Structure of the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii and its relation to other homotrimeric dUTPases

The bifunctional dCTP deaminase-dUTPase (DCD-DUT) from Methanocaldococcus jannaschii catalyzes the deamination of the cytosine moiety in dCTP and the hydrolysis of the triphosphate moiety forming dUMP, thereby preventing uracil from being incorporated into DNA. The crystal structure of DCD-DUT has been determined to 1.88-A resolution and represents the first known structure of an enzyme catalyzing dCTP deamination. The functional form of DCD-DUT is a homotrimer wherein the subunits are composed of a central distorted beta-barrel surrounded by two beta-sheets and four helices. The trimeric DCD-DUT shows structural similarity to trimeric dUTPases at the tertiary and quaternary levels. There are also additional structural elements in DCD-DUT compared with dUTPase because of a longer primary structure. Four of the five conserved sequence motifs that create the active sites in dUTPase are found in structurally equivalent positions in DCD-DUT. The last 25 C-terminal residues of the 204-residue-long DCD-DUT are not visible in the electron density map, but, analogous to dUTPases, the C terminus is probably ordered, closing the active site upon catalysis. Unlike other enzymes catalyzing the deamination of cytosine compounds, DCD-DUT is not exploiting an enzyme-bound metal ion such as zinc or iron for nucleophile generation. The active site contains two water molecules that are engaged in hydrogen bonds to the invariant residues Ser118, Arg122, Thr130, and Glu145. These water molecules are potential nucleophile candidates in the deamination reaction.     

The monomeric dUTPase from Epstein-Barr virus mimics trimeric dUTPases

Deoxyuridine 5'-triphosphate pyrophosphatases (dUTPases) are ubiquitous enzymes cleaving dUTP into dUMP and pyrophosphate. They occur as monomeric, dimeric, or trimeric molecules. The trimeric and monomeric enzymes both contain the same five characteristic sequence motifs but in a different order, whereas the dimeric enzymes are not homologous. Monomeric dUTPases only occur in herpesviruses, such as Epstein-Barr virus (EBV). Here, we describe the crystal structures of EBV dUTPase in complex with the product dUMP and a substrate analog alpha,beta-imino-dUTP. The molecule consists of three domains forming one active site that has a structure extremely similar to one of the three active sites of trimeric dUTPases. The three domains functionally correspond to the subunits of the trimeric form. Domains I and II have the dUTPase fold, but they differ considerably in the regions that are not involved in the formation of the unique active site, whereas domain III has only little secondary structure.     

Structures of dCTP deaminase from Escherichia coli with bound substrate and product: reaction mechanism and determinants of mono- and bifunctionality for a family of enzymes

dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg(115) and Glu(138), were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg(2+) that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.     

An intersubunit active site between supercoiled parallel beta helices in the trimeric tailspike endorhamnosidase of Shigella flexneri Phage Sf6

Sf6 belongs to the Podoviridae family of temperate bacteriophages that infect gram-negative bacteria by insertion of their double-stranded DNA. They attach to their hosts specifically via their tailspike proteins. The 1.25 A crystal structure of Shigella phage Sf6 tailspike protein (Sf6 TSP) reveals a conserved architecture with a central, right-handed beta helix. In the trimer of Sf6 TSP, the parallel beta helices form a left-handed, coiled-beta coil with a pitch of 340 A. The C-terminal domain consists of a beta sandwich reminiscent of viral capsid proteins. Further crystallographic and biochemical analyses show a Shigella cell wall O-antigen fragment to bind to an endorhamnosidase active site located between two beta-helix subunits each anchoring one catalytic carboxylate. The functionally and structurally related bacteriophage, P22 TSP, lacks sequence identity with Sf6 TSP and has its active sites on single subunits. Sf6 TSP may serve as an example for the evolution of different host specificities on a similar general architecture.     

