sedimentation-equilibrium-small-oligomer-forming-membrane-protein

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and ...

Nanyang Technological University

Coronavirus envelope (E) proteins are short (~100 residues) polypeptides that contain at least one transmembrane (TM) domain and a cluster of 2-3 juxtamembrane cysteines. These proteins are involved in viral morphogenesis and tropism, and their absence leads in some cases to aberrant virions, or to viral attenuation. In common to other viroporins, coronavirus envelope proteins increase membrane permeability to ions. Although an NMR-based model for the TM domain of the E protein in the severe acute respiratory syndrome virus (SARS-CoV E) has been reported, structural data and biophysical studies of full length E proteins are not available because efficient expression and purification methods for these proteins are lacking. Herein we have used a novel fusion protein consisting of a modified β-barrel to purify both wild type and cysteine-less mutants of two representatives of coronavirus E proteins: the shortest (76 residues), from SARS-CoV E, and one of the longest (109 residues), from the infectious bronchitis virus (IBV E). The fusion construct was subsequently cleaved with cyanogen bromide and all polypeptides were obtained with high purity. This is an approach that can be used in other difficult hydrophobic peptides.

Expression and purification of coronavirus envelope proteins using a modified β-barrel construct.

The accepted model for the interaction of α and β integrins in the transmembrane (TM) domain is based on the pair αIIbβ3. This involves the so-called outer and inner membrane association clasps (OMC and IMC, respectively). In the α chain, the OMC involves a GxxxG-like motif, whereas in the IMC a conserved juxtamembrane GFFKR motif experiences a backbone reversal that partially fills the void generated by TM separation towards the cytoplasmic half. However, the GFFKR motif of several α integrin cytoplasmic tails in non-bicelle environments has been shown to adopt an α-helical structure that is not membrane-embedded and which was shown to bind a variety of cytoplasmic proteins. Thus it is not known if a membrane-embedded backbone reversal is a conserved structural feature in α integrins. We have studied the system αLβ2 because of its importance in leukocytes, where integrin deactivation is particularly important. Herein we show that the backbone reversal feature is not only present in αIIb but also in αL-TM when reconstituted in bicelles. Additionally, titration with β2 TM showed eight residues clustering along one side of αL-TM, forming a plausible interacting face with β2. The latter orientation is consistent with a previously predicted reported polar interaction between αL Ser-1071 and β2 Thr-686.

Transmembrane and Juxtamembrane Structure of αL Integrin in Bicelles.

The p7 protein from the hepatitis C virus (HCV) is a 63 amino acid long polypeptide that is essential for replication, and is involved in protein trafficking and proton transport. Therefore, p7 is a possible target for antivirals. The consensus model for the channel formed by p7 protein is a hexameric or heptameric oligomer of α-helical hairpin monomers, each having two transmembrane domains, TM1 and TM2, where the N-terminal TM1 would face the lumen of this channel. A reported high-throughput functional assay to search for p7 channel inhibitors is based on carboxyfluorescein (CF) release from liposomes after p7 addition. However, the rationale for the dual ability of p7 to serve as an ion or proton channel in the infected cell, and to permeabilize membranes to large molecules like CF is not clear. We have recreated both activities in vitro, examining the conformation present in these assays using infrared spectroscopy. Our results indicate that an α-helical form of p7, which can transport protons, is not able to elicit CF release. In contrast, membrane permeabilization to CF is observed when p7 contains a high percentage of β-structure, or when using a C-terminal fragment of p7, encompassing TM2. We propose that the reported inhibitory effect of some small compounds, e.g., rimantadine, on both CF release and proton transport can be explained via binding to the membrane-inserted C-terminal half of p7, increasing its rigidity, in a similar way to the influenza A M2-rimantadine interaction.

Two different conformations in hepatitis C virus p7 protein account for proton transport and dye release.

