RESUMO
The emergence and spread of antibiotic resistance represent a growing threat to public health. Of particular concern is the appearance of ß-lactamases, which are capable to hydrolyze and inactivate the most important class of antibiotics, the ß-lactams. Effective ß-lactamase inhibitors and mechanistic insights into their action are central in overcoming this type of resistance, and in this context boronate-based ß-lactamase inhibitors were just recently approved to treat multidrug-resistant bacteria. Using boric acid as a simplified inhibitor model, time-resolved serial crystallography was employed to obtain mechanistic insights into binding to the active site serine of ß-lactamase CTX-M-14, identifying a reaction time frame of 80-100 ms. In a next step, the subsequent 1,2-diol boric ester formation with glycerol in the active site was monitored proceeding in a time frame of 100-150 ms. Furthermore, the displacement of the crucial anion in the active site of the ß-lactamase was verified as an essential part of the binding mechanism of substrates and inhibitors. In total, 22 datasets of ß-lactamase intermediate complexes with high spatial resolution of 1.40-2.04 Å and high temporal resolution range of 50-10,000 ms were obtained, allowing a detailed analysis of the studied processes. Mechanistic details captured here contribute to the understanding of molecular processes and their time frames in enzymatic reactions. Moreover, we could demonstrate that time-resolved crystallography can serve as an additional tool for identifying and investigating enzymatic reactions.
RESUMO
Emerging RNA viruses, including SARS-CoV-2, continue to be a major threat. Cell entry of SARS-CoV-2 particles via the endosomal pathway involves cysteine cathepsins. Due to ubiquitous expression, cathepsin L (CatL) is considered a promising drug target in the context of different viral and lysosome-related diseases. We characterized the anti-SARS-CoV-2 activity of a set of carbonyl- and succinyl epoxide-based inhibitors, which were previously identified as inhibitors of cathepsins or related cysteine proteases. Calpain inhibitor XII, MG-101, and CatL inhibitor IV possess antiviral activity in the very low nanomolar EC50 range in Vero E6 cells and inhibit CatL in the picomolar Ki range. We show a relevant off-target effect of CatL inhibition by the coronavirus main protease α-ketoamide inhibitor 13b. Crystal structures of CatL in complex with 14 compounds at resolutions better than 2 Å present a solid basis for structure-guided understanding and optimization of CatL inhibitors toward protease drug development.
Assuntos
Antivirais , Catepsina L , SARS-CoV-2 , Catepsina L/antagonistas & inibidores , Catepsina L/metabolismo , Antivirais/farmacologia , Antivirais/química , Antivirais/síntese química , Animais , Chlorocebus aethiops , Células Vero , SARS-CoV-2/efeitos dos fármacos , Humanos , Relação Estrutura-Atividade , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/síntese química , Cristalografia por Raios X , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/síntese química , Inibidores de Proteases/metabolismo , Modelos MolecularesRESUMO
Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin's efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections.
Assuntos
COVID-19 , Humanos , SARS-CoV-2/metabolismo , Catepsinas , Antivirais/farmacologia , Antivirais/uso terapêutico , Antivirais/química , Inibidores de Proteases/farmacologia , Cisteína Endopeptidases/metabolismoRESUMO
TetR/AcrR-like transcription regulators enable bacteria to sense a wide variety of chemical compounds and to dynamically adapt the expression levels of specific genes in response to changing growth conditions. Here, we describe the structural characterisation of SCO3201, an atypical TetR/AcrR family member from Streptomyces coelicolor that strongly represses antibiotic production and morphological development under conditions of overexpression. We present crystal structures of SCO3201 in its ligand-free state as well as in complex with an unknown inducer, potentially a polyamine. In the ligand-free state, the DNA-binding domains of the SCO3201 dimer are held together in an unusually compact conformation and, as a result, the regulator cannot span the distance between the two half-sites of its operator. Interaction with the ligand coincides with a major structural rearrangement and partial conversion of the so-called hinge helix (α4) to a 310 -conformation, markedly increasing the distance between the DNA-binding domains. In sharp contrast to what was observed for other TetR/AcrR-like regulators, the increased interdomain distance might facilitate rather than abrogate interaction of the dimer with the operator. Such a 'reverse' induction mechanism could expand the regulatory repertoire of the TetR/AcrR family and may explain the dramatic impact of SCO3201 overexpression on the ability of S. coelicolor to generate antibiotics and sporulate.