Self-complementary motifs (SCM) in alpha-crystallin small heat shock proteins

Small heat shock proteins (sHsp) have been implicated in many cell processes involving the dynamics of protein-protein interactions. Two unusual sequences containing self-complementary motifs (SCM) have been identified within the conserved alpha-crystallin domain of sHsps. When two SCMs are aligned in an anti-parallel direction (N to C and C to N), the charged or polar residues form either salt bridges or hydrogen bonds while the non-polar residues participate in hydrophobic interactions. When aligned in reverse order, the residues of these motifs in alpha-crystallin subunits form either hydrophobic and/or polar interactions. Homology based molecular modeling of the C-terminal domain of alpha-crystallin subunits using the crystal structure of MjHSP16.5 suggests that SCM1 and 2 participate in stabilizing secondary structure and subunit interactions. Also there is overwhelming evidence that these motifs are important in the chaperone-like activity of alpha-crystallin subunits. These sequences are conserved and appear to be characteristic of the entire sHsp superfamily. Similar motifs are also present in the Hsp70 family and the immunoglobulin superfamily.     

Conformational flexibility of the C terminus with implications for substrate binding and catalysis revealed in a new crystal form of deacetoxycephalosporin C synthase

Deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus catalyses the oxidative ring expansion of the penicillin nucleus into the nucleus of cephalosporins. The reaction requires dioxygen and 2-oxoglutarate as co-substrates to create a reactive iron-oxygen intermediate from a ferrous iron in the active site. The active enzyme is monomeric in solution. The structure of DAOCS was determined earlier from merohedrally twinned crystals where the last four C-terminal residues (308-311) of one molecule penetrate the active site of a neighbouring molecule, creating a cyclic trimeric structure in the crystal. Shortening the polypeptide chain from the C terminus by more than four residues diminishes activity. Here, we describe a new crystal form of DAOCS in which trimer formation is broken and the C-terminal arm is free. These crystals show no signs of twinning, and were obtained from DAOCS labelled with an N-terminal His-tag. The modified DAOCS is catalytically active. The free C-terminal arm protrudes into the solvent, and the C-terminal domain (residues 268-299) is rotated by about 16 degrees towards the active site. The last 12 residues (300-311) are disordered. Structures for various enzyme-substrate and enzyme-product complexes in the new crystal form confirm overlapping binding sites for penicillin and 2-oxoglutarate. The results support the notion that 2-oxoglutarate and dioxygen need to react first to produce an oxidizing iron species, followed by reaction with the penicillin substrate. The position of the penicillin nucleus is topologically similar in the two crystal forms, but the penicillin side-chain in the new non-twinned crystals overlaps with the position of residues 304-306 of the C-terminal arm in the twinned crystals. An analysis of the interactions between the C-terminal region and residues in the active site indicates that DAOCS could also accept polypeptide chains as ligands, and these could bind near the iron.     

Norovirus proteinase-polymerase and polymerase are both active forms of RNA-dependent RNA polymerase

In vitro mapping studies of the MD145 norovirus (Caliciviridae) ORF1 polyprotein identified two stable cleavage products containing the viral RNA-dependent RNA polymerase (RdRp) domains: ProPol (a precursor comprised of both the proteinase and polymerase) and Pol (the mature polymerase). The goal of this study was to identify the active form (or forms) of the norovirus polymerase. The recombinant ProPol (expressed as Pro(-)Pol with an inactivated proteinase domain to prevent autocleavage) and recombinant Pol were purified after synthesis in bacteria and shown to be active RdRp enzymes. In addition, the mutant His-E1189A-ProPol protein (with active proteinase but with the natural ProPol cleavage site blocked) was active as an RdRp, confirming that the norovirus ProPol precursor could possess two enzymatic activities simultaneously. The effects of several UTP analogs on the RdRp activity of the norovirus and feline calicivirus Pro(-)Pol enzymes were compared and found to be similar. Our data suggest that the norovirus ProPol is a bifunctional enzyme during virus replication. The availability of this recombinant ProPol enzyme might prove useful in the development of antiviral drugs for control of the noroviruses associated with acute gastroenteritis.     

