Crystal structure of human Dickkopf 4 cysteine-rich domain 1 and evaluation of conformational rigidity

Abstract

Dickkopf (DKK) family proteins (DKK1–DKK4), which function as extracellular modulators of Wnt signaling, contain two cysteine-rich domains: CRD1 and CRD2. In DKK1, CRD1 modulates interaction with its receptor low-density lipoprotein receptor-related protein (LRP) 5/6, whereas CRD2 directly binds to LRP5/6. The crystal structure of human DKK4-CRD1 was determined at 1.83 Å resolution. Crystals were obtained from refolded protein expressed as inclusion bodies and belonged to space group P2₁, with two molecules in the asymmetric unit. Initial molecular-replacement attempts using the solution NMR structure were unsuccessful, whereas an AlphaFold2-predicted model provided a clear solution. The refined structure reveals a compact fold comprising N- and C-subdomains connected by a linker region and stabilized by five conserved disulfide bonds. The crystal structure closely resembles the AlphaFold2 model, but shows larger deviations from the NMR ensemble. ANSURR analysis and hydrogen-bond comparisons indicate that the NMR models underestimate structural rigidity, particularly in β-sheet regions, owing to fewer stabilizing hydrogen bonds. Notably, enhanced conformational variability is observed in the N-subdomain, suggesting a potential role for structural plasticity in ligand recognition.