Assuntos
Proteínas Repressoras , Streptomyces coelicolor , Proteínas Repressoras/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/química , Streptomyces coelicolor/metabolismo , Antibacterianos/farmacologia , Domínios Proteicos , DNA , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Regulação Bacteriana da Expressão GênicaRESUMO
The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by a reverse Michael addition. The overall fold and the constitution of the active site of the enzyme completely differ from the well-characterised chalcone isomerase of plants. For (+)-taxifolin, CHI catalyses the intramolecular ring contraction to alphitonin. In this study, Fwe perform crystal structure analyses of CHI and its active site mutant His33Ala in the presence of the substrate taxifolin at 2.15 and 2.8 Å resolution, respectively. The inactive enzyme binds the substrate (+)-taxifolin as well defined, whereas the electron density maps of the native CHI show a superposition of substrate, product alphitonin, and most probably also the reaction intermediate taxifolin chalcone. Evidently, His33 mediates the stereospecific acid-base reaction by abstracting a proton from the flavonoid scaffold. The stereospecificity of the product is discussed.
Assuntos
Eubacterium , Liases Intramoleculares , Liases Intramoleculares/genéticaRESUMO
SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host's innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.
Assuntos
Antivirais , Tratamento Farmacológico da COVID-19 , Sítio Alostérico , Antivirais/farmacologia , Proteases Semelhantes à Papaína de Coronavírus , Humanos , Papaína/metabolismo , Peptídeo Hidrolases/metabolismo , SARS-CoV-2RESUMO
ß-lactamases are a major cause of rapidly emerging and spreading antibiotic resistance. Currently ß-lactamase inhibitors (BLIs) in clinical use act only on Ambler Class A, C and some class D lactamases. The urgent need to identify new BLIs recently lead to FDA approval of boron-based compounds BLIs, e.g. Vaborbactam. The boron-based proteasome inhibitors Bortezomib and Ixazomib are used in cancer therapy as multiple myeloma drugs but they also bind to Ser-/Thr- proteases. In this study we show the crystal structures of the ß-lactamase CTX-M-14 with covalently bound Bortezomib and Ixazomib at high resolutions of 1.3 and 1.1 Å, respectively. Ixazomib is well defined in electron density whereas Bortezomib show some disorder which corresponds to weaker inhibition efficiency observed for Ixazomib. Both inhibitors mimic the deacylation transition state of ß-lactam hydrolysis, because they replace the deacylating water molecule. We further investigate differences in binding of Bortezomib/Ixazomib to CTX-M-14 and its target proteases as well as known ß-lactamase drugs. Our findings can help to use Bortezomib/Ixazomib as lead compounds for development of new BLIs.
Assuntos
Inibidores de Proteassoma , Inibidores de beta-Lactamases , Boro , Compostos de Boro , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Glicina/análogos & derivados , Inibidores de Proteassoma/farmacologia , Inibidores de beta-Lactamases/farmacologiaRESUMO
The papain-like protease (PLpro) of SARS-CoV-2 is essential for viral propagation and, additionally, dysregulation of the host innate immune system. Using a library of 40 potential metal-chelating compounds we performed an X-ray crystallographic screening against PLpro. As outcome we identified six compounds binding to the target protein. Here we describe the interaction of one hydrazone (H1) and five thiosemicarbazone (T1-T5) compounds with the two distinct natural substrate binding sites of PLpro for ubiquitin and ISG15. H1 binds to a polar groove at the S1 binding site by forming several hydrogen bonds with PLpro. T1-T5 bind into a deep pocket close to the polyubiquitin and ISG15 binding site S2. Their interactions are mainly mediated by multiple hydrogen bonds and further hydrophobic interactions. In particular compound H1 interferes with natural substrate binding by sterical hindrance and induces conformational changes in protein residues involved in substrate binding, while compounds T1-T5 could have a more indirect effect. Fluorescence based enzyme activity assay and complementary thermal stability analysis reveal only weak inhibition properties in the high micromolar range thereby indicating the need for compound optimization. Nevertheless, the unique binding properties involving strong hydrogen bonding and the various options for structural optimization make the compounds ideal lead structures. In combination with the inexpensive and undemanding synthesis, the reported hydrazone and thiosemicarbazones represent an attractive scaffold for further structure-based development of novel PLpro inhibitors by interrupting protein-protein interactions at the S1 and S2 site.