The structure of human lymphotoxin (tumor necrosis factor-beta) at 1.9-A resolution.

The three-dimensional structure of recombinant human lymphotoxin (residues 24-171 of the mature protein) has been determined by x-ray crystallography at 1.9-A resolution (Rcryst = 0.215 for I greater than 3 sigma (I)). Phases were derived by molecular replacement using tumor necrosis factor (TNF-alpha) as a search model. Like TNF-alpha, lymphotoxin (LT) folds to form a "jellyroll" beta-sheet sandwich. Three-fold related LT subunits form a trimer stabilized primarily by hydrophobic interactions. A cluster of 6 basic residues around the 3-fold axis may account for the acid lability of the trimer. Although the structural cores of TNF-alpha and LT are similar, insertions and deletions relative to TNF-alpha occur in loops at the "top" of the LT trimer and significantly alter the local structure and the overall shape trimer is highly conserved. The sites of two mutations (Asp-50 and Tyr-108) that abolish the cytotoxicity of LT are contained within poorly ordered loops of polypeptide chain that flank the cleft between neighboring subunits at the base of the molecule, suggesting that the receptor recognizes an intersubunit binding site.     

Expression, purification, and structural analysis of the trimeric form of the catalytic domain of the Escherichia coli dihydrolipoamide succinyltransferase

The dihydrolipoamide succinyltransferase (E2o) component of the alpha-ketoglutarate dehydrogenase complex catalyzes the transfer of a succinyl group from the S-succinyldihydrolipoyl moiety to coenzyme A. E2o is normally a 24-mer, but is found as a trimer when E2o is expressed with a C-terminal [His]6 tag. The crystal structure of the trimeric form of the catalytic domain (CD) of the Escherichia coli E2o has been solved to 3.0 A resolution using the Molecular Replacement method. The refined model contains an intact trimer in the asymmetric unit and has an R-factor of 0.257 (Rfree = 0.286) for 18,699 reflections between 10.0 and 3.0 A resolution. The core of tE2oCD (residues 187-396) superimposes onto that of the cubic E2oCD with an RMS difference of 0.4 A for all main-chain atoms. The C-terminal end of tE2oCD (residues 397-404) rotates by an average of 37 degrees compared to cubic E2oCD, disrupting the normal twofold interface. Despite the alteration of quaternary structure, the active site of tE2oCD shows no significant differences from that of the cubic E2oCD, although several side chains in the active site are more ordered in the trimeric form of E2oCD. Analysis of the available sequence data suggests that the majority of E2 components have active sites that resemble that of E. coli E2oCD. The remaining E2 components can be divided into three groups based on active-site sequence similarity. Analysis of the surface properties of both crystal forms of E. coli E2oCD suggests key residues that may be involved in the protein-protein contacts that occur between the catalytic and lipoyl domains of E2o.     

Molecular shape and prominent role of β-strand swapping in organization of dUTPase oligomers

Most dUTP pyrophosphatases (dUTPases) are homotrimers with interfaces formed between subunit surfaces, in the central channel, and by C-terminal β-strand swapping. Analysis of intersubunit interactions reveals an important cohesive role for the C-terminus. This is reflected in the crystal structure of fruitfly dUTPase displaying a dimeric organization in crystals grown in alcohol solution, where only β-strand swapping interactions between subunits are retained from the usual trimer structure. Mutations of a suggested hinge proline destabilize human and Escherichia coli dUTPases without preventing trimeric organization. Trimer formation was, however, prevented in the human enzyme by truncating the C-terminus before the swapping arm. The molecular shape of full-length enzymes in solution reveals the localization and variation in flexibility of N- and C-terminal segments.

Structured summary

MINT-6946477:dUTPase (uniprotkb:Q9V3I1) and dUTPase (uniprotkb:Q9V3I1) bind (MI:0407) by X-ray crystallography (MI:0114)     

Crosslinking-MS analysis reveals RNA polymerase I domain architecture and basis of rRNA cleavage

RNA polymerase (Pol) I contains a 10-subunit catalytic core that is related to the core of Pol II and includes subunit A12.2. In addition, Pol I contains the heterodimeric subcomplexes A14/43 and A49/34.5, which are related to the Pol II subcomplex Rpb4/7 and the Pol II initiation factor TFIIF, respectively. Here we used lysine-lysine crosslinking, mass spectrometry (MS) and modeling based on five crystal structures, to extend the previous homology model of the Pol I core, to confirm the location of A14/43 and to position A12.2 and A49/34.5 on the core. In the resulting model of Pol I, the C-terminal ribbon (C-ribbon) domain of A12.2 reaches the active site via the polymerase pore, like the C-ribbon of the Pol II cleavage factor TFIIS, explaining why the intrinsic RNA cleavage activity of Pol I is strong, in contrast to the weak cleavage activity of Pol II. The A49/34.5 dimerization module resides on the polymerase lobe, like TFIIF, whereas the A49 tWH domain resides above the cleft, resembling parts of TFIIE. This indicates that Pol I and also Pol III are distantly related to a Pol II-TFIIS-TFIIF-TFIIE complex.     

Atomic structure of the major capsid protein of rotavirus: implications for the architecture of the virion

The structural protein VP6 of rotavirus, an important pathogen responsible for severe gastroenteritis in children, forms the middle layer in the triple-layered viral capsid. Here we present the crystal structure of VP6 determined to 2 A resolution and describe its interactions with other capsid proteins by fitting the atomic model into electron cryomicroscopic reconstructions of viral particles. VP6, which forms a tight trimer, has two distinct domains: a distal beta-barrel domain and a proximal alpha-helical domain, which interact with the outer and inner layer of the virion, respectively. The overall fold is similar to that of protein VP7 from bluetongue virus, with the subunits wrapping about a central 3-fold axis. A distinguishing feature of the VP6 trimer is a central Zn(2+) ion located on the 3-fold molecular axis. The crude atomic model of the middle layer derived from the fit shows that quasi-equivalence is only partially obeyed by VP6 in the T = 13 middle layer and suggests a model for the assembly of the 260 VP6 trimers onto the T = 1 viral inner layer.     

Pregenomic RNA is required for efficient incorporation of pol polyprotein into foamy virus capsids

The foamy virus (FV) Pol polyprotein is translated independently of Gag from a spliced mRNA. This method of expression raises the question of how Pol is associated with the viral particle. Using a transient FV vector transfection system, it is shown that pregenomic RNA is required for efficient virion incorporation of functionally active Pol and that protein-protein interactions of Pol with Gag are not sufficient to complete particle assembly.     

Structure of the foot-and-mouth disease virus leader protease: a papain-like fold adapted for self-processing and eIF4G recognition.

The leader protease of foot-and-mouth disease virus, as well as cleaving itself from the nascent viral polyprotein, disables host cell protein synthesis by specific proteolysis of a cellular protein: the eukaryotic initiation factor 4G (eIF4G). The crystal structure of the leader protease presented here comprises a globular catalytic domain reminiscent of that of cysteine proteases of the papain superfamily, and a flexible C-terminal extension found intruding into the substrate-binding site of an adjacent molecule. Nevertheless, the relative disposition of this extension and the globular domain to each other supports intramolecular self-processing. The different sequences of the two substrates cleaved during viral replication, the viral polyprotein (at LysLeuLys/GlyAlaGly) and eIF4G (at AsnLeuGly/ArgThrThr), appear to be recognized by distinct features in a narrow, negatively charged groove traversing the active centre. The structure illustrates how the prototype papain fold has been adapted to the requirements of an RNA virus. Thus, the protein scaffold has been reduced to a minimum core domain, with the active site being modified to increase specificity. Furthermore, surface features have been developed which enable C-terminal self-processing from the viral polyprotein.     

