Crystal structure of a novel viral protease with a serine/lysine catalytic dyad mechanism

The blotched snakehead virus (BSNV), an aquatic birnavirus, encodes a polyprotein (NH2-pVP2-X-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease (VP4) to liberate itself and the viral proteins pVP2, X and VP3. The protein pVP2 is further processed by VP4 to give rise to the capsid protein VP2 and four structural peptides. We report here the crystal structure of a VP4 protease from BSNV, which displays a catalytic serine/lysine dyad in its active site. This is the first crystal structure of a birnavirus protease and the first crystal structure of a viral protease that utilizes a lysine general base in its catalytic mechanism. The topology of the VP4 substrate binding site is consistent with the enzymes substrate specificity and a nucleophilic attack from the si-face of the substrates scissile bond. Despite low levels of sequence identity, VP4 shows similarities in its active site to other characterized Ser/Lys proteases such as signal peptidase, LexA protease and Lon protease. Together, the structure of VP4 provides insights into the mechanism of a recently characterized clan of serine proteases that utilize a lysine general base and reveals the structure of potential targets for antiviral therapy, especially for other related and economically important viruses, such as infectious bursal disease virus in poultry and infectious pancreatic necrosis virus in aquaculture.     

The structural basis for catalysis and substrate specificity of a rhomboid protease

Rhomboids are intramembrane proteases that use a catalytic dyad of serine and histidine for proteolysis. They are conserved in both prokaryotes and eukaryotes and regulate cellular processes as diverse as intercellular signalling, parasitic invasion of host cells, and mitochondrial morphology. Their widespread biological significance and consequent medical potential provides a strong incentive to understand the mechanism of these unusual enzymes for identification of specific inhibitors. In this study, we describe the structure of Escherichia coli rhomboid GlpG covalently bound to a mechanism‐based isocoumarin inhibitor. We identify the position of the oxyanion hole, and the S₁‐ and S₂′‐binding subsites of GlpG, which are the key determinants of substrate specificity. The inhibitor‐bound structure suggests that subtle structural change is sufficient for catalysis, as opposed to large changes proposed from previous structures of unliganded GlpG. Using bound inhibitor as a template, we present a model for substrate binding at the active site and biochemically test its validity. This study provides a foundation for a structural explanation of rhomboid specificity and mechanism, and for inhibitor design.     

Converting a DNA damage checkpoint effector (UmuD2C) into a lesion bypass polymerase (UmuD'2C)

During the SOS response of Escherichia coli to DNA damage, the umuDC operon is induced, producing the trimeric protein complexes UmuD2C, a DNA damage checkpoint effector, and UmuD'2C (DNA polymerase V), which carries out translesion synthesis, the basis of 'SOS mutagenesis'. UmuD'2, the homodimeric component of DNA pol V, is produced from UmuD by RecA-facilitated self-cleavage, which removes the 24 N-terminal residues of UmuD. We report the solution structure of UmuD'2 (PDB ID 1I4V) and interactions within UmuD'-UmuD, a heterodimer inactive in translesion synthesis. The overall shape of UmuD'2 in solution differs substantially from the previously reported crystal structure, even though the topologies of the two structures are quite similar. Most significantly, the active site residues S60 and K97 do not point directly at one another in solution as they do in the crystal, suggesting that self-cleavage of UmuD might require RecA to assemble the active site. Structural differences between UmuD'2 and UmuD'- UmuD suggest that UmuD'2C and UmuD2C might achieve their different biological activities through distinct interactions with RecA and DNA pol III.     

