This study describes the development of a general strategy to retain the prefusion conformation of the MERS-CoV spike (S) protein, which is crucial for receptor binding and membrane fusion. The strategy involves introducing two consecutive proline substitutions at the beginning of the central helix, leading to increased expression levels and conformational homogeneity of the prefusion-stabilized S protein. This approach resulted in the production of high-titer neutralizing antibodies in mice, demonstrating enhanced immunogenicity compared to monomeric S1 protein and wild-type S trimer. The study also characterized the interaction of G4, a stem-directed neutralizing antibody, with the prefusion-stabilized MERS-CoV S protein. High-resolution structures of the trimeric MERS-CoV S ectodomain in complex with G4 revealed that G4 recognizes a glycosylated loop in the S2 connector domain, which is variable among coronaviruses. The structures identified four conformational states of the trimer, each with a different arrangement of the receptor-binding domains (RBDs). These findings provide insights into the mechanisms of MERS-CoV entry and antibody-mediated neutralization, and suggest potential strategies for the design of broadly protective coronavirus vaccines.This study describes the development of a general strategy to retain the prefusion conformation of the MERS-CoV spike (S) protein, which is crucial for receptor binding and membrane fusion. The strategy involves introducing two consecutive proline substitutions at the beginning of the central helix, leading to increased expression levels and conformational homogeneity of the prefusion-stabilized S protein. This approach resulted in the production of high-titer neutralizing antibodies in mice, demonstrating enhanced immunogenicity compared to monomeric S1 protein and wild-type S trimer. The study also characterized the interaction of G4, a stem-directed neutralizing antibody, with the prefusion-stabilized MERS-CoV S protein. High-resolution structures of the trimeric MERS-CoV S ectodomain in complex with G4 revealed that G4 recognizes a glycosylated loop in the S2 connector domain, which is variable among coronaviruses. The structures identified four conformational states of the trimer, each with a different arrangement of the receptor-binding domains (RBDs). These findings provide insights into the mechanisms of MERS-CoV entry and antibody-mediated neutralization, and suggest potential strategies for the design of broadly protective coronavirus vaccines.