DOI： 10.1038/s41428-025-01046-4

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Biological Chemistry  

Protein phosphorylation is a crucial process in various cellular functions, and its irregularities have been implicated in several diseases, including cancer. Antibodies are commonly employed to detect protein phosphorylation in research. However, unlike the extensive studies on recognition mechanisms of the phosphate group by proteins such as kinases and phosphatases, only a few studies have explored antibody mechanisms. In this study, we produced and characterized two rabbit monoclonal antibodies that recognize a monophosphorylated Akt peptide. Through crystallography, thermodynamic mutational analyses, and molecular dynamics simulations, we investigated the unique recognition mechanism that enables higher binding affinity and selectivity of the antibodies compared to other generic proteins with lower binding affinity to phosphorylated epitopes. Our results demonstrate that molecular dynamics simulations provide novel insights into the dynamic aspects of molecular recognition of posttranslational modifications by proteins beyond static crystal structures, highlighting how specific atomic level interactions drive the exceptional affinity and selectivity of antibodies.

DOI： 10.1016/j.jbc.2024.107989

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

<jats:p>Recent years have seen an uptick in the use of computational applications in antibody engineering. These tools have enhanced our ability to predict interactions with antigens and immunogenicity, facilitate humanization, and serve other critical functions. However, several studies highlight the concern of potential trade-offs between antibody affinity and stability in antibody engineering. In this study, we analyzed anti-measles virus antibodies as a case study, to examine the relationship between binding affinity and stability, upon identifying the binding hotspots. We leverage <jats:italic>in silico</jats:italic> tools like Rosetta and FoldX, along with molecular dynamics (MD) simulations, offering a cost-effective alternative to traditional <jats:italic>in vitro</jats:italic> mutagenesis. We introduced a pattern in identifying key residues in pairs, shedding light on hotspots identification. Experimental physicochemical analysis validated the predicted key residues by confirming significant decrease in binding affinity for the high-affinity antibodies to measles virus hemagglutinin. Through the nature of the identified pairs, which represented the relative hydropathy of amino acid side chain, a connection was proposed between affinity and stability. The findings of the study enhance our understanding of the interactions between antibody and measles virus hemagglutinin. Moreover, the implications of the observed correlation between binding affinity and stability extend beyond the field of anti-measles virus antibodies, thereby opening doors for advancements in antibody research.</jats:p>

DOI： 10.3389/fmolb.2023.1302737

Language：English   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Oral presentation (general)  

Language：Japanese   Presentation type：Oral presentation (general)  

Language：English