丙型肝炎病毒NS3蛋白酶在酵母系统中的可溶性表达

利用毕赤酵母系统表达具有催化活性的丙型肝炎病毒 (HCV)NS3蛋白酶 .将PCR直接扩增的病毒NS3丝氨酸蛋白酶基因和重组的带有辅酶的单链NS3 NS4A蛋白酶基因 ,分别插入表达载体pPICZαA的EcoRⅠ和XbaⅠ克隆位点 ,转化毕赤酵母GS115 ,可溶性表达NS3蛋白酶和单链NS3 4A蛋白酶 ;ELISA法测定表达蛋白酶的抗原性 ;原核高效表达载体pBVIL1表达酶切底物NS5A B片段 ,体外与蛋白酶共同温育 ,SDS PAGE鉴定蛋白酶催化活性 .载体测序结果表明 ,重组载体pPICZαA NS3和pPICZαA NS3 4A中的目的基因序列插入正确 ;SDS PAGE结果显示 ,培养物上清中存在分泌型目的蛋白带 ;ELISA结果证实 ,表达蛋白与HCV阳性血清有结合活性 ;蛋白酶与底物NS5A B片段不同作用时间的SDS PAGE ,看到约 2 4kD处底物条带的分解 .说明用毕赤酵母表达系统成功地表达了可溶性HCVNS3和单链NS3 4A蛋白酶 ;两种结构形式的蛋白酶在体外系统中都有催化活性 ,同时也都具有抗原性 .该研究为大量和方便地获得有催化活性的HCVNS3蛋白酶提供了有效途径 .     

A model for the structure of the Escherichia coli SOS-regulated UmuD2 protein

The ubiquitous Y-family of DNA polymerases, exemplified by the Escherichia coli UmuC protein (the catalytic subunit of DNA Pol V), possess the remarkable ability to replicate imperfect DNA templates that cannot be replicated by other types of DNA polymerases. Since this ability comes at the cost of a reduced fidelity, it is important that organisms manage these unique polymerases to coordinate their actions with those of the replication machinery. In E. coli, it is becoming evident that a sophisticated series of protein-protein interactions involving the two forms of the umuD gene product, UmuD and UmuD' and components of the replicative DNA polymerase serve to manage the actions of the umuC-encoded DNA polymerase. The purpose of this study was to better understand how structural differences between UmuD2 and UmuD2' help to determine which biological role the umuDC gene products will play; the UmuD2C complex functions as a DNA damage checkpoint effector, while the UmuD2'C complex participates in translesion DNA synthesis, which serves as the mechanistic basis for most chemical and UV light mutagenesis. Based on the results of a combination of disulfide cross-linking experiments, measurements of solvent accessibility and electron paramagnetic spin resonance (EPR) studies, we have developed a refined model for the structure of the UmuD2 homodimer. In the model that we are proposing, the N-terminal arms of UmuD (residues 1-39) form an extended interface in the UmuD2 homodimer by folding down over the globular domains of their intradimer partners. As a result, significant portions of the surface of each globular domain are buried in the UmuD2 homodimer. Based on the structure of the UmuD2' homodimer, both in the crystal and in solution, these same surfaces are exposed. Implications of these structural differences between the UmuD2 and the UmuD2' homodimers with respect to their roles in managing the actions of the umuC-encoded DNA polymerase are discussed.     

Mechanistic role of NS4A and substrate in the activation of HCV NS3 protease

Hepatitis C virus (HCV) NS3 protease is the key enzyme for its maturation. Three hypotheses have been advanced in the literature to demonstrate the mechanism of the activation of the HCV NS3 protease. A virus-encoded protein NS4A and substrate are proposed to be involved in the activation of the HCV NS3 protease. However, the three hypotheses are not completely consistent with one another. Multiple molecular dynamics simulations were performed on various NS3 protease systems: free NS3 protease, NS3/4A, NS3/inhibitor, and NS3/4A/inhibitor complexes, to further unravel the mechanism of the activation of the NS3 protease. Simulation results suggest that the binding of NS4A induces a classic serine protease conformation of the catalytic triad of the NS3 protease. NS4A rearranges the secondary structure of both the N-terminus and catalytic site of the NS3 protease, reduces the mobility of the global structure of the NS3 protease, especially the catalytic site, and provides a rigid and tight structure, except for the S1 pocket, for the binding and hydrolysis of substrates. The binding of substrate also contributes to the activation of the NS3 protease by an induced-fit of the classic serine protease catalytic triad. However, the global structure of the NS3 protease is still loose and highly flexible without stable secondary structural elements, such as helix α0 at the N-terminus and helix α1 and β-sheet E1-F1 at the catalytic site. The structure of the NS3 protease without NS4A is not suitable for the binding and hydrolysis of substrates.     

