Abstract
Hepatoma-derived growth factor (hHDGF) stimulates cell proliferation on both sides of plasma membrane by either binding to membrane receptor as a growth factor or binding to DNA in nucleus as a transcriptional factor. Secreted hHDGF recognizes cell surface heparan sulfate to promote its internalization and the N-terminal PWWP/HATH domain have been proved to be responsible for the heparan sulfate binding. The PWWP/HATH domains are highly conserved among the HDGF-related proteins (HRPs) with high identity > 70% and all domains contain the characteristic structural motif, PWWP motif. The PWWP motifs in HRPs can be classified into two types due to the difference of the first residue. One is with sequence of PHWP (Pro-His-Trp-Pro) and the other is AHWP. In order to realize the significance of the first residue of PWWP motif in mediating protein function and structure, we chose HDGF as models and examined the protein stability and heparin binding by replacing the Pro residue to Ala (mutation P24A). As shown by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), the binding affinity of heparin (KD ≈ 1×10-6 M) was less related to the residue. However, the substitution significantly reduced PWWP/HATH domain stability as reflected in NMR H/D exchange experiment and circular dichroism (CD) melting temperature measurement. In addition, we also detected NMR dynamic parameters on the local region to illustrate the dynamic differences derived from the mutation. Nevertheless, the phenomenon well corresponds to the severe aggregation tendency previously observed in AHWP-type HRPs. In summary, using site-direct mutagenesis and solution NMR method, we identified that the first residue of PWWP motif plays the key role in mediating PWWP domain stability.