Fig. 4: Structural elucidation and interaction profiling of PgAFP and AfAFP with Flag-Strep-tagged variants in A. flavus.

A The overall crystal structure of PgAFP, obtained through recombinant expression methods, is presented as a gray-colored cartoon representation. The predicted three-dimensional structure of AfAFP is shown. B, C The three-dimensional structures of PgAFP-Flag-Strep and AfAFP-Flag-Strep fusion proteins are illustrated, showing the molecular architecture. D Tandem affinity purification coupled with mass spectrometry (TAP-MS) was used with PgAFP-Flag-Strep and AfAFP-Flag-Strep as bait protein to isolate potential interacting proteins of AFPs in A. flavus. A total of 76 and 58 candidate protein targets were identified, respectively. A subsequent comparative analysis of the proteomes identified 38 proteins potentially influenced by the AFPs. E The 38 candidate interacting proteins of AFPs in A. flavus were systematically categorized. F, G Analysis of the binding interactions between Ntp1^546–588. E The 38 candidate interacting proteins of AFPs in A. flavus were systematically categorized. F, G Analysis of the binding interactions between Ntp1546–588 (colored with gray) and the Flag-Strep tagged variants of PgAFP (yellow) and AfAFP (yellow) suggested that the Flag-Strep tag did not contribute to the recognition process of Ntp1 by the AFPs. Furthermore, the predicted protein-protein interaction structures are categorized by four levels of confidence intervals. High confidence: regions with pLDDT > 90, indicating high modeling accuracy. Moderate confidence: regions with pLDDT between 70 and 90, generally reflect accurate backbone predictions. Low confidence: regions with pLDDT between 50 and 70, which should be interpreted cautiously. Structural disorder: regions with pLDDT < 50, suggesting potential structural disorder.