Functional Consequences of the Kaposi's Sarcoma-Associated Herpesvirus Protease Structure:  Regulation of Activity and Dimerization by Conserved Structural Elements<sup>,</sup>

Reiling, K. Kinkead; Pray, Todd; Craik, Charles S.; Stroud, Robert M.

doi:10.1021/bi001019h

Cited by 38 publications

(54 citation statements)

References 30 publications

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“…Like all herpesviruses, KSHV expresses and requires a virally encoded protease, KSHV Pr (24,25,29,37). Like those of other herpesvirus proteases, the dimer interface of KSHV Pr is composed primarily of helix ␣5.…”

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confidence: 99%

Herpesvirus Protease Inhibition by Dimer Disruption

Shimba

Nomura

Marnett

et al. 2004

J Virol

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Kaposi's sarcoma-associated herpesvirus (KSHV), like all herpesviruses, encodes a protease (KSHV Pr), which is necessary for the viral lytic cycle. Herpesvirus proteases function as obligate dimers; however, each monomer has an intact, complete active site which does not interact directly with the other monomer across the dimer interface. Protein grafting of an interfacial KSHV Pr ␣-helix onto a small stable protein, avian pancreatic polypeptide, generated a helical 30-amino-acid peptide designed to disrupt the dimerization of KSHV Pr. The chimeric peptide was optimized through protein modeling of the KSHV Pr-peptide complex. Circular dichroism analysis and gel filtration chromatography revealed that the rationally designed peptide adopts a helical conformation and is capable of disrupting KSHV Pr dimerization, respectively. Additionally, the optimized peptide inhibits KSHV Pr activity by 50% at a ϳ200-fold molar excess of peptide to KSHV Pr, and the dissociation constant was estimated to be 300 M. Mutagenesis of the interfacial residue M197 to a leucine resulted in an inhibitory concentration which was twofold higher for KSHV Pr M197L than for KSHV Pr, in agreement with the model that the dimer interface is involved in peptide binding. These results indicate that the dimer interface, as well as the active sites, of herpesvirus proteases is a viable target for inhibiting enzyme activity.

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confidence: 99%

Herpesvirus Protease Inhibition by Dimer Disruption

Shimba

Nomura

Marnett

et al. 2004

J Virol

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“…As in dsDNA bacteriophages, the proteolytic processing of capsid protein precursor by a virus-encoded enzyme is essential for producing infectious virions (17,20,(37)(38)(39), and several structural and mechanistic parallels between the maturation of phage heads and herpesvirus capsids have been noted (14,31). So far, the UL26 gene products are the only known proteases encoded by herpesvirus genomes, and the three-dimensional structures of UL26 proteases from all three herpesvirus subfamilies have been determined (3,6,15,25,26,28,32,35) (Fig. 1A).…”

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confidence: 99%

Displacements of Prohead Protease Genes in the Late Operons of Double-Stranded-DNA Bacteriophages

Liu

Mushegian

2004

J Bacteriol

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Most of the known prohead maturation proteases in double-stranded-DNA bacteriophages are shown, by computational methods, to fall into two evolutionarily independent clans of serine proteases, herpesvirus assemblin-like and ClpP-like. Phylogenetic analysis suggests that these two types of phage prohead protease genes displaced each other multiple times while preserving their exact location within the late operons of the phage genomes.Capsid maturation in double-stranded-DNA (dsDNA) bacteriophages requires proteolytic cleavage by a prohead protease. The MEROPS database (27; http://merops.sanger.ac.uk) currently places phage prohead proteases into four families, two of which exclusively consist of phage and prophage prohead proteases with uncharacterized catalytic mechanisms. They are family U9 (prohead proteases from T4-like Myoviridae) and family U35 (prohead proteases from HK97 and related phages of the Siphoviridae family). The other two, serine protease families S14 and S49, are typified by ClpP protease and Escherichia coli peptidase IV, respectively, and contain phage prohead proteases such as gpC protein of phage lambda and prohead protease of Pseudomonas aeruginosa phage D3 (both phages are members of the Siphoviridae).Prohead protease is an essential factor in phage capsid morphogenesis, as first demonstrated for phage T4 (2). The maturational cleavage of the capsid protein precursors confers stability to the nucleic acid-free prohead and triggers a conformational switch of prohead, which allows phage genomic DNA to enter and initiate the DNA packaging process. A similar capsid maturation mechanism has been reported for various dsDNA phages from the Siphoviridae family, such as HK97 (8), PVL (7), and D3 (12), suggesting that the proteolytic cleavage of structural proteins during phage capsid assembly is common in many dsDNA phages and that the prohead protease is the key enzyme in the process. The prohead protease is generally encoded next to the gene for the capsid protein that it cleaves and may be fused to it in some cases (e.g., Siphoviridae psiM2 and psiM100). Although the capsid assembly and maturation processes in phages HK97, from the lambda-like Siphoviridae group, and T4, from the Myoviridae family, are tightly regulated and share many common features, no sequence similarity or evolutionary connection between their prohead proteases has been reported. In this study, we use computational analysis to show that virtually all known prohead proteases from dsDNA phages belong to one of the two superfamilies of serine proteases and are unrelated to each other but apparently functionally interchangeable.To examine the evolutionary relationship between the prohead proteases from phages T4 and HK97, we searched the NCBI nonredundant protein database (7 November 2003; 1,537,641 sequences; 502,645,420 total letters) with the PSI-BLAST program (1) using members of the U9 and U35 families as queries, with the threshold for inclusion set at a BLAST E value of 0.02. Starting with the prohead protease from HK97...

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“…Epstein-Barr virus protease (EBV Pr), a related γ-herpesvirus protease, is also composed of 29% R-helix at low micromolar concentrations (26). Analysis of the X-ray dimeric structures of KSHV Pr and EBV Pr shows that, when completely dimeric, both proteases should possess 31% R-helicity, consistent with the R-helical content of dimeric, inhibited KSHV Pr as measured by CD (25,26).…”

Section: Quantification Of Structural Differences Between the Monomermentioning

confidence: 83%

“…(25). Helix 1 contains greater than average chemical shift differences throughout the length of the helix, which extends from Ser73 to Arg91.…”

Section: Quantification Of Structural Differences Between the Monomermentioning

confidence: 99%

“…All herpesvirus proteases share a common structure composed of a seven-stranded -barrel surrounded by six R-helices (19)(20)(21)(22)(23)(24)(25)(26). Herpesvirus proteases require dimerization for activity (17,18,(27)(28)(29)(30)(31) with binding affinities in the low micromolar to high nanomolar range; however, the dimeric X-ray structures all show the presence of intact active sites in each monomer, which are separate from the dimer interface.…”

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One Functional Switch Mediates Reversible and Irreversible Inactivation of a Herpesvirus Protease

et al. 2006

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Distinct mechanisms have evolved to regulate the function of proteolytic enzymes. Viral proteases in particular have developed novel regulatory mechanisms, presumably due to their comparatively rapid life cycles and responses to constant evolutionary pressure. Herpesviruses are a family of human pathogens that require a viral protease with a concentration-dependent zymogen activation involving folