Dimerization-Induced Allosteric Changes of the Oxyanion-Hole Loop Activate the Pseudorabies Virus Assemblin pUL26N,a Herpesvirus Serine Protease

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.     

Activation of retinal guanylyl cyclase-1 by Ca2+-binding proteins involves its dimerization.

Retinal guanylyl cyclase-1 (retGC-1), a key enzyme in phototransduction, is activated by guanylyl cyclase-activating proteins (GCAPs) if [Ca2+] is less than 300 nM. The activation is believed to be essential for the recovery of photoreceptors to the dark state; however, the molecular mechanism of the activation is unknown. Here, we report that dimerization of retGC-1 is involved in its activation by GCAPs. The GC activity and the formation of a 210-kDa cross-linked product of retGC-1 were monitored in bovine rod outer segment homogenates, GCAPs-free bovine rod outer segment membranes and recombinant bovine retGC-1 expressed in COS-7 cells. In addition to recombinant bovine GCAPs, constitutively active mutants of GCAPs that activate retGC-1 in a [Ca2+]-independent manner and bovine brain S100b that activates retGC-1 in the presence of approximately 10 microM [Ca2+] were used to investigate whether these activations take place through a similar mechanism, and whether [Ca2+] is directly involved in the dimerization. We found that a monomeric form of retGC-1 ( approximately 110 kDa) was mainly observed whenever GC activity was at basal or low levels. However, the 210-kDa product was increased whenever the GC activity was stimulated by any Ca2+-binding proteins used. We also found that [Ca2+] did not directly regulate the formation of the 210-kDa product. The 210-kDa product was detected in a purified GC preparation and did not contain GCAPs even when the formation of the 210-kDa product was stimulated by GCAPs. These data strongly suggest that the 210-kDa cross-linked product is a homodimer of retGC-1. We conclude that inactive retGC-1 is predominantly a monomeric form, and that dimerization of retGC-1 may be an essential step for its activation by active forms of GCAPs.     

Mechanism for controlling the dimer-monomer switch and coupling dimerization to catalysis of the severe acute respiratory syndrome coronavirus 3C-like protease

Unlike 3C protease, the severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CLpro) is only enzymatically active as a homodimer and its catalysis is under extensive regulation by the unique extra domain. Despite intense studies, two puzzles still remain: (i) how the dimer-monomer switch is controlled and (ii) why dimerization is absolutely required for catalysis. Here we report the monomeric crystal structure of the SARS-CoV 3CLpro mutant R298A at a resolution of 1.75 A. Detailed analysis reveals that Arg298 serves as a key component for maintaining dimerization, and consequently, its mutation will trigger a cooperative switch from a dimer to a monomer. The monomeric enzyme is irreversibly inactivated because its catalytic machinery is frozen in the collapsed state, characteristic of the formation of a short 3(10)-helix from an active-site loop. Remarkably, dimerization appears to be coupled to catalysis in 3CLpro through the use of overlapped residues for two networks, one for dimerization and another for the catalysis.     

Solution structure of the mature HIV-1 protease monomer: insight into the tertiary fold and stability of a precursor

We present the first solution structure of the HIV-1 protease monomer spanning the region Phe1-Ala95 (PR1-95). Except for the terminal regions (residues 1-10 and 91-95) that are disordered, the tertiary fold of the remainder of the protease is essentially identical to that of the individual subunit of the dimer. In the monomer, the side chains of buried residues stabilizing the active site interface in the dimer, such as Asp25, Asp29, and Arg87, are now exposed to solvent. The flap dynamics in the monomer are similar to that of the free protease dimer. We also show that the protease domain of an optimized precursor flanked by 56 amino acids of the N-terminal transframe region is predominantly monomeric, exhibiting a tertiary fold that is quite similar to that of PR1-95 structure. This explains the very low catalytic activity observed for the protease prior to its maturation at its N terminus as compared with the mature protease, which is an active stable dimer under identical conditions. Adding as few as 2 amino acids to the N terminus of the mature protease significantly increases its dissociation into monomers. Knowledge of the protease monomer structure and critical features of its dimerization may aid in the screening and design of compounds that target the protease prior to its maturation from the Gag-Pol precursor.     