Terminase Subunits from the Pseudomonas-Phage E217

Pseudomonas phages are increasingly important biomedicines for phage therapy, but little is known about how these viruses package DNA. This paper explores the terminase subunits from the Myoviridae E217, a Pseudomonas-phage used in an experimental cocktail to eradicate P. aeruginosa in vitro and in animal models. We identified the large (TerL) and small (TerS) terminase subunits in two genes ～58 kbs away from each other in the E217 genome. TerL presents a classical two-domain architecture, consisting of an N-terminal ATPase and C-terminal nuclease domain arranged into a bean-shaped tertiary structure. A 2.05 Å crystal structure of the C-terminal domain revealed an RNase H-like fold with two magnesium ions in the nuclease active site. Mutations in TerL residues involved in magnesium coordination had a dominant-negative effect on phage growth. However, the two ions identified in the active site were too far from each other to promote two-metal-ion catalysis, suggesting a conformational change is required for nuclease activity. We also determined a 3.38 Å cryo-EM reconstruction of E217 TerS that revealed a ring-like decamer, departing from the most common nonameric quaternary structure observed thus far. E217 TerS contains both N-terminal helix-turn-helix motifs enriched in basic residues and a central channel lined with basic residues large enough to accommodate double-stranded DNA. Overexpression of TerS caused a more than a 4-fold reduction of E217 burst size, suggesting a catalytic amount of the protein is required for packaging. Together, these data expand the molecular repertoire of viral terminase subunits to Pseudomonas-phages used for phage therapy.     

Unusual Conformation of the SxN Motif in the Crystal Structure of Penicillin-Binding Protein A from Mycobacterium tuberculosis

PBPA from Mycobacterium tuberculosis is a class B-like penicillin-binding protein (PBP) that is not essential for cell growth in M. tuberculosis, but is important for proper cell division in Mycobacterium smegmatis. We have determined the crystal structure of PBPA at 2.05 Å resolution, the first published structure of a PBP from this important pathogen. Compared to other PBPs, PBPA has a relatively small N-terminal domain, and conservation of a cluster of charged residues within this domain suggests that PBPA is more related to class B PBPs than previously inferred from sequence analysis. The C-terminal domain is a typical transpeptidase fold and contains the three conserved active-site motifs characterisitic of penicillin-interacting enzymes. Whilst the arrangement of the SxxK and KTG motifs is similar to that observed in other PBPs, the SxN motif is markedly displaced away from the active site, such that its serine (Ser281) is not involved in hydrogen bonding with residues of the other two motifs. A disulfide bridge between Cys282 (the “x” of the SxN motif) and Cys266, which resides on an adjacent loop, may be responsible for this unusual conformation. Another interesting feature of the structure is a relatively long connection between β5 and α11, which restricts the space available in the active site of PBPA and suggests that conformational changes would be required to accommodate peptide substrate or β-lactam antibiotics during acylation. Finally, the structure shows that one of the two threonines postulated to be targets for phosphorylation is inaccessible (Thr362), whereas the other (Thr437) is well placed on a surface loop near the active site.     

Structural platform for the autolytic activity of an intact NS2B-NS3 protease complex from dengue virus

Dengue virus completes its protein synthesis inside human cells on the endoplasmic reticulum membrane by processing the single-chain polyprotein precursor into 10 functional proteins. This vital process relies on the two-component virus-encoded protease complex; nonstructural protein 3 (NS3) possesses the proteolytic activity in its N-terminus, and NS2B acts as a fundamental activator and membrane-anchoring subunit. The membrane-associated NS2B-NS3 complex has essentially not yet been isolated or studied. We describe here a useful protocol for the preparation of the full-length NS2B-NS3 complex from dengue serotype 2 virus by utilizing a Mistic-fusion expression cassette in Escherichia coli. The protease complex was successfully solubilized and stabilized from the bacterial membrane and purified with the use of fos-choline-14 detergent. The detergent-solubilized protease complex retained autolytic activity and, intriguingly, exists as a robust trimer, implying a molecular assembly in the membrane. We further conducted a random mutagenesis study to efficiently scan for entire residues and motifs contributing to autocleavage and provide evidence of the importance of the two distal β-hairpins in the activity of the viral protease. Our results provide the first comprehensive view of an active dengue protease in the membrane-bound form.     