Crystal structure of a bacterial signal peptidase apoenzyme: implications for signal peptide binding and the Ser-Lys dyad mechanism.

We report here the x-ray crystal structure of a soluble catalytically active fragment of the Escherichia coli type I signal peptidase (SPase-(Delta2-75)) in the absence of inhibitor or substrate (apoenzyme). The structure was solved by molecular replacement and refined to 2.4 A resolution in a different space group (P4(1)2(1)2) from that of the previously published acyl-enzyme inhibitor-bound structure (P2(1)2(1)2) (Paetzel, M., Dalbey, R.E., and Strynadka, N.C.J. (1998) Nature 396, 186-190). A comparison with the acyl-enzyme structure shows significant side-chain and main-chain differences in the binding site and active site regions, which result in a smaller S1 binding pocket in the apoenzyme. The apoenzyme structure is consistent with SPase utilizing an unusual oxyanion hole containing one side-chain hydroxyl hydrogen (Ser-88 OgammaH) and one main-chain amide hydrogen (Ser-90 NH). Analysis of the apoenzyme active site reveals a potential deacylating water that was displaced by the inhibitor. It has been proposed that SPase utilizes a Ser-Lys dyad mechanism in the cleavage reaction. A similar mechanism has been proposed for the LexA family of proteases. A structural comparison of SPase and members of the LexA family of proteases reveals a difference in the side-chain orientation for the general base lysine, both of which are stabilized by an adjacent hydroxyl group. To gain insight into how signal peptidase recognizes its substrates, we have modeled a signal peptide into the binding site of SPase. The model is built based on the recently solved crystal structure of the analogous enzyme LexA (Luo, Y., Pfuetzner, R. A., Mosimann, S., Paetzel, M., Frey, E. A., Cherney, M., Kim, B., Little, J. W., and Strynadka, N. C. J. (2001) Cell 106, 1-10) with its bound cleavage site region.     

Mutational evidence of transition state stabilization by serine 88 in Escherichia coli type I signal peptidase

Type I signal peptidase (SPase I) catalyzes the hydrolytic cleavage of the N-terminal signal peptide from translocated preproteins. SPase I belongs to a novel class of Ser proteases that utilize a Ser/Lys dyad catalytic mechanism instead of the classical Ser/His/Asp triad found in most Ser proteases. Recent X-ray crystallographic studies indicate that the backbone amide nitrogen of the catalytic Ser 90 and the hydroxyl side chain of Ser 88 might participate as H-bond donors in the transition-state oxyanion hole. In this work, contribution of the side-chain Ser 88 in Escherichia coli SPase I to the stabilization of the transition state was investigated through in vivo and in vitro characterizations of Ala-, Cys-, and Thr-substituted mutants. The S88T mutant maintains near-wild-type activity with the substrate pro-OmpA nuclease A. In contrast, substitution with Cys at position 88 results in more than a 740-fold reduction in activity (k(cat)) whereas S88A retains much less activity (>2440-fold decrease). Measurements of the kinetic constants of the individual mutant enzymes indicate that these decreases in activity are attributed mainly to decreases in k(cat) while effects on K(m) are minimal. Thermal inactivation and CD spectroscopic analyses indicate no global conformational perturbations of the Ser 88 mutants relative to the wild-type E. coli SPase I enzyme. These results provide strong evidence for the stabilization by Ser 88 of the oxyanion intermediate during catalysis by E. coli SPase I.     

The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis

The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.     

Protease II from Escherichia coli: sequencing and expression of the enzyme gene and characterization of the expressed enzyme.