RESUMO
Bradyrhizobium diazoefficiens, a bacterial symbiont of soybean and other leguminous plants, enters a nodulation-promoting genetic programme in the presence of host-produced flavonoids and related signalling compounds. Here, we describe the crystal structure of an isoflavonoid-responsive regulator (FrrA) from Bradyrhizobium, as well as cocrystal structures with inducing and noninducing ligands (genistein and naringenin, respectively). The structures reveal a TetR-like fold whose DNA-binding domain is capable of adopting a range of orientations. A single molecule of either genistein or naringenin is asymmetrically bound in a central cavity of the FrrA homodimer, mainly via C-H contacts to the π-system of the ligands. Strikingly, however, the interaction does not provoke any conformational changes in the repressor. Both the flexible positioning of the DNA-binding domain and the absence of structural change upon ligand binding are corroborated by small-angle X-ray scattering (SAXS) experiments in solution. Together with a model of the promoter-bound state of FrrA our results suggest that inducers act as a wedge, preventing the DNA-binding domains from moving close enough together to interact with successive positions of the major groove of the palindromic operator.
Assuntos
Proteínas de Ligação a DNA/genética , Flavonoides/genética , Glycine max/genética , Proteínas Ribossômicas/genética , Sítios de Ligação/genética , Bradyrhizobium/genética , Bradyrhizobium/patogenicidade , Cristalografia por Raios X , Proteínas de Ligação a DNA/ultraestrutura , Flavonoides/biossíntese , Regulação Bacteriana da Expressão Gênica/genética , Ligantes , Ligação Proteica/genética , Conformação Proteica , Proteínas Ribossômicas/ultraestrutura , Glycine max/microbiologiaRESUMO
Previously, we reported the isolation of a quorum quenching protein (QQ), designated GqqA, from Komagataeibacter europaeus CECT 8546 that is highly homologous to prephenate dehydratases (PDT) (Valera et al. in Microb Cell Fact 15, 88. https://doi.org/10.1186/s12934-016-0482-y , 2016). GqqA strongly interfered with N-acyl-homoserine lactone (AHL) quorum sensing signals from Gram-negative bacteria and affected biofilm formation in its native host strain Komagataeibacter europaeus. Here we present and discuss data identifying GqqA as a novel acylase. ESI-MS-MS data showed unambiguously that GqqA hydrolyzes the amide bond of the acyl side-chain of AHL molecules, but not the lactone ring. Consistent with this observation the protein sequence does not carry a conserved Zn2+ binding motif, known to be essential for metal-dependent lactonases, but in fact harboring the typical periplasmatic binding protein domain (PBP domain), acting as catalytic domain. We report structural details for the native structure at 2.5 Å resolution and for a truncated GqqA structure at 1.7 Å. The structures obtained highlight that GqqA acts as a dimer and complementary docking studies indicate that the lactone ring of the substrate binds within a cleft of the PBP domain and interacts with polar residues Y16, S17 and T174. The biochemical and phylogenetic analyses imply that GqqA represents the first member of a novel type of QQ family enzymes.
Assuntos
Acetobacteraceae/enzimologia , Proteínas de Bactérias/metabolismo , Prefenato Desidratase/metabolismo , Acetobacteraceae/classificação , Acetobacteraceae/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrólise , Modelos Moleculares , Prefenato Desidratase/química , Prefenato Desidratase/genética , Conformação Proteica , Percepção de Quorum , Especificidade por SubstratoRESUMO
Allosteric regulation of the Tet repressor (TetR) homodimer relies on tetracycline binding that abolishes the affinity for the DNA operator. Previously, interpretation of circular dichroism data called for unfolding of the α-helical DNA-binding domains in absence of binding to DNA or tetracycline. Our small angle X-ray scattering of TetR(D) in solution contradicts this unfolding as a physiological process. Instead, in the core domain crystal structures analyses show increased immobilisation of helix α9 and two C-terminal turns of helix α8 upon tetracycline binding. Tetracycline complexes of TetR(D) and four single-site alanine variants were characterised by isothermal titration calorimetry, fluorescence titration, X-ray crystal structures, and melting curves. Five crystal structures confirm that Thr103 is a key residue for the allosteric events of induction, with the T103A variant lacking induction by any tetracycline. The T103A variant shows anti-cooperative inducer binding, and a melting curve of the tetracycline complex different to TetR(D) and other variants. For the N82A variant inducer binding is clearly anti-cooperative but triggers the induced conformation.
