RESUMEN
Infection with human noroviruses requires attachment to histo blood group antigens (HBGAs) via the major capsid protein VP1 as a primary step. Several crystal structures of VP1 protruding domain dimers, so called P-dimers, complexed with different HBGAs have been solved to atomic resolution. Corresponding binding affinities have been determined for HBGAs and other glycans exploiting different biophysical techniques, with mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy being most widely used. However, reported binding affinities are inconsistent. At the extreme, for the same system MS detects binding whereas NMR spectroscopy does not, suggesting a fundamental source of error. In this short essay, we will explain the reason for the observed differences and compile reliable and reproducible binding affinities. We will then highlight how a combination of MS techniques and NMR experiments affords unique insights into the process of HBGA binding by norovirus capsid proteins.
Asunto(s)
Antígenos de Grupos Sanguíneos , Norovirus , Sitios de Unión , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Humanos , Norovirus/química , Norovirus/metabolismo , Polisacáridos/metabolismo , Unión ProteicaRESUMEN
The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo-microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. Molecular dynamics simulations indicate that this unique architecture generates specificity for SPs based on the length of their hydrophobic segments.
Asunto(s)
Retículo Endoplásmico/enzimología , Péptido Hidrolasas/metabolismo , Señales de Clasificación de Proteína , Serina Endopeptidasas/metabolismo , Células A549 , Dominio Catalítico , Microscopía por Crioelectrón , Células HEK293 , Células Hep G2 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Simulación de Dinámica Molecular , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Proteómica , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Relación Estructura-Actividad , Especificidad por Sustrato , Espectrometría de Masas en Tándem , Células U937RESUMEN
Glycan-protein interactions are highly specific yet transient, rendering glycans ideal recognition signals in a variety of biological processes. In human norovirus (HuNoV) infection, histo-blood group antigens (HBGAs) play an essential but poorly understood role. For murine norovirus infection (MNV), sialylated glycolipids or glycoproteins appear to be important. It has also been suggested that HuNoV capsid proteins bind to sialylated ganglioside head groups. Here, we study the binding of HBGAs and sialoglycans to HuNoV and MNV capsid proteins using NMR experiments. Surprisingly, the experiments show that none of the norovirus P-domains bind to sialoglycans. Notably, MNV P-domains do not bind to any of the glycans studied, and MNV-1 infection of cells deficient in surface sialoglycans shows no significant difference compared to cells expressing respective glycans. These findings redefine glycan recognition by noroviruses, challenging present models of infection.