Protease II gene of Escherichia coli HB101 was cloned and expressed in E. coli JM83. The transformant harboring a hybrid plasmid, pPROII-12, with a 2.4 kbp fragment showed 90-fold higher enzyme activity than the host. The whole nucleotide sequence of the inserted fragment of plasmid pPROII-12 was clarified by the dideoxy chain-terminating method. The sequence that encoded the mature enzyme protein was found to start at an ATG codon, as judged by comparison with amino terminal protein sequencing. The molecular weight of the enzyme was estimated to be 81,858 from the nucleotide sequence. The reactive serine residue of protease II was identified as Ser-532 with tritium DFP. The sequence around the serine residue is coincident with the common sequence of Gly-X-Ser-X-Gly, which has been found in the active site of serine proteases. Except for this region, protease II showed no significant sequence homology with E. coli serine proteases, protease IV and protease La (lon gene), or other known families of serine proteases. However, 25.3% homology was observed between protease II and prolyl endopeptidase from porcine brain. Although the substrate specificities of these two enzymes are quite different, it seems possible to classify protease II as a member of the prolyl endopeptidase family from the structural point of view.     

丝氨酸蛋白酶抑制剂Ea的表达纯化与活性分析

Ea是一种植物来源的丝氨酸蛋白酶抑制剂,分子量为18kD。利用其与丝氨酸蛋白酶家族成员的结合特性,可用于丝氨酸蛋白酶的结构与功能研究,也可作为亲和层析的配体而用于丝氨酸蛋白酶的纯化。将Ea基因插入大肠杆菌表达载体pET11a,在BL21(DE3)菌中以包涵体形式表达出重组蛋白质,表达量可占菌体蛋白质总量的30%。将包涵体变性、复性,得到具有天然抑制活性的rEa。经两步纯化所得rEa的纯度达到967%以上。活性分析表明,rEa对胰蛋白酶和人组织型纤溶酶原激活剂均有抑制作用。制备成rEaSepharose亲和柱可有效结合胰蛋白酶。     

Molecular structure of a novel membrane protease specific for a stomatin homolog from the hyperthermophilic archaeon Pyrococcus horikoshii

Membrane-bound proteases are involved in various regulatory functions. Our previous study indicated that the N-terminal region of an open reading frame, PH1510 (residues 16-236, designated as 1510-N) from the hyperthermophilic archaeon Pyrococcus horikoshii, is a serine protease with a catalytic Ser-Lys dyad that specifically cleaves the C-terminal hydrophobic residues of a membrane protein, the stomatin-homolog PH1511. In humans, an absence of stomatin is associated with a form of hemolytic anemia known as hereditary stomatocytosis, but the function of stomatin is not fully understood. Here, we report the crystal structure of 1510-N in dimeric form. Each active site of 1510-N is rich in hydrophobic residues, which accounts for the substrate-specificity. The monomer of 1510-N shows structural similarity to one monomer of Escherichia coli ClpP, an ATP-dependent tetradecameric protease. But, their oligomeric forms are different. Major contributors to dimeric interaction in 1510-N are the alpha7 helix and beta9 strand, both of which are missing from ClpP. While the long handle region of ClpP contributes to the stacking of two heptameric rings, the corresponding L2 loop of 1510-N is disordered because the region has little interaction with other residues of the same molecule. The catalytic Ser97 of 1510-N is in almost the same location as the catalytic Ser97 of E.coli ClpP, whereas another residue, Lys138, presumably forming the catalytic dyad, is located in the disordered L2 region of 1510-N. These findings suggest that the binding of the substrate to the catalytic site of 1510-N induces conformational changes in a region that includes loop L2 so that Lys138 approaches the catalytic Ser97.     

The dimeric SOS mutagenesis protein UmuD is active as a monomer

The homodimeric umuD gene products play key roles in regulating the cellular response to DNA damage in Escherichia coli. UmuD(2) is composed of 139-amino acid subunits and is up-regulated as part of the SOS response. Subsequently, damage-induced RecA·ssDNA nucleoprotein filaments mediate the slow self-cleavage of the N-terminal 24-amino acid arms yielding UmuD'(2). UmuD(2) and UmuD'(2) make a number of distinct protein-protein contacts that both prevent and facilitate mutagenic translesion synthesis. Wild-type UmuD(2) and UmuD'(2) form exceptionally tight dimers in solution; however, we show that the single amino acid change N41D generates stable, active UmuD and UmuD' monomers that functionally mimic the dimeric wild-type proteins. The UmuD N41D monomer is proficient for cleavage and interacts physically with DNA polymerase IV (DinB) and the β clamp. Furthermore, the N41D variants facilitate UV-induced mutagenesis and promote overall cell viability. Taken together, these observations show that a monomeric form of UmuD retains substantial function in vivo and in vitro.     

Selective disruption of the DNA polymerase III α-β complex by the umuD gene products

DNA polymerase III (DNA pol III) efficiently replicates the Escherichia coli genome, but it cannot bypass DNA damage. Instead, translesion synthesis (TLS) DNA polymerases are employed to replicate past damaged DNA; however, the exchange of replicative for TLS polymerases is not understood. The umuD gene products, which are up-regulated during the SOS response, were previously shown to bind to the α, β and ε subunits of DNA pol III. Full-length UmuD inhibits DNA replication and prevents mutagenic TLS, while the cleaved form UmuD' facilitates mutagenesis. We show that α possesses two UmuD binding sites: at the N-terminus (residues 1-280) and the C-terminus (residues 956-975). The C-terminal site favors UmuD over UmuD'. We also find that UmuD, but not UmuD', disrupts the α-β complex. We propose that the interaction between α and UmuD contributes to the transition between replicative and TLS polymerases by removing α from the β clamp.     

Identification of specific amino acid residues in the E. coli beta processivity clamp involved in interactions with DNA polymerase III, UmuD and UmuD'

Variants of a pentapeptide sequence (QL[S/F]LF), referred to as the eubacterial clamp-binding motif, appear to be required for certain proteins to bind specifically to the Escherichia coli beta sliding clamp, apparently by making contact with a hydrophobic pocket located at the base of the C-terminal tail of each beta protomer. Although both UmuC (DNA pol V) and the alpha catalytic subunit of DNA polymerase III (pol III) each bear a reasonable match to this motif, which appears to be required for their respective interactions with the clamp, neither UmuD not UmuD' do. As part of an ongoing effort to understand how interactions involving the different E. coli umuDC gene products and components of DNA polymerase III help to coordinate DNA replication with a DNA damage checkpoint control and translesion DNA synthesis (TLS) following DNA damage, we characterized the surfaces on beta important for its interactions with the two forms of the umuD gene product. We also characterized the surface of beta important for its interaction with the alpha catalytic subunit of pol III. Our results indicate that although UmuD, UmuD' and alpha share some common contacts with beta, each also makes unique contacts with the clamp. These findings suggest that differential interactions of UmuD and UmuD' with beta impose a DNA damage-responsive conditionality on how beta interacts with the translesion DNA polymerase UmuC. This is formally analogous to how post-translational modification of the eukaryotic PCNA clamp influences mutagenesis. We discuss the implications of our findings in terms of how E. coli might coordinate the actions of the umuDC gene products with those of pol III, as well as for how organisms in general might manage the actions of their multiple DNA polymerases.     

Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate–peptide complex structures

The mechanisms of intramembrane proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid proteases, we have synthesised a series of novel peptidyl-chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl-CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the protease. The S1 subsite is prominent and merges into the ‘water retention site’, suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid-like protein superfamily may have similar substrate or client-protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG.     

Structure and function of the virulence-associated high-temperature requirement A of Mycobacterium tuberculosis

The high-temperature requirement A (HtrA) family of serine proteases has been shown to play an important role in the environmental and cellular stress damage control system in Escherichia coli. Mycobacterium tuberculosis ( Mtb) has three putative HtrA-like proteases, HtrA1, HtrA2, and HtrA3. The deletion of htrA2 gives attenuated virulence in a mouse model of TB. Biochemical analysis reveals that HtrA2 can function both as a protease and as a chaperone. The three-dimensional structure of HtrA2 determined at 2.0 A resolution shows that the protease domains form the central core of the trimer and the PDZ domains extend to the periphery. Unlike E. coli DegS and DegP, the protease is naturally active due to the formation of the serine protease-like catalytic triad and its uniquely designed oxyanion hole. Both protease and PDZ binding pockets of each HtrA2 molecule are occupied by autoproteolytic peptide products and reveal clues for a novel autoregulatory mechanism that might have significant importance in HtrA-associated virulence of Mtb.     