Assuntos
Proteínas de Bactérias/química , Proteínas Repressoras/química , Tetraciclina/química , Termodinâmica , Regulação Alostérica , Sequência de Aminoácidos , Cristalografia por Raios X , Proteínas de Ligação a DNA , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Tetraciclina/farmacologia , Difração de Raios X , Ecrans Intensificadores para Raios XRESUMO
X-ray crystallographic analysis of a phosin (PptA) from Steptomyces chartreusis reveals a metal-associated, lozenge-shaped fold featuring a 5-10 Å wide, positively charged tunnel that traverses the protein core. Two distinct metal-binding sites were identified in which the predominant metal ion was Cu2+ . In solution, PptA forms stable homodimers that bind with nanomolar affinity to polyphosphate, a stress-related biopolymer acting as a phosphate and energy reserve in conditions of nutrient depletion. A single protein dimer interacts with 14-15 consecutive phosphate moieties within the polymer. Our observations suggest that PptA plays a role in polyphosphate metabolism, mobilisation or sensing, possibly by acting in concert with polyphosphate kinase (Ppk). Like Ppk, phosins may influence antibiotic synthesis by streptomycetes.
Assuntos
Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/metabolismo , Polifosfatos/metabolismo , Streptomyces/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Dimerização , Ferro/metabolismo , Modelos Moleculares , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Espalhamento a Baixo ÂnguloRESUMO
During assembly of herpesvirus capsids, a protein scaffold self-assembles to ring-like structures forming the scaffold of the spherical procapsids. Proteolytic activity of the herpesvirus maturational protease causes structural changes that result in angularization of the capsids. In those mature icosahedral capsids, the packaging of viral DNA into the capsids can take place. The strictly regulated protease is called assemblin. It is inactive in its monomeric state and activated by dimerization. The structures of the dimeric forms of several assemblins from all herpesvirus subfamilies have been elucidated in the last two decades. They revealed a unique serine-protease fold with a catalytic triad consisting of a serine and two histidines. Inhibitors that disturb dimerization by binding to the dimerization area were found recently. Additionally, the structure of the monomeric form of assemblin from pseudorabies virus and some monomer-like structures of Kaposi's sarcoma-associated herpesvirus assemblin were solved. These findings are the proof-of-principle for the development of new anti-herpesvirus drugs. Therefore, the most important information on this fascinating and unique class of proteases is summarized here.
Assuntos
Infecções por Herpesviridae/virologia , Herpesviridae/enzimologia , Serina Endopeptidases/metabolismo , Proteínas Virais/metabolismo , Animais , Capsídeo/metabolismo , Herpesviridae/classificação , Herpesviridae/genética , Herpesviridae/fisiologia , Humanos , Serina Endopeptidases/química , Serina Endopeptidases/genética , Proteínas Virais/química , Proteínas Virais/genética , Montagem de VírusRESUMO
The Gram-negative bacterium Aeromonas salmonicida is a fish pathogen for various fish species worldwide. Aeromonas salmonicida subsp. achromogenes produces the extracellular, toxic zinc endopeptidase AsaP1. Crystal structure analyses at 2.0 Å resolution of two proteolytically inactive AsaP1 variants show the polypeptide folding of the protease domain and the propeptide domain. These first crystal structure analyses of a precursor of a deuterolysin-like aspzincin protease provide insights into propeptide function, and specific substrate binding. A lysine side chain of the propeptide binds in the hydrophobic S1'-pocket interacting with three carboxylate side chains. An AsaP1 variant with a lysine to alanine exchange identifies the chaperone function of the propeptide.
Assuntos
Proteínas de Bactérias/química , Metaloendopeptidases/química , Dobramento de Proteína , Aeromonas salmonicida/enzimologia , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Metaloendopeptidases/metabolismo , Ligação ProteicaRESUMO
UNLABELLED: Induction of the tetracycline repressor (TetR) results from antibiotic-dependent changes in the relative positioning of the DNA-binding domains within the promoter-associated repressor dimer, but the key determinants of this allosteric effect remain poorly characterised. Intriguingly, previous mutational analyses of the tetracycline-interacting site revealed a lack of correlation between residual affinity and induction propensity, suggesting that some of the residues in contact with the antibiotic primarily act in ligand recognition and retention, whereas others are required to transmit the allosteric signal. Here, we provide a structural basis for these observations via crystallographic analysis of the point mutants N82A, H100A, T103A and E147A in complex with the inducer 5a,6-anhydrotetracycline. In conjunction with the available functional data, the four structures demonstrate that a trigger-like movement of the region between helices α6 and α7 towards and into the binding site plays a decisive role in the intramolecular communication process. In sharp contrast, residues lining the binding cavity proper have little or no influence on the allosteric mechanism as such. This nearly complete physical separation of ligand recognition and allostery will have allowed diverging TetR-like repressors to bind novel effectors while the existing induction mechanism remained intact. Consequently, the modularity described here may have been a key factor in the evolutionary success of the widespread and highly diversified repressor class. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 5FKK, 5FKL, 5FKM, 5FKN and 5FKO.
