RESUMEN
The inhibition of human angiotensin I converting enzyme (ACE) has been regarded as a promising approach for the treatment of hypertension. Despite research attempts over many years, our understanding the mechanisms of activation and inhibition of ACE is still far from complete. Here, we present results of all atom molecular dynamics simulations of ACE with and without ligands. Two types of inhibitors, competitive and mixed non-competitive, were used to model the ligand bound forms. In the absence of a ligand the simulation showed spontaneous large hinge-bending motions of multiple conversions between the closed and open states of ACE, while the ligand bound forms were stable in the closed state. Our simulation results imply that the equilibrium between pre-existing backbone conformations shifts in the presence of a ligand. The hinge-bending motion of ACE is considered as an essential to the enzyme function. A mechanistic model of activation and the inhibition may provide valuable information for novel inhibitors of ACE.
Asunto(s)
Hipertensión/tratamiento farmacológico , Peptidil-Dipeptidasa A/química , Unión Proteica/efectos de los fármacos , Conformación Proteica , Sitios de Unión/efectos de los fármacos , Humanos , Hipertensión/genética , Ligandos , Simulación de Dinámica Molecular , Peptidil-Dipeptidasa A/efectos de los fármacos , Peptidil-Dipeptidasa A/ultraestructura , TermodinámicaRESUMEN
The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral , Receptores Virales/metabolismo , Anticuerpos de Dominio Único/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Secuencia de Aminoácidos , Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Neutralizantes/ultraestructura , Anticuerpos Antivirales/metabolismo , Anticuerpos Antivirales/ultraestructura , Afinidad de Anticuerpos , Reacciones Antígeno-Anticuerpo/inmunología , Betacoronavirus/metabolismo , Unión Competitiva , COVID-19 , Microscopía por Crioelectrón , Cristalografía por Rayos X , Epítopos/inmunología , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/inmunología , Modelos Moleculares , Biblioteca de Péptidos , Peptidil-Dipeptidasa A/ultraestructura , Unión Proteica , Conformación Proteica , Receptores Virales/ultraestructura , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , SARS-CoV-2 , Homología de Secuencia de Aminoácido , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/ultraestructura , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/ultraestructuraRESUMEN
Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for severe acute respiratory syndrome-coronavirus (SARS-CoV) and the new coronavirus (SARS-CoV-2) that is causing the serious coronavirus disease 2019 (COVID-19) epidemic. Here, we present cryo-electron microscopy structures of full-length human ACE2 in the presence of the neutral amino acid transporter B0AT1 with or without the receptor binding domain (RBD) of the surface spike glycoprotein (S protein) of SARS-CoV-2, both at an overall resolution of 2.9 angstroms, with a local resolution of 3.5 angstroms at the ACE2-RBD interface. The ACE2-B0AT1 complex is assembled as a dimer of heterodimers, with the collectrin-like domain of ACE2 mediating homodimerization. The RBD is recognized by the extracellular peptidase domain of ACE2 mainly through polar residues. These findings provide important insights into the molecular basis for coronavirus recognition and infection.