Breaking and forming peptidyl bonds are fundamental biochemical reactions in protein chemistry. Unlike proteases that are abundantly available, fast-acting ligases are rare. OaAEP1 is an enzyme isolated from the cyclotide-producing plant oldenlandia affinis that displayed weak peptide cyclase activity, despite having a similar structural fold with other asparaginyl endopeptidases (AEP). Here we report the first atomic structure of OaAEP1, at a resolution of 2.56 Å, in its preactivation form. Our structure and biochemical analysis of this enzyme reveals its activation mechanism as well as structural features important for its ligation activity. Importantly, through structure-based mutagenesis of OaAEP1, we obtained an ultrafast variant having hundreds of times faster catalytic kinetics, capable of ligating well-folded protein substrates using only a submicromolar concentration of enzyme. In contrast, the protein-protein ligation activity in the original wild-type OaAEP1 enzyme described previously is extremely weak. Thus, the structure-based engineering of OaAEP1 described here provides a unique and novel recombinant tool that can now be used to conduct various protein labeling and modifications that were extremely challenging before.

Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptideproducing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1′ sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs. peptide ligase | data mining | ligase-activity determinant

The Spike (S) protein is the main handle for SARS-CoV-2 to enter host cells via surface ACE2 receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, using amide hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations, we have mapped the S:ACE2 interaction interface and uncovered long-range allosteric propagation of ACE2 binding to sites necessary for host-mediated proteolysis of S protein, critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking protease docking site ~27 Å away while dampening dynamics of the stalk hinge (central helix and heptad repeat) regions ~130 Å away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the pre-fusion state. Our findings provide a dynamics map of the S:ACE2 interface in solution and also offer mechanistic insights into how ACE2 binding is allosterically coupled to distal proteolytic processing sites and viral-host membrane fusion. Our findings highlight protease docking sites flanking the S1/S2 cleavage site, fusion peptide and heptad repeat 1 (HR1) as alternate allosteric hotspot targets for potential therapeutic development.

Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs.

SUMMARY Dengue is the most common vector-borne viral disease, causing nearly 400 million infections yearly. Currently there are no approved therapies. Antibody epitopes that elicit weak humoral responses may not be accessible by conventional B cell panning methods. To demonstrate an alternative strategy to generating a therapeutic antibody, we employed a non-immunodominant, but functionally relevant, epitope in domain III of the E protein, and engineered by structure-guided methods an antibody directed to it. The resulting antibody, Ab513, exhibits high-affinity binding to, and broadly neutralizes, multiple genotypes within all four serotypes. To assess therapeutic relevance of Ab513, activity against important human clinical features of dengue was investigated. Ab513 mitigates thrombocytopenia in a humanized mouse model, resolves vascular leakage, reduces viremia to nearly undetectable levels, and protects mice in a maternal transfer model of lethal antibody-mediated enhancement. The results demonstrate that Ab513 may reduce the public health burden from dengue.