Receptor binding and proteolysis do not induce large conformational changes in the SARS-CoV spike

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 as a highly transmissible pathogenic human betacoronavirus. The viral spike glycoprotein (S) utilizes angiotensin-converting enzyme 2 (ACE2) as a host protein receptor and mediates fusion of the viral and host membranes, making S essential to viral entry into host cells and host species tropism. As SARS-CoV enters host cells, the viral S undergoes two proteolytic cleavages at S1/S2 and S2’ sites necessary for efficient membrane fusion. Here, we present a cryo-EM analysis of the trimeric SARS-CoV S interactions with ACE2 and of the trypsin-cleaved S. Surprisingly, neither binding to ACE2 nor cleavage by trypsin at the S1/S2 cleavage site impart large conformational changes within S or expose the secondary cleavage site, S2’. These observations suggest that S2’ cleavage does not occur in the S prefusion conformation and that additional triggers may be required.

Biosynthesis and Transport of the Lantibiotic Mutacin 1140 Produced by Streptococcus mutans

ABSTRACTLantibiotics are ribosomally synthesized peptide antibiotics composed of an N-terminal leader peptide that is cleaved to yield the active antibacterial peptide. Significant advancements in molecular tools that promote the study of lantibiotic biosynthesis can be used inStreptococcus mutans. Herein, we further our understanding of leader peptide sequence and core peptide structural requirements for the biosynthesis and transport of the lantibiotic mutacin 1140. Our study on mutacin 1140 biosynthesis shows a dedicated secondary cleavage site within the leader peptide and the dependency of transport on core peptide posttranslational modifications (PTMs). The secondary cleavage site on the leader peptide is found at the −9 position, and secondary cleavage occurs before the core peptide is transported out of the cell. The coordinated cleavage at the −9 position was absent in alanTdeletion strain, suggesting that the core peptide interaction with the LanT transporter enables uniform cleavage at the −9 position. Following transport, the LanP protease was found to be tolerant to a wide variety of amino acid substitutions at the primary leader peptide cleavage site, with the exception of arginine at the −1 position. Several leader and core peptide mutations produced core peptide variants that had intermediate stages of PTM enzyme modifications, supporting the concept that PTM enzyme modifications, secondary cleavage, and transport are occurring in a highly coordinated fashion.IMPORTANCEMutacin 1140 belongs to the class I lantibiotic family of ribosomally synthesized and posttranslationally modified peptides (RiPPs). The biosynthesis of mutacin 1140 is a highly efficient process which does not lead to a discernible level of production of partially modified core peptide variants. The products isolated from an extensive mutagenesis study on the leader and core peptides of mutacin 1140 show that the posttranslational modifications (PTMs) on the core peptide occur under a highly coordinated dynamic process. PTMs are dictated by the distance of the core peptide modifiable residues from PTM enzyme active sites. The formation of lanthionine rings aids in the formation of successive PTMs, as was observed in a peptide variant lacking a C-terminal decarboxylation.

Damage and Fracture Mechanism of a Nickel-Based Single Crystal Superalloy during Creep at Moderate Temperature

By means of creep properties measurement and microstructure observation, an investigation has been made into the damage and fracture mechanism of a nickel-based single crystal superalloy during creep at moderate temperature. Results show that the deformation mechanism of the alloy in the latter stage of creep is that the primary-secondary slipping systems are alternately activated, and the micro-crack is firstly initiated on the γ′/γ phases interface in the intersection regions of two slip systems. As creep goes on, the micro-crack is propagated along the γ′/γ interface, which is perpendicular to stress axis, to form the square-like cleavage plane on the (001) plane. Thereinto, the propagation of the cracks on (001) plane is intersected with {111} cleavage plane which is secondly activated, which may terminate the propagation of the crack to form the cleavage plane with square-like feature on (001) plane along the <110> directions. Due to the multi-cracks may be propagated on different cross-section of the alloy during creep, and the tearing edge or secondary cleavage plane are formed along the direction with bigger shearing stress at the crack tip, which makes the multi-cracks connected each other until the occurrence of creep fracture, this is thought to be the main reason of the creep fracture having the uneven and multi-level cleavage characteristics.

In vitro maturation of Drosophila melanogaster Spätzle protein with refolded Easter reveals a novel cleavage site within the prodomain

Dorsoventral patterning during Drosophila melanogaster embryogenesis is mediated by a well-defined gradient of the mature NGF-like ligand Spätzle. Easter, the ultimate protease of a ventrally-restricted serine protease cascade, plays a key role in the regulation of the morphogenic gradient, catalyzing the activation cleavage of proSpätzle. As a result of alternative splicing, proSpätzle exists in multiple isoforms, almost all of which differ only in their prodomain. Although this domain is unstructured in isolation, it has a stabilizing influence on the mature cystine knot domain and is involved in the binding to the Toll receptor. Here, we report the expression and refolding of Easter, and show that the renatured enzyme performs the activation cleavage of two Spätzle isoforms. We determine the affinity of the prodomain for the cystine knot domain, and show that Easter performs a previously unknown secondary cleavage in each prodomain.

Reconstitution of human azurocidin catalytic triad and proteolytic activity by site-directed mutagenesis

Azurocidin belongs to the serprocidin family, but it is devoid of proteolytic activity due to a substitution of His and Ser residues in the catalytic triad. The aim of this study was to reconstitute the active site of azurocidin by site-directed mutagenesis, analyze its processing and restored proteolytic activity. Azurocidin expressed inSf9 insect cells possessing the reconstituted His41-Asp89-Ser175 triad exhibited significant proteolytic activity toward casein with a pH optimum of approximately 8–9, but a reconstitution of only one active site amino acid did not result in proteolytically active protein. Enzymatically active recombinant azurocidin caused cleavage of the C-terminal fusion tag with the primary cleavage site after lysine at Lys-Leu and after alanine at Ala-Ala, and the secondary cleavage site after arginine at Arg-Gln, as well as with low efficiency caused cleavage of insulin chain B after leucine at Leu-Tyr and Leu-Cys, and after alanine at Ala-Leu. We demonstrate that cleavage of the azurocidin C-terminal tripeptide is not necessary for its enzymatic activity. The first isoleucine present in mature azurocidin can be replaced by similar amino acids, such as leucine or valine, but its substitution by histidine or arginine decreases proteolytic activity.