The study investigates the molecular basis of human coronavirus HKU1 binding to the transmembrane protease serine 2 (TMPRSS2) receptor, which is crucial for viral entry. The researchers designed an active human TMPRSS2 construct for high-yield recombinant production and determined a cryo-electron microscopy (cryoEM) structure of the HKU1 receptor-binding domain (RBD) bound to human TMPRSS2. This structure reveals the interactions that support viral entry and explain the specificity of HKU1 for TMPRSS2 among human type 2 transmembrane serine proteases. Key residues in the HKU1 RBD that govern host receptor usage were identified, and the role of these residues in TMPRSS2 binding was validated. The study also found that serum antibodies targeting the HKU1 RBD TMPRSS2 binding site are essential for neutralization, and HKU1 uses conformational masking and glycan shielding to balance immune evasion and receptor engagement. The findings provide insights into the molecular mechanisms of HKU1 infection and inform vaccine design.The study investigates the molecular basis of human coronavirus HKU1 binding to the transmembrane protease serine 2 (TMPRSS2) receptor, which is crucial for viral entry. The researchers designed an active human TMPRSS2 construct for high-yield recombinant production and determined a cryo-electron microscopy (cryoEM) structure of the HKU1 receptor-binding domain (RBD) bound to human TMPRSS2. This structure reveals the interactions that support viral entry and explain the specificity of HKU1 for TMPRSS2 among human type 2 transmembrane serine proteases. Key residues in the HKU1 RBD that govern host receptor usage were identified, and the role of these residues in TMPRSS2 binding was validated. The study also found that serum antibodies targeting the HKU1 RBD TMPRSS2 binding site are essential for neutralization, and HKU1 uses conformational masking and glycan shielding to balance immune evasion and receptor engagement. The findings provide insights into the molecular mechanisms of HKU1 infection and inform vaccine design.