Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes the disease COVID-19, produces replicase polyproteins 1a and 1ab that contain, respectively, 11 or 16 nonstructural proteins (nsp). Nsp5 is the main protease (Mpro) responsible for cleavage at eleven positions along these polyproteins, including at its own N- and C-terminal boundaries, representing essential processing events for subsequent viral assembly and maturation. We have determined X-ray crystallographic structures of this cysteine protease in its wild-type free active site state at 1.8 Å resolution, in its acyl-enzyme intermediate state with the native C-terminal autocleavage sequence at 1.95 Å resolution and in its product bound state at 2.0 Å resolution by employing an active site mutation (C145A). We characterize the stereochemical features of the acyl-enzyme intermediate including critical hydrogen bonding distances underlying catalysis in the Cys/His dyad and oxyanion hole. We also identify a highly ordered water molecule in a position compatible for a role as the deacylating nucleophile in the catalytic mechanism and characterize the binding groove conformational changes and dimerization interface that occur upon formation of the acyl-enzyme. Collectively, these crystallographic snapshots provide valuable mechanistic and structural insights for future antiviral therapeutic development including revised molecular docking strategies based on Mpro inhibition.

CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in the synthesis of phosphatidylcholine, the most abundant phospholipid in eukaryotic cell membranes. The CCT-catalyzed transfer of a cytidylyl group from CTP to phosphocholine to form CDP-choline is regulated by a membrane lipid-dependent mechanism imparted by its C-terminal membrane binding domain. We present the first analysis of a crystal structure of a eukaryotic CCT. A deletion construct of rat CCT␣ spanning residues 1-236 (CCT236) lacks the regulatory domain and as a result displays constitutive activity. The 2.2-Å structure reveals a CCT236 homodimer in complex with the reaction product, CDP-choline. Each chain is composed of a complete catalytic domain with an intimately associated N-terminal extension, which together with the catalytic domain contributes to the dimer interface. Although the CCT236 structure reveals elements involved in binding cytidine that are conserved with other members of the cytidylyltransferase superfamily, it also features nonconserved active site residues, His-168 and Tyr-173, that make key interactions with the ␤-phosphate of CDP-choline. Mutagenesis and kinetic analyses confirmed their role in phosphocholine binding and catalysis. These results demonstrate structural and mechanistic differences in a broadly conserved protein fold across the cytidylyltransferase family. Comparison of the CCT236 structure with those of other nucleotidyltransferases provides evidence for substrate-induced active site loop movements and a disorder-to-order transition of a loop element in the catalytic mechanism.A key rate-limiting step in the synthesis of phosphatidylcholine in animal cells is the formation of the headgroup donor, CDP-choline, by transfer of a cytidylyl group from CTP to phosphocholine. This reaction is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT 4 ; EC 2.7.7.15), an enzyme subject to many layers of regulation (1-4). The ubiquitous and best studied isoform of mammalian CCT (CCT␣, 367 residues) has been described as having four domains (Fig. 1A). An N-terminal domain (ϳ75 residues) housing its nuclear localization signal (NLS) sequence is followed by an ϳ150-residue catalytic domain, an ϳ60-residue membrane binding domain (domain M), and an unstructured phosphorylated tail (ϳ50 residues) (2, 4). CCT functions as a homodimer (5).CCT activation requires transformation of the enzyme from a soluble form to a membrane lipid-bound form. When the full-length soluble CCT interacts with anionic membrane surfaces, domain M transforms from a mixture of structural elements into an amphipathic ␣-helix (6 -8). Domain M appears to act as an autoinhibitory device, whose interaction with phosphatidylcholine-deficient membranes releases an inhibitory constraint at the active site to enhance k cat by 2 orders of magnitude (9). The primary evidence for this model is the constitutive activity of a construct lacking domain M, CCT236 (9).To elucidate the mechanism whereby membrane binding activates this important re...

Background:The CCT M-domain contains an autoinhibitory (AI) segment, but its mechanism was obscure. Results: The AI helix partly occludes active site access and engages a mobile loop (L2), impeding the dynamics of key catalytic lysine 122. Conclusion: Loop L2 is a key target of the AI clamp. Significance: This work reveals the nature of a regulatory off-switch in an enzyme regulated by membrane binding.