Structure and active site residues of PglD, an N-acetyltransferase from the bacillosamine synthetic pathway required for N-glycan synthesis in Campylobacter jejuni

Campylobacter jejuni is highly unusual among bacteria in forming N-linked glycoproteins. The heptasaccharide produced by its pgl system is attached to protein Asn through its terminal 2,4-diacetamido-2,4,6-trideoxy-d-Glc (QuiNAc4NAc or N,N'-diacetylbacillosamine) moiety. The crucial, last part of this sugar's synthesis is the acetylation of UDP-2-acetamido-4-amino-2,4,6-trideoxy-d-Glc by the enzyme PglD, with acetyl-CoA as a cosubstrate. We have determined the crystal structures of PglD in CoA-bound and unbound forms, refined to 1.8 and 1.75 A resolution, respectively. PglD is a trimer of subunits each comprised of two domains, an N-terminal alpha/beta-domain and a C-terminal left-handed beta-helix. Few structural differences accompany CoA binding, except in the C-terminal region following the beta-helix (residues 189-195), which adopts an extended structure in the unbound form and folds to extend the beta-helix upon binding CoA. Computational molecular docking suggests a different mode of nucleotide-sugar binding with respect to the acetyl-CoA donor, with the molecules arranged in an "L-shape", compared with the "in-line" orientation in related enzymes. Modeling indicates that the oxyanion intermediate would be stabilized by the NH group of Gly143', with His125' the most likely residue to function as a general base, removing H+ from the amino group prior to nucleophilic attack at the carbonyl carbon of acetyl-CoA. Site-specific mutations of active site residues confirmed the importance of His125', Glu124', and Asn118. We conclude that Asn118 exerts its function by stabilizing the intricate hydrogen bonding network within the active site and that Glu124' may function to increase the pKa of the putative general base, His125'.     

Crystal structure of imidazole glycerol-phosphate dehydratase: duplication of an unusual fold

Imidazole glycerol-phosphate dehydratase (IGPD) catalyzes the sixth step of histidine biosynthesis. The enzyme is of fundamental biochemical interest, because it catalyzes removal of a non-acidic hydrogen atom in the dehydration reaction. It is also a potential target for development of herbicides. IGPD is a metalloenzyme in which transition metals induce aggregation and are required for catalysis. Addition of 1 equivalent of Mn(2+)/subunit is shown by analytical ultracentrifugation to induce the formation of 24-mers from trimeric IGPD. Two histidine-rich motifs may participate in metal binding and aggregation. The 2.3-A crystal structure of metal-free trimeric IGPD from the fungus Filobasidiella neoformans reveals a novel fold containing an internal repeat, apparently the result of gene duplication. The 95-residue alpha/beta half-domain occurs in a few other proteins, including the GHMP kinase superfamily (galacto-homoserine-mevalonate-phosphomevalonate), but duplication to form a compact domain has not been seen elsewhere. Conserved residues cluster at two types of sites in the trimer, each site containing a conserved histidine-rich motif. A model is proposed for the intact, active 24-mer in which all highly conserved residues, including the histidine-rich motifs in both the N- and C-terminal halves of the polypeptide, cluster at a common site between trimers. This site is a candidate for the active site and also for metal binding leading to aggregation of trimers. The structure provides a basis for further studies of enzyme function and mechanism and for development of more potent and specific herbicides.     

A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral M^pro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. The lack of structural information for intermediary forms of M^pro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 M^pro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the M^pro bound to its endogenous N and C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.