Dimerization inhibitors of HIV-1 protease

By targeting the highly conserved antiparallel beta-sheet formed by the interdigitation of the N- and C-terminal strands of each monomer, dimerization inhibitors of HIV-1 protease may be useful to overcome the drug resistance observed with current active-site directed antiproteases. Sequestration of the monomer by the inhibitor (or disruption of the dimer interface) prevents the correct assembly of the inactive monomers to active enzyme. Strategies for the design of drugs targeting the dimer interface are described. Various dimerization inhibitors are reported including N- and C-terminal mimetics, lipopeptides and cross-linked interface peptides.     

The fully-active and structurally-stable form of the mitochondrial ATP synthase of Polytomella sp. is dimeric

Mitochondrial F₁F_O-ATP synthase of chlorophycean algae is a stable dimeric complex of 1,600 kDa. It lacks the classic subunits that constitute the peripheral stator-stalk and the orthodox polypeptides involved in the dimerization of the complex. Instead, it contains nine polypeptides of unknown evolutionary origin named ASA1 to ASA9. The isolated enzyme exhibited a very low ATPase activity (0.03 Units/mg), that increased upon heat treatment, due to the release of the F₁ sector. Oligomycin was found to stabilize the dimeric structure of the enzyme, providing partial resistance to heat dissociation. Incubation in the presence of low concentrations of several non-ionic detergents increased the oligomycin-sensitive ATPase activity up to 7.0–9.0 Units/mg. Incubation with 3% (w/v) taurodeoxycholate monomerized the enzyme. The monomeric form of the enzyme exhibited diminished activity in the presence of detergents and diminished oligomycin sensitivity. Cross-linking experiments carried out with the dimeric and monomeric forms of the ATP synthase suggested the participation of the ASA6 subunit in the dimerization of the enzyme. The dimeric enzyme was more resistant to heat treatment, high hydrostatic pressures, and protease digestion than the monomeric enzyme, which was readily disrupted by these treatments. We conclude that the fully-active algal mitochondrial ATP synthase is a stable catalytically active dimer; the monomeric form is less active and less stable. Monomer-monomer interactions could be mediated by the membrane-bound subunits ASA6 and ASA9, and may be further stabilized by other polypeptides such as ASA1 and ASA5. Alexa Villavicencio-Queijeiro and Miriam Vázquez-Acevedo have contributed equally to this work.     

Ectodomain shedding and intramembrane cleavage of mammalian Notch proteins is not regulated through oligomerization

Intramembrane cleaving proteases such as site 2 protease, gamma-secretase, and signal peptide peptidase hydrolyze peptide bonds within the transmembrane domain (TMD) of signaling molecules such as SREBP, Notch, and HLA-E, respectively. All three enzymes require a prior cleavage at the juxtamembrane region by another protease. It has been proposed that removing the extracellular domain allows dissociation of substrate TMD, held together by the extracellular domain or loop. Using gamma-secretase as a model intramembrane cleaving protease and Notch as a model substrate, we investigated whether activating and inactivating mutations in Notch modulate gamma-secretase cleavage through changes in oligomerization. We find that although the Notch epidermal growth factor repeats can promote dimer formation, most surface Notch molecules in mammalian cells are monomeric as are constitutively active or inactive Notch1 proteins. Using a bacterial assay for TM dimerization, we find that the isolated TMD of Notch and amyloid precursor protein self-associate and that mutations affecting Notch cleavage by gamma-secretase cleavage do not alter TMD dimerization. Our results indicate that ligand-induced reversal of controlled TMD dimerization by the Notch extracellular domain is unlikely to underlie the regulatory mechanism of intramembranous cleavage.     