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The activity of CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme in phosphatidylcholine synthesis, is regulated by reversible interactions of a lipid-inducible amphipathic helix (domain M) with membrane phospholipids. When dissociated from membranes, a portion of the M domain functions as an auto-inhibitory (AI) element to suppress catalysis. The AI helix from each subunit binds to a pair of α helices (αE) that extend from the base of the catalytic dimer to create a four-helix bundle. The bound AI helices make intimate contact with loop L2, housing a key catalytic residue, Lys The impacts of the AI helix on active-site dynamics and positioning of Lys are unknown. Extensive MD simulations with and without the AI helix revealed that backbone carbonyl oxygens at the point of contact between the AI helix and loop L2 can entrap the Lys side chain, effectively competing with the substrate, CTP. , removal of the AI helices dramatically increased αE dynamics at a predicted break in the middle of these helices, enabling them to splay apart and forge new contacts with loop L2. cross-linking confirmed the reorganization of the αE element upon membrane binding of the AI helix. Moreover, when αE bending was prevented by disulfide engineering, CCT activation by membrane binding was thwarted. These findings suggest a novel two-part auto-inhibitory mechanism for CCT involving capture of Lys and restraint of the pliable αE helices. We propose that membrane binding enables bending of the αE helices, bringing the active site closer to the membrane surface.

Glucoamylase from Aspergillus niger is an industrially important biocatalyst that is utilized in the mass production of glucose from raw starch or soluble oligosaccharides. The G1 isoform consists of a catalytic domain and a starch-binding domain connected by a heavily glycosylated linker region. The amino-terminal catalytic domain of the G1 isoform generated by subtilisin cleavage has been crystallized at pH 8.5, which is a significantly higher pH condition than used for previously characterized glucoamylase crystals. The refined structure at 1.9 Å resolution reveals the active site of the enzyme in complex with both Tris and glycerol molecules. The ligands display both unique and analogous interactions with the substrate-binding site when compared with previous structures of homologous enzymes bound to inhibitors.

Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus that infects salmonid fish, encodes a large polyprotein (NH 2 -pVP2-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease, VP4, to release the proteins pVP2 and VP3. pVP2 is further processed to give rise to the capsid protein VP2 and three peptides that are incorporated into the virion. Reported here are two crystal structures of the IPNV VP4 protease solved from two different crystal symmetries. The electron density at the active site in the triclinic crystal form, refined to 2.2-Å resolution, reveals the acyl-enzyme complex formed with an internal VP4 cleavage site. The complex was generated using a truncated enzyme in which the general base lysine was substituted. Inside the complex, the nucleophilic Ser 633 O␥ forms an ester bond with the main-chain carbonyl of the C-terminal residue, Ala 716 , of a neighboring VP4. The structure of this substrate-VP4 complex allows us to identify the S1, S3, S5, and S6 substrate binding pockets as well as other substrate-VP4 interactions and therefore provides structural insights into the substrate specificity of this enzyme. The structure from the hexagonal crystal form, refined to 2.3-Å resolution, reveals the free-binding site of the protease. Three-dimensional alignment with the VP4 of blotched snakehead virus, another birnavirus, shows that the overall structure of VP4 is conserved despite a low level of sequence identity (ϳ19%). The structure determinations of IPNV VP4, the first of an acyl-enzyme complex for a Ser/Lys dyad protease, provide insights into the catalytic mechanism and substrate recognition of this type of protease.Birnaviruses are non-enveloped viruses of ϳ60 -70 nm in diameter, which replicate in the cytoplasm of their host cells (1) and are characterized by their two double-stranded RNA genomic segments (A and B) (2). Segment A displays two overlapping reading frames, the larger one encoding a polyprotein (NH 2 -pVP2-VP4-VP3-COOH; Fig. 1). The polyprotein is processed through the proteolytic activity of VP4 to generate the proteins pVP2 and VP3 as well as VP4 (3). During virus assembly, pVP2 is further processed by VP4 to generate the capsid protein VP2 and several structural peptides. The crystal structure of the infectious bursal disease virus particle (an avian birnavirus) solved at a resolution of 7 Å revealed the VP2 protein incorporated into an iscosahedral viral capsid with triangulation T ϭ 13 (4).The Birnaviridae virus family includes three genera, Aquabirnavirus, Avibirnavirus, and Entomobirnavirus, which infect fish, birds, and insects, respectively (5, 6). Infectious pancreatic necrosis virus (IPNV) 3 is a well known pathogen in salmonid fish. It is responsible for infectious pancreatic necrosis, a disease characterized by severe damage to the internal organs and tissues (7). Because of the high mortality rate it induces and its widespread distribution, this virus is a major economical and ecological threat to the aquaculture and sea farmin...