Abstract
Protein N-phosphorylation represents one of the principal forms of protein phosphorylation modifications, which has been extensively identified in both prokaryotes and eukaryotes. However, significant knowledge gaps persist regarding its functional implications, sequence motifs, and structural characteristics. Through integrated functional, sequential, and structural analyses of experimentally verified large-scale N-phosphorylation datasets, we found that protein N-phosphorylation broadly participates in diverse biological processes and exhibits close associations with human diseases, particularly cancer. And the high motif conservation between N- and O-phosphorylation in eukaryotes offers novel research perspectives for investigating N-phosphorylation through the lens of O-phosphorylation. Moreover, the structural-sequence pattern analyses demonstrate that relative solvent accessibility at modification sites provides more distinctive characterization of phosphorylation sites than other structural-sequence descriptors. These findings provide evidence supporting the potential role of N-phosphorylation in biological processes and contribute to its further development in biological sciences, analytical chemistry, and medical research.