This study describes the rational design of a prefusion-stabilized MERS-CoV spike (S) protein antigen that elicits high neutralizing antibody titers. The S protein, which mediates receptor recognition and membrane fusion, was engineered to remain in the prefusion conformation using structure-based design. The engineered immunogen, MERS S-2P, was shown to retain high-affinity binding to its receptor DPP4 and a panel of neutralizing antibodies. Cryo-EM structures of MERS S-2P in complex with a stem-directed neutralizing antibody, G4, revealed that G4 recognizes a glycosylated loop in the S2 connector domain and avoids the glycosylation via its angle of approach. The structures also defined four conformational states of the trimer, with each receptor-binding domain either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation. These findings suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines. The study also highlights the importance of the S2 stem as a conserved target for neutralizing antibodies and demonstrates that prefusion-stabilized S proteins can elicit more robust neutralizing antibody responses than other forms of the S protein. The results have implications for the development of broadly protective vaccines against coronaviruses, including MERS-CoV and SARS-CoV.This study describes the rational design of a prefusion-stabilized MERS-CoV spike (S) protein antigen that elicits high neutralizing antibody titers. The S protein, which mediates receptor recognition and membrane fusion, was engineered to remain in the prefusion conformation using structure-based design. The engineered immunogen, MERS S-2P, was shown to retain high-affinity binding to its receptor DPP4 and a panel of neutralizing antibodies. Cryo-EM structures of MERS S-2P in complex with a stem-directed neutralizing antibody, G4, revealed that G4 recognizes a glycosylated loop in the S2 connector domain and avoids the glycosylation via its angle of approach. The structures also defined four conformational states of the trimer, with each receptor-binding domain either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation. These findings suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines. The study also highlights the importance of the S2 stem as a conserved target for neutralizing antibodies and demonstrates that prefusion-stabilized S proteins can elicit more robust neutralizing antibody responses than other forms of the S protein. The results have implications for the development of broadly protective vaccines against coronaviruses, including MERS-CoV and SARS-CoV.