Crystal (Cry) toxins are widely used for insect control, but their mechanism of toxicity is still uncertain. These toxins can form lytic pores in vitro, and water soluble tetrameric pre-pore intermediates have been reported. Even the precise oligomeric state of the toxin in membranes, trimeric or tetrameric, is still a debated issue. Based on previous reports, we have assumed that interactions between toxin monomers in solution are at least partly mediated by domain I, and we have analyzed in silico the homo-oligomerization tendencies of the domain I α-helices individually. Using many homologous sequences for each α-helix, our strategy allows selection of evolutionarily conserved interactions. These interactions appeared only in helices α3 and α5, but only α3 produced a suitably oriented or α-helical sample in lipid bilayers, forming homotetramers in C14-betaine, and allowing determination of its rotational orientation in lipid bilayers using site-specific infrared dichroism (SSID). The determined orientation in the tetrameric model is in agreement with only one of the evolutionarily conserved models. In addition mutation R99E, which was found to inhibit oligomerization experimentally, greatly destabilized the tetramer in molecular dynamic simulations. In this model, helix 3 is able to form inter-monomer interactions without significant rearrangements of domain I, which is compatible with the available crystal structure of Cry toxins in solution. The model presented here at least partially explains the reported tetrameric oligomerization of Cry toxins in solution and the inhibition of this oligomerization by a synthetic α3 peptide.

A conserved tetrameric interaction of cry toxin helix α3 suggests a functional role for toxin oligomerization.

Coronavirus envelope (CoV E) proteins are ∼100-residue polypeptides with at least one channel-forming α-helical transmembrane (TM) domain. The extramembrane C-terminal tail contains a completely conserved proline, at the center of a predicted β-coil-β motif. This hydrophobic motif has been reported to constitute a Golgi-targeting signal or a second TM domain. However, no structural data for this or other extramembrane domains in CoV E proteins is available. Herein, we show that the E protein in the severe acute respiratory syndrome virus has only one TM domain in micelles, whereas the predicted β-coil-β motif forms a short membrane-bound α-helix connected by a disordered loop to the TM domain. However, complementary results suggest that this motif is potentially poised for conformational change or in dynamic exchange with other conformations.

Structure of a conserved Golgi complex-targeting signal in coronavirus envelope proteins.

The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by the human respiratory syncytial virus (hRSV). SH protein has a single α-helical transmembrane (TM) domain that forms pentameric ion channels. Herein, we report the first inhibitor of the SH protein channel, pyronin B, and we have mapped its binding site to a conserved surface of the RSV SH pentamer, at the C-terminal end of the transmembrane domain. The validity of the SH protein structural model used has been confirmed by using a bicellar membrane-mimicking environment. However, in bicelles the α-helical stretch of the TM domain extends up to His-51, and by comparison with previous models both His-22 and His-51 adopt an interhelical/lumenal orientation relative to the channel pore. Neither His residue was found to be essential for channel activity although His-51 protonation reduced channel activity at low pH, with His-22 adopting a more structural role. The latter results are in contrast with previous patch clamp data showing channel activation at low pH, which could not be reproduced in the present work. Overall, these results establish a solid ground for future drug development targeting this important viroporin. Importance: The human respiratory syncytial virus (hRSV) is responsible for 64 million reported cases of infection and 160,000 deaths each year. Lack of adequate antivirals fuels the search for new targets for treatment. The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by hRSV and other paramyxoviruses, and its absence leads to viral attenuation in vivo and early apoptosis in infected cells. SH protein forms pentameric ion channels that may constitute novel drug targets, but no inhibitor for this channel activity has been reported so far. A small-molecule inhibitor, pyronin B, can reduce SH channel activity, and its likely binding site on the SH protein channel has been identified. Black lipid membrane (BLM) experiments confirm that protonation of both histidine residues reduces stability and channel activity. These results contrast with previous patch clamp data that showed low-pH activation, which we have not been able to reproduce.

Inhibition of the human respiratory syncytial virus small hydrophobic protein and structural variations in a bicelle environment.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly identified pathogen able of human transmission that causes a mortality of almost 40%. As in the case of SARS-CoV, MERS virus lacking E protein represents a potential vaccine. In both cases, abolishment of channel activity may be a contributor to the attenuation observed in E-deleted viruses. Herein, we report that purified MERS-CoV E protein, like SARS-CoV E protein, is almost fully α-helical, has a single α-helical transmembrane domain, and forms pentameric ion channels in lipid bilayers. Based on these similarities, and the proposed involvement of channel activity as virulence factor in SARS-CoV E protein, MERS-CoV E protein may constitute a potential drug target.

Wahyu Surya

School of Biological Sciences

Nanyang Technological University

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability