Human DKK4-CRD1 crystal structure reveals compact fold, five disulfide bonds, and N-subdomain flexibility
Background
Dickkopf (DKK) family proteins (DKK1-DKK4) are crucial extracellular modulators of Wnt signaling, a pathway implicated in diverse processes from embryonic development to disease pathogenesis, including head and neck squamous cell carcinoma (HNSCC) and chronic inflammation. DKK1's cysteine-rich domain 1 (CRD1) modulates interaction with its receptor, low-density lipoprotein receptor-related protein (LRP) 5/6. However, the precise structural details of DKK4-CRD1, particularly its conformational dynamics, remained elusive, hindering a complete understanding of its regulatory mechanisms and potential therapeutic targeting.
Study Design
Researchers expressed and refolded human DKK4-CRD1 from inclusion bodies to obtain purified protein. They then crystallized the protein and determined its crystal structure at 1.83 Å resolution. Initial molecular-replacement attempts using solution NMR structures were unsuccessful, prompting the use of an AlphaFold2-predicted model, which provided a clear solution. The team performed ANSURR analysis and hydrogen-bond comparisons to evaluate structural rigidity against NMR ensembles.
Results
The refined crystal structure of DKK4-CRD1 revealed a compact fold, comprising distinct N- and C-subdomains connected by a linker region. This structure is robustly stabilized by five conserved disulfide bonds. The determined crystal structure closely resembled the AlphaFold2 model, demonstrating its predictive accuracy, but showed larger deviations from previously published NMR ensembles. The crystals belonged to space group P21, with two molecules observed in the asymmetric unit.
ANSURR analysisand hydrogen-bond comparisons indicated that the NMR models significantly underestimated structural rigidity, particularly within the β-sheet regions, due to fewer stabilizing hydrogen bonds. Notably, enhanced conformational variability was observed within the N-subdomain, suggesting a potential role for this structural plasticity in specific ligand recognition events.
Key Findings
- Human DKK4-CRD1 crystal structure determined at 1.83 Å resolution.
- Structure reveals a compact fold with N- and C-subdomains, stabilized by five conserved disulfide bonds.
- Crystal structure closely matches AlphaFold2 model, but deviates from NMR ensembles.
- NMR models underestimated structural rigidity, particularly in β-sheet regions.
- Enhanced conformational variability observed in the N-subdomain, suggesting a role in ligand recognition.
Why It Matters
This detailed structural elucidation of DKK4-CRD1 provides a fundamental blueprint for understanding its role in modulating Wnt signaling and offers a critical foundation for rational drug design. The identified conformational flexibility in the N-subdomain could represent a novel target for developing small-molecule modulators or peptide mimetics aimed at regulating DKK4's activity. Furthermore, the successful use of an AlphaFold2-predicted model for molecular replacement highlights the increasing utility and accuracy of AI-driven protein structure prediction, potentially accelerating future structural biology efforts and drug discovery pipelines.
dkk4
wnt-signaling
protein-structure
crystallography
alphafold2
in-vitro