Without its N-finger, the main protease of severe acute respiratory syndrome coronavirus can form a novel dimer through its C-terminal domain

The main protease (M(pro)) of severe acute respiratory syndrome coronavirus (SARS-CoV) plays an essential role in the extensive proteolytic processing of the viral polyproteins (pp1a and pp1ab), and it is an important target for anti-SARS drug development. It was found that SARS-CoV M(pro) exists in solution as an equilibrium of both monomeric and dimeric forms, and the dimeric form is the enzymatically active form. However, the mechanism of SARS-CoV M(pro) dimerization, especially the roles of its N-terminal seven residues (N-finger) and its unique C-terminal domain in the dimerization, remain unclear. Here we report that the SARS-CoV M(pro) C-terminal domain alone (residues 187 to 306; M(pro)-C) is produced in Escherichia coli in both monomeric and dimeric forms, and no exchange could be observed between them at room temperature. The M(pro)-C dimer has a novel dimerization interface. Meanwhile, the N-finger deletion mutant of SARS-CoV M(pro) also exists as both a stable monomer and a stable dimer, and the dimer is formed through the same C-terminal-domain interaction as that in the M(pro)-C dimer. However, no C-terminal domain-mediated dimerization form can be detected for wild-type SARS-CoV M(pro). Our study results help to clarify previously published controversial claims about the role of the N-finger in SARS-CoV M(pro) dimerization. Apparently, without the N-finger, SARS-CoV M(pro) can no longer retain the active dimer structure; instead, it can form a new type of dimer which is inactive. Therefore, the N-finger of SARS-CoV M(pro) is not only critical for its dimerization but also essential for the enzyme to form the enzymatically active dimer.     

Molecular mechanism for dimerization to regulate the catalytic activity of human cytomegalovirus protease

Biochemical studies indicate that dimerization is required for the catalytic activity of herpesvirus proteases, whereas structural studies show a complete active site in each monomer, away from the dimer interface. Here we report kinetic, biophysical and crystallographic characterizations of structure-based mutants in the dimer interface of human cytomegalovirus (HCMV) protease. Such mutations can produce a 1,700-fold reduction in the kcat while having minimal effects on the K(m). Dimer stability is not affected by these mutations, suggesting that dimerization itself is insufficient for activity. There are large changes in monomer conformation and dimer organization of the apo S225Y mutant enzyme. However, binding of an activated peptidomimetic inhibitor induced a conformation remarkably similar to the wild type protease. Our studies suggest that appropriate dimer formation may be required to indirectly stabilize the protease oxyanion hole, revealing a novel mechanism for dimerization to regulate enzyme activity.     

Role of proteolysis in caspase-8 activation and stabilization

Caspase-8 is an apoptotic protease that is activated at the cytosolic face of the cell membrane. Activation relies on adaptor-induced dimerization of monomeric caspase-8 and is followed by specific limited autoproteolysis of the linker which separates the two subunits of the catalytic domain. However, the role of this autoproteolysis, which directly activates executioner caspases-3 and -7, is unknown for the apical caspase-8. We have generated linker mutants of caspase-8 that can be proteolyzed in a controlled manner by thrombin or tobacco etch mosaic virus protease, and we use these to define the role of proteolysis in the activation and stability of the enzyme. We show that proteolysis is insufficient for generating enzymatic activity in recombinant caspase-8. Kinetic activation studies using Hoffmeister salts demonstrate that activation is the result of caspase dimerization. However, linker proteolysis significantly enhances the equilibrium for caspase-8 dimerization, thereby increasing the stability of the dimer. Kinetic and fluorescence measurements demonstrate that caspase-8 activation by Hoffmeister salts is at least a two-step event, with the required step being dimerization, followed by an intramolecular event that further stabilizes the catalytic conformation. Autoproteolysis of caspase-8 may be a mechanism for increasing the lifetime of the dimeric enzyme following dissociation from its activating complex at the cell membrane.     

Potent inhibition of HIV-1 replication by novel non-peptidyl small molecule inhibitors of protease dimerization