Assuntos
Proteínas de Escherichia coli/química , Proteínas Repressoras/química , Tetraciclinas/química , Substituição de Aminoácidos/genética , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Ligantes , Mutação Puntual , Regiões Promotoras Genéticas/genética , Estrutura Secundária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Tetraciclinas/metabolismoRESUMO
Venezuelan equine encephalitis virus (VEEV) is an Alphavirus from the family Togaviridae that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for the calculation of copy numbers of viral genome of Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and VEEV. The new assay provides a reliable high-throughput method for the detection and quantitation of VEEV RNA in clinical and field samples and allows a rapid differentiation from potentially cocirculating EEEV and WEEV strains. The capability to detect all known VEEV variants was experimentally demonstrated and makes this assay suitable especially for the surveillance of VEEV.
Assuntos
Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Encefalomielite Equina Venezuelana/diagnóstico , Encefalomielite Equina Venezuelana/genética , RNA Viral/genética , Alphavirus/genética , Animais , Vírus da Encefalite Equina do Leste/genética , Vírus da Encefalite Equina do Leste/isolamento & purificação , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina do Oeste/genética , Vírus da Encefalite Equina do Oeste/isolamento & purificação , Encefalomielite Equina Venezuelana/virologia , Cavalos/virologia , Humanos , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , América do SulRESUMO
Homologs of the eukaryotic transcription coactivator PC4, which also functions in DNA repair and oxidative stress, were recently identified in prokaryotes. Crystallographic analysis of BPSL1147, a putative homolog from the pathogen Burkholderia pseudomallei K96243, reveals a highly conserved core structure and suggests a nucleic acid binding mode similar to that of PC4. Knock-out and complementation experiments do not reveal distinguishing phenotypes under normal growth conditions or in the presence of H2O2, arguing against a critical role in repair or the oxidative stress response of Burkholderia. These results may reflect redundancy or point at a bacteriophage origin of Burkholderia PC4 homologs.
Assuntos
Proteínas de Bactérias/química , Burkholderia pseudomallei/genética , Fatores de Transcrição/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Burkholderia pseudomallei/metabolismo , Dados de Sequência Molecular , Mutação , Estresse Oxidativo , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
Herpesviruses encode a characteristic serine protease with a unique fold and an active site that comprises the unusual triad Ser-His-His. The protease is essential for viral replication and as such constitutes a promising drug target. In solution, a dynamic equilibrium exists between an inactive monomeric and an active dimeric form of the enzyme, which is believed to play a key regulatory role in the orchestration of proteolysis and capsid assembly. Currently available crystal structures of herpesvirus proteases correspond either to the dimeric state or to complexes with peptide mimetics that alter the dimerization interface. In contrast, the structure of the native monomeric state has remained elusive. Here, we present the three-dimensional structures of native monomeric, active dimeric, and diisopropyl fluorophosphate-inhibited dimeric protease derived from pseudorabies virus, an alphaherpesvirus of swine. These structures, solved by X-ray crystallography to respective resolutions of 2.05, 2.10 and 2.03 Å, allow a direct comparison of the main conformational states of the protease. In the dimeric form, a functional oxyanion hole is formed by a loop of 10 amino-acid residues encompassing two consecutive arginine residues (Arg136 and Arg137); both are strictly conserved throughout the herpesviruses. In the monomeric form, the top of the loop is shifted by approximately 11 Å, resulting in a complete disruption of the oxyanion hole and loss of activity. The dimerization-induced allosteric changes described here form the physical basis for the concentration-dependent activation of the protease, which is essential for proper virus replication. Small-angle X-ray scattering experiments confirmed a concentration-dependent equilibrium of monomeric and dimeric protease in solution.