Insight derived from molecular dynamics simulation into dynamics and molecular motions of cuticle-degrading serine protease Ver112

Cuticle-degrading serine protease Ver112, which derived from a nematophagous fungus Lecanicillium psalliotae, has been exhibited to have high cuticle-degrading and nematicidal activities. We have performed molecular dynamics (MD) simulation based on the crystal structure of Ver112 to investigate its dynamic properties and large-scale concerted motions. The results indicate that the structural core of Ver112 shows a small fluctuation amplitude, whereas the substrate binding sites, and the regions close to and opposite the substrate binding sites experience significant conformational fluctuations. The large concerted motions obtained from essential dynamics (ED) analysis of MD trajectory can lead to open or close of the substrate binding sites, which are proposed to be linked to the functional properties of Ver112, such as substrate binding, orientation, catalytic, and release. The significant motion in the loop regions that is located opposite the binding sites are considered to play an important role in modulating the dynamics of the substrate binding sites. Furthermore, the bottom of free energy landscape (FEL) of Ver112 are rugged, which is mainly caused by the fluctuations of substrate binding regions and loops located opposite the binding site. In addition, the mechanism underlying the high flexibility and catalytic activity of Ver112 was also discussed. Our simulation study complements the biochemical and structural studies, and provides insight into the dynamics-function relationship of cuticle-degrading serine protease Ver112.     

Active-site mobility in human immunodeficiency virus, type 1, protease as demonstrated by crystal structure of A28S mutant.

The mutation Ala28 to serine in human immunodeficiency virus, type 1, (HIV-1) protease introduces putative hydrogen bonds to each active-site carboxyl group. These hydrogen bonds are ubiquitous in pepsin-like eukaryotic aspartic proteases. In order to understand the significance of this difference between HIV-1 protease and homologous, eukaryotic aspartic proteases, we solved the three-dimensional structure of A28S mutant HIV-1 protease in complex with a peptidic inhibitor U-89360E. The structure has been determined to 2.0 A resolution with an R factor of 0.194. Comparison of the mutant enzyme structure with that of the wild-type HIV-1 protease bound to the same inhibitor (Hong L, Treharne A, Hartsuck JA, Foundling S, Tang J, 1996, Biochemistry 35:10627-10633) revealed double occupancy for the Ser28 hydroxyl group, which forms a hydrogen bond either to one of the oxygen atoms of the active-site carboxyl or to the carbonyl oxygen of Asp30. We also observed marked changes in orientation of the Asp25 catalytic carboxyl groups, presumably caused by the new hydrogen bonds. These observations suggest that catalytic aspartyl groups of HIV-1 protease have significant conformational flexibility unseen in eukaryotic aspartic proteases. This difference may provide an explanation for some unique catalytic properties of HIV-1 protease.     

Subunit-specific degradation of the UmuD/D' heterodimer by the ClpXP protease: the role of trans recognition in UmuD' stability

The Escherichia coli UmuD' protein is a subunit of the recently described error-prone DNA polymerase, pol V. UmuD' is initially synthesized as an unstable and mutagenically inactive pro-protein, UmuD. Upon processing, UmuD' assumes a relatively stable conformation and becomes mutagenically active. While UmuD and UmuD' by themselves exist in vivo as homodimers, when together they preferentially interact to form heterodimers. Quite strikingly, it is in this context that UmuD' becomes susceptible to ClpXP-mediated proteolysis. Here we report a novel targeting mechanism designed for degrading the mutagenically active UmuD' subunit of the UmuD/D' heterodimer complex, while leaving the UmuD protein intact. Surprisingly, a signal that is essential and sufficient for targeting UmuD' for degradation was found to reside on UmuD not UmuD'. UmuD was also shown to be capable of channeling an excess of UmuD' to ClpXP for degradation, thereby providing a mechanism whereby cells can limit error-prone DNA replication.