RESUMO
Chloroplast signal recognition particle (cpSRP) is a heterodimer composed of an evolutionarily conserved 54-kDa GTPase (cpSRP54) and a unique 43-kDa subunit (cpSRP43) responsible for delivering light-harvesting chlorophyll binding protein to the thylakoid membrane. While a nearly complete three-dimensional structure of cpSRP43 has been determined, no high-resolution structure is yet available for cpSRP54. In this study, we developed and examined an in silico three-dimensional model of the structure of cpSRP54 by homology modeling using cytosolic homologs. Model selection was guided by single-molecule Förster resonance energy transfer experiments, which revealed the presence of at least two distinct conformations. Small angle x-ray scattering showed that the linking region among the GTPase (G-domain) and methionine-rich (M-domain) domains, an M-domain loop, and the cpSRP43 binding C-terminal extension of cpSRP54 are predominantly disordered. Interestingly, the linker and loop segments were observed to play an important role in organizing the domain arrangement of cpSRP54. Further, deletion of the finger loop abolished loading of the cpSRP cargo, light-harvesting chlorophyll binding protein. These data highlight important structural dynamics relevant to cpSRP54's role in the post- and cotranslational signaling processes.
Assuntos
GTP Fosfo-Hidrolases/química , Partícula de Reconhecimento de Sinal/química , Animais , Arabidopsis , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cloroplastos/metabolismo , Cães , Escherichia coli , Transferência Ressonante de Energia de Fluorescência , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Methanocaldococcus , Simulação de Dinâmica Molecular , Mutação , Domínios Proteicos , Espalhamento a Baixo Ângulo , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismo , Homologia Estrutural de Proteína , Sulfolobus solfataricus , Thermus , Difração de Raios XRESUMO
New SARS-CoV-2 variants that have accumulated multiple mutations in the spike (S) glycoprotein enable increased transmission and resistance to neutralizing antibodies. Here, we study the antigenic and structural impacts of the S protein mutations from four variants, one that was involved in transmission between minks and humans, and three that rapidly spread in human populations and originated in the United Kingdom, Brazil or South Africa. All variants either retained or improved binding to the ACE2 receptor. The B.1.1.7 (UK) and B.1.1.28 (Brazil) spike variants showed reduced binding to neutralizing NTD and RBD antibodies, respectively, while the B.1.351 (SA) variant showed reduced binding to both NTD- and RBD-directed antibodies. Cryo-EM structural analyses revealed allosteric effects of the mutations on spike conformations and revealed mechanistic differences that either drive inter-species transmission or promotes viral escape from dominant neutralizing epitopes. HIGHLIGHTS: Cryo-EM structures reveal changes in SARS-CoV-2 S protein during inter-species transmission or immune evasion.Adaptation to mink resulted in increased ACE2 binding and spike destabilization.B.1.1.7 S mutations reveal an intricate balance of stabilizing and destabilizing effects that impact receptor and antibody binding.E484K mutation in B.1.351 and B.1.1.28 S proteins drives immune evasion by altering RBD conformation.S protein uses different mechanisms to converge upon similar solutions for altering RBD up/down positioning.