Dimerization of HIV-1 protease subunits is essential for its proteolytic activity, which plays a critical role in HIV-1 replication. Hence, the inhibition of protease dimerization represents a unique target for potential intervention of HIV-1. We developed an intermolecular fluorescence resonance energy transfer-based HIV-1-expression assay employing cyan and yellow fluorescent protein-tagged protease monomers. Using this assay, we identified non-peptidyl small molecule inhibitors of protease dimerization. These inhibitors, including darunavir and two experimental protease inhibitors, blocked protease dimerization at concentrations of as low as 0.01 microm and blocked HIV-1 replication with IC(50) values of 0.0002-0.48 microm. These agents also inhibited the proteolytic activity of mature protease. Other approved anti-HIV-1 agents examined except tipranavir, a CCR5 inhibitor, and soluble CD4 failed to block the dimerization event. Once protease monomers dimerize to become mature protease, mature protease is not dissociated by this dimerization inhibition mechanism, suggesting that these agents block dimerization at the nascent stage of protease maturation. The proteolytic activity of mature protease that managed to undergo dimerization despite the presence of these agents is likely to be inhibited by the same agents acting as conventional protease inhibitors. Such a dual inhibition mechanism should lead to highly potent inhibition of HIV-1.     

Interchain proteolysis, in the absence of a dimerization stimulus, can initiate apoptosis-associated caspase-8 activation

Caspases coordinate the internal demolition of the cell that is seen during apoptosis. Proteolytic processing of caspases is observed during apoptosis, and this correlates with conversion of inactive caspase proenzymes into their active two-chain forms. However, recent studies have suggested that caspase-8 is activated through dimerization and that interchain proteolysis is not sufficient for activation of this caspase. This proposal casts doubt upon whether caspase-8 is productively activated by granzyme B during granule-dependent cytotoxic T lymphocyte or natural killer cell-mediated killing, for example. Contrary to the dimerization model, we show that direct proteolysis of caspase-8 by the cytotoxic T lymphocyte protease granzyme B, or by caspase-6, produces an active enzyme that displays robust proteolytic activity toward synthetic as well as natural caspase-8 substrates. These data suggest that enforced dimerization of caspase-8 zymogens by scaffold proteins such as Fas-associated protein with death domain (FADD), although important in certain contexts, is not a prerequisite for activation of this protease.     

An aspartic protease analogue: intermolecular catalysis of peptide hydrolysis by carboxyl groups

Two aspartic carboxyl groups act as key catalytic groups in the active site of an aspartic protease. We synthesized an aspartic protease analogue by positioning three salicylate residues in close proximity on a cross-linked polystyrene. The immobile artificial protease effectively hydrolyzed albumin into many small fragments by the catalytic action of carboxyl groups contained in the active site. The artificial protease manifested optimum activity at pH 3 just as aspartic proteases.     

Long-range cooperative interactions modulate dimerization in SARS 3CLpro

Severe acute respiratory syndrome (SARS) is an infectious disease caused by the human coronavirus, SARS-CoV. The main viral protease, SARS 3CLpro, is a validated target for the development of antiviral therapies. Since the enzyme is a homodimer and the individual monomers are inactive, two approaches are being used to develop inhibitors: enzyme activity inhibitors that target the active site and dimerization inhibitors. Dimerization inhibitors are usually targeted to the dimerization interface and need to compete with the attractive forces between subunits to be effective. In this paper, we show that the dimerization of SARS 3CLpro is also under allosteric control and that additional and energetically more favorable target sites away from the dimerization interface may also lead to subunit dissociation. We previously identified a cluster of conserved serine residues (Ser139, Ser144, and Ser147) located adjacent to the active site of 3CLpro that could effectively be targeted to inactivate the protease [Bacha, U et al. (2004) Biochemistry 43, 4906-4912]. Mutation of any of these serine residues to alanine had a debilitating effect on the catalytic activity of 3CLpro. In particular, the mutation of Ser147, which does not make any contact with the opposing subunit and is located approximately 9 A away from the dimer interface, totally inhibited dimerization and resulted in a complete loss of enzymatic activity. The finding that residues away from the dimer interface are able to control dimerization defines alternative targets for the design of dimerization inhibitors.     

