1, red ovals) to give 13a (see scheme S1 for synthesis). Further, to increase the solubility of the compound in plasma and to reduce its binding to plasma proteins, we replaced the hydrophobic cinnamoyl moiety by the somewhat less hydrophobic Boc group ( Fig. 1, green ovals) in the expectation that this might prevent cellular proteases from accessing this bond and cleaving it. To improve the half-life of the compound in plasma, we modified 11r by hiding the P3-P2 amide bond within a pyridone ring ( Fig. 1) showed an half-maximal effective concentration (EC 50) of 400 pM against Middle East respiratory syndrome–coronavirus (MERS-CoV) in Huh7 cells as well as low-μM EC 50 values against SARS-CoV and a whole range of enteroviruses in various cell lines, although the antiviral activity seemed to depend to a great extent on the cell type used in the experiments ( 6). The results uncovered that the stability profile of the neuronal SNARE complex reflects its potency to drive fast and robust membrane fusion and provided potential guiding principle in rationally design artificial SNARE complexes and remold functional SNARE complexes with desirable stability.Previously, we designed and synthesized peptidomimetic α-ketoamides as broad-spectrum inhibitors of the main proteases of betacoronaviruses and alphacoronaviruses as well as the 3C proteases of enteroviruses ( 6). This study focused on one of the most well-known SNARE complexes, the neuronal SNARE complex, and investigated the residues in the SNARE motif in maintaining stability and generating the half-zippered conformation by multiple biophysical approaches. Resolving the functional mechanisms of the SNARE complex endowed by these thermodynamic features in driving efficient membrane fusion is a fundamental task for biophysical research. However, the multiple assembly conformation seems to thermodynamically conflict with the nature of high stability. The SNARE complexes usually possess high thermostability and multiple assembly conformations, which is thought to be crucial for their abilities in catalyzing most types of membrane fusion processes. Based on these results, we also developed a new parameter, the stability factor ( F s), to characterize the overall stability of the neuronal SNARE complex and resolved a linear correlation between the stability and inter-residue coulombic interactions of the neuronal SNARE complex, which would help rationally design artificial SNARE complexes and remold functional SNARE complexes with desirable stability. Overall, these results uncovered that the stability profile of the neuronal SNARE complex reflects its potency to drive fast and robust membrane fusion.

Furthermore, our data confirmed a strong coupling of folding energy between the N- and C-half assembly of the neuronal SNARE complex, which rationalizes the strong potency of the half-zippered conformation to conduct robust and fast fusion. Our data showed that the core residues predominantly contribute to the complex stability to meet a basal requirement for SNARE-mediated membrane fusion, while, the core-side residues exert an unbalanced effect on the N- and C-half bundle stability that determines the half-zippered conformation of the neuronal SNARE complex, which would accommodate essential regulations by complexins and Synaptotagmins for fast Ca 2+-triggered membrane fusion. We reclassified the amino acids of the SNARE motif into four sub-groups (core, core-side I and II, and non-contact). In this study, we have employed temperature-dependent disassociation assays and single-molecule FRET experiments to analyze the stability and conformation of the neuronal SNARE complex. However, these two features seem to be thermodynamically mutual exclusive. The neuronal SNARE complex is featured by its high stability and half-zippered conformation required for driving robust and fast synaptic exocytosis. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) form the SNARE complex to mediate most fusion events of the secretory pathway.

Expi293 membrane pro crystal structure