Human coronavirus HKU1 recognizes the TMPRSS2 host receptor to initiate infection. This study designed an active human TMPRSS2 construct for high-yield recombinant production, enabling detailed biochemical and structural analysis. A cryo-electron microscopy structure of the HKU1 RBD bound to human TMPRSS2 revealed the molecular interactions supporting viral entry and explaining the specificity for TMPRSS2 among human type 2 transmembrane serine proteases. Key residues governing host receptor usage were identified, and data showed that serum antibodies targeting the HKU1 RBD TMPRSS2 binding site are critical for neutralization. HKU1 uses conformational masking and glycan shielding to balance immune evasion and receptor engagement. HKU1 is a beta-coronavirus that circulates seasonally in humans. It was first identified in 2005 and has been found in multiple continents. Although often considered a common cold virus, it can cause severe illness, particularly in vulnerable populations. Its high seroprevalence indicates continuous circulation and endemicity. Coronavirus infections begin with the trimeric spike (S) glycoprotein interacting with a host cell receptor, leading to membrane fusion and genome delivery. Only a few proteinaceous receptors have been identified as promoting coronavirus infection, all being host transmembrane proteases. TMPRSS2 is a key entry receptor for HKU1, likely engaged upon S conformational changes triggered by 9-O-acetylated disialosides binding. The study designed an active human TMPRSS2 construct for high-yield recombinant production, enabling detailed biochemical and structural analysis. The cryo-EM structure of the HKU1 RBD bound to human TMPRSS2 provided insights into the interactions mediating receptor engagement, specificity, and host receptor tropism. Analysis of serum antibodies revealed that the TMPRSS2-binding site is a key vulnerability for HKU1, becoming exposed upon sialoglycan-mediated RBD opening. The study also identified that several TMPRSS2 SNPs may affect HKU1 binding, with some mutations reducing susceptibility to HKU1 infection. The molecular determinants of HKU1 utilization of human TMPRSS2 and host receptor tropism were elucidated, revealing that rat, hamster, and camel TMPRSS2 orthologs support HKU1 S-mediated entry into cells. Glycan shielding and conformational masking mediate immune evasion by masking the receptor-binding motif (RBM) and reducing targeting by neutralizing antibodies. The study showed that RBM-directed serum antibodies are key for neutralization, and that the removal of the N355 glycan could improve antibody responses elicited by HKU1 S. The study provides a molecular blueprint for understanding the evolution of RBM residues and their impact on receptor binding and immune evasion. The identification of the RBM as a key site of vulnerability suggests that removal of the N355 glycan could be a suitable vaccineHuman coronavirus HKU1 recognizes the TMPRSS2 host receptor to initiate infection. This study designed an active human TMPRSS2 construct for high-yield recombinant production, enabling detailed biochemical and structural analysis. A cryo-electron microscopy structure of the HKU1 RBD bound to human TMPRSS2 revealed the molecular interactions supporting viral entry and explaining the specificity for TMPRSS2 among human type 2 transmembrane serine proteases. Key residues governing host receptor usage were identified, and data showed that serum antibodies targeting the HKU1 RBD TMPRSS2 binding site are critical for neutralization. HKU1 uses conformational masking and glycan shielding to balance immune evasion and receptor engagement. HKU1 is a beta-coronavirus that circulates seasonally in humans. It was first identified in 2005 and has been found in multiple continents. Although often considered a common cold virus, it can cause severe illness, particularly in vulnerable populations. Its high seroprevalence indicates continuous circulation and endemicity. Coronavirus infections begin with the trimeric spike (S) glycoprotein interacting with a host cell receptor, leading to membrane fusion and genome delivery. Only a few proteinaceous receptors have been identified as promoting coronavirus infection, all being host transmembrane proteases. TMPRSS2 is a key entry receptor for HKU1, likely engaged upon S conformational changes triggered by 9-O-acetylated disialosides binding. The study designed an active human TMPRSS2 construct for high-yield recombinant production, enabling detailed biochemical and structural analysis. The cryo-EM structure of the HKU1 RBD bound to human TMPRSS2 provided insights into the interactions mediating receptor engagement, specificity, and host receptor tropism. Analysis of serum antibodies revealed that the TMPRSS2-binding site is a key vulnerability for HKU1, becoming exposed upon sialoglycan-mediated RBD opening. The study also identified that several TMPRSS2 SNPs may affect HKU1 binding, with some mutations reducing susceptibility to HKU1 infection. The molecular determinants of HKU1 utilization of human TMPRSS2 and host receptor tropism were elucidated, revealing that rat, hamster, and camel TMPRSS2 orthologs support HKU1 S-mediated entry into cells. Glycan shielding and conformational masking mediate immune evasion by masking the receptor-binding motif (RBM) and reducing targeting by neutralizing antibodies. The study showed that RBM-directed serum antibodies are key for neutralization, and that the removal of the N355 glycan could improve antibody responses elicited by HKU1 S. The study provides a molecular blueprint for understanding the evolution of RBM residues and their impact on receptor binding and immune evasion. The identification of the RBM as a key site of vulnerability suggests that removal of the N355 glycan could be a suitable vaccine