and reduce LH activity. Ectopic L230 expression in tumors promotes collagen cross-linking and metastasis. These insights suggest inhibitor targets for fibrosis and cancer.

Highlights

Work in pancreatic cancer models has shown that intratumoral fibrosis exerts mechanical forces that activate tumor cell invasive properties and restrict the influx of immune cells and vascular cells, creating an immunosuppressive, hypoxic environment that favors metastasis and inhibits antitumor drug delivery[1,2,3,4]
Subsequent findings by our group showed that metastatic lung adenocarcinomas produce a stable type of collagen cross-link driven by high expression of lysyl hydroxylase 2 (LH2); LH2 depletion in tumor cells led to a reduction in tumor stiffness and metastatic activity and a change in the type, but not the total amount, of collagen cross-links as manifested by a decrease in hydroxylysine (Hyl) aldehyde-derived collagen cross-links (HLCCs) and a reciprocal increase in Lys aldehyde-derived collagen cross-links (LCCs)[10]
Other side chains facing the 2-OG-binding pocket are conserved in human LH1-3 but not invertebrate LHs (Supplementary Fig. 3 and Supplementary Table 2), except L230 Ala[879], which is glycine in human LHs (Fig. 1b, c)

Summary

Introduction

Work in pancreatic cancer models has shown that intratumoral fibrosis exerts mechanical forces that activate tumor cell invasive properties and restrict the influx of immune cells and vascular cells, creating an immunosuppressive, hypoxic environment that favors metastasis and inhibits antitumor drug delivery[1,2,3,4]. The collagen LHs include three vertebrate (LH1-3) and one invertebrate gene that are members of a large family of Fe2+- and 2-oxoglutarate (2-OG)-dependent oxygenases These oxygenases utilize mononuclear non-heme iron and the co-substrates 2-OG and oxygen to oxidize substrate and generate succinate and CO212. Our findings reveal features of the L230 active site and dimerization interface that are conserved in human LHs, show that L230 dimer assemblies are stabilized by Fe2+-binding, and demonstrate that L230 has telopeptidyl LH activity and promotes cancer metastasis. These findings suggest that LHs have evolved a dimerization mode that adapts to the unique structural properties of collagen

Methods

Results

Conclusion