Structure of SARS-CoV-2 membrane protein essential for virus assembly

新型コロナウイルスのウイルス形成に必須の膜タンパク質の構造を解明. 京都大学プレスリリース. 2022-08-08.The coronavirus membrane protein (M) is the most abundant viral structural protein and plays a central role in virus assembly and morphogenesis. However, the process of M protein-driven virus assembly are largely unknown. Here, we report the cryo-electron microscopy structure of the SARS-CoV-2 M protein in two different conformations. M protein forms a mushroom-shaped dimer, composed of two transmembrane domain-swapped three-helix bundles and two intravirion domains. M protein further assembles into higher-order oligomers. A highly conserved hinge region is key for conformational changes. The M protein dimer is unexpectedly similar to SARS-CoV-2 ORF3a, a viral ion channel. Moreover, the interaction analyses of M protein with nucleocapsid protein (N) and RNA suggest that the M protein mediates the concerted recruitment of these components through the positively charged intravirion domain. Our data shed light on the M protein-driven virus assembly mechanism and provide a structural basis for therapeutic intervention targeting M protein

Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion

タンパク質の抗体ラベリング技術を改良し、構造解析をアシスト --電子顕微鏡やX線結晶解析による構造決定を加速化--. 京都大学プレスリリース. 2021-04-20.Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab

Binding and Unbinding Pathways of Peptide Substrates on the SARS-CoV‑2 3CL Protease

Based on many crystal structures of ligand complexes, much study has been devoted to understanding the molecular recognition of SARS-CoV-2 3C-like protease (3CLpro), a potent drug target for COVID-19. In this research, to extend this present static view, we examined the kinetic process of binding/unbinding of an eight-residue substrate peptide to/from 3CLpro by evaluating the path ensemble with the weighted ensemble simulation. The path ensemble showed the mechanism of how a highly flexible peptide folded into the bound form. At the early stage, the dominant motion was the diffusion on the protein surface showing a broad distribution, whose center was led into the cleft of the chymotrypsin fold. We observed a definite sequential formation of the hydrogen bonds at the later stage occurring in the cleft, initiated between Glu166 (3CLpro) and P3_Val (peptide), followed by binding to the oxyanion hole and completed by the sequence-specific recognition at P1_Gln

TLR3 forms a laterally aligned multimeric complex along double-stranded RNA for efficient signal transduction

TLR3 activates a potent immune response by binding to dsRNA. Here the authors report cryo-EM analyses to show that TLR3 dimers laterally form a higher multimeric complex along dsRNA, providing the basis for cooperative binding and efficient signal transduction

Apo- and Antagonist-Binding Structures of Vitamin D Receptor Ligand-Binding Domain Revealed by Hybrid Approach Combining Small-Angle X‑ray Scattering and Molecular Dynamics

Vitamin D receptor (VDR) controls the expression of numerous genes through the conformational change caused by binding 1α,25-dihydroxyvitamin D₃. Helix 12 in the ligand-binding domain (LBD) is key to regulating VDR activation. The structures of apo VDR-LBD and the VDR-LBD/antagonist complex are unclear. Here, we reveal their unprecedented structures in solution using a hybrid method combining small-angle X-ray scattering and molecular dynamics simulations. In apo rat VDR-LBD, helix 12 is partially unraveled, and it is positioned around the canonical active position and fluctuates. Helix 11 greatly bends toward the outside at Q396, creating a kink. In the rat VDR-LBD/antagonist complex, helix 12 does not generate the activation function 2 surface, and loop 11–12 is remarkably flexible compared to that in the apo rat VDR-LBD. On the basis of these structural insights, we propose a “folding-door model” to describe the mechanism of agonism/antagonism of VDR-LBD

A synthetic ion channel with anisotropic ligand response

Transmembrane proteins are important for cellular functions and synthetic analogues are of interest. Here the authors report on the design and testing of a synthetic multipass transmembrane channel which shows anisotropic responses to agonistic and antagonistic ligands