Methionyl-tRna synthetase from wheat germ. Effect of an endogenous protease and correlations between structural characteristics and catalytic properties

Methionyl-tRNA synthetase (MetRS) has been described as a free monomeric or oligomeric enzyme; or included in a multienzyme complex. Moreover, on limited tryptic digestion, it can generate shorter forms. So, when purified from wheat-germ lysate, the possible presence of proteases able to hydrolyse this enzyme was investigated. When extraction was performed with sulfhydryl-blocking reagents, an active monomeric MetRS of Mr 105,000 was purified. This enzyme form was identical to the structure exhibiting methionyl-tRNA synthetase activity in multienzyme complexes. Without this inhibitor, MetRS was purified as an active dimeric form of Mr 165,000 with identical subunits of Mr 82,000. A protease inhibited by sulfhydryl-blocking reagents and included in a complex of Mr 2.10(6) was isolated from this wheat-germ lysate. This protease was able to hydrolyse different proteins (albumin, casein), but was without activity for a trypsin substrate, such as N-alpha-benzoyl-DL-arginine p-nitroanilide. When added to a solution of Mr-105,000 MetRS, it yielded an inactive peptide of Mr 20,000, containing numerous charged amino acids and a protein of Mr 82,000, able to give an active dimeric enzyme of Mr 165,000. Amino acid analysis of this last form, indicated an identical structure with the active dimeric MetRS of Mr 165,000, purified in the absence of protease inhibitors. Moreover, the affinity for methionine was the same for the monomeric enzyme of Mr 105,000 and the dimeric form of Mr 165,000, probably because proteolysis did not affect the catalytic domain. When enzymic activity of the proteolyzed form (Mr 2 x 82,000) was studied versus enzyme concentration, a decrease in specific activity, at low concentrations, was seen. This phenomenon was analysed on the basis of the existence of an equilibrium between an active dimer and two inactive monomers. With the active monomeric form of Mr 105,000, no change in specific activity with decreasing enzyme concentration occurred.     

Staphylococcus aureus sortase A exists as a dimeric protein in vitro

We report the first direct observation of the self-association behavior of the Staphylococcus aureus sortase A (SrtA) transpeptidase. Formation of a SrtA dimer was observed under native conditions by polyacrylamide gel electrophoresis and fast protein liquid chromatography (FPLC). Subsequent peptide mass fingerprinting and protein sequencing experiments confirmed the dimeric form of the SrtA protein. Furthermore, SrtA can be selectively cross-linked both in vitro and in Escherichia coli. Multiple samples of enzyme were subjected to analytical sedimentation equilibrium ultracentrifugation to obtain an apparent Kd for dimer formation of about 55 microM. Finally, enzyme kinetic studies suggested that the dimeric form of SrtA is more active than the monomeric enzyme. Discovery of SrtA dimerization may have significant implications for understanding microbial physiology and developing new antibiotics.     

Dimerization-induced activation of soluble insulin/IGF-1 receptor kinases: an alternative mechanism of activation.

To study the role of kinase dimerization in the activation of the insulin receptor (IR) and the insulin-like growth factor receptor-1 (IGF-1R), we have cloned, expressed, and purified monomeric and dimeric forms of the corresponding soluble kinase domains via the baculovirus expression system. Dimerization of the kinases was achieved by fusion of the kinase domains to the homodimeric glutathione S-transferase (GST). Kinetic analyses revealed that kinase dimerization results in substantial increases (10-100-fold) in the phosphotransferase activity in both the auto- and substrate phosphorylation reactions. Furthermore, kinase dimerization rendered the autophosphorylation reaction concentration-independent. However, whereas dimerization was required for the rapid autophosphorylation of the kinases, it was not essential for the enhanced kinase activity in substrate phosphorylation reactions. Comparison of HPLC-phosphopeptide maps of the monomeric and dimeric kinases revealed that dimerization leads to an increased phosphorylation of the regulatory activation loop of the kinases, strongly suggesting that bis- and trisphosphorylation of the activation loop are mediated by transphosphorylation within the kinase dimers. Most strikingly, limited proteolysis revealed that GST-mediated dimerization by itself had a major impact on the conformation of the activation loop by stabilizing a conformation that corresponds to the active, phosphorylated form of the kinase. Thus, in analogy to the insulin/IGF-1-ligated holoreceptors, the dimeric GST-kinases are primed to rapid autophosphorylation by an increase in the local concentration of both phosphoryl donor and phosphoryl acceptor sites and by a dimerization-induced conformational change of the activation loop that leads to an efficient transphosphorylation of the regulatory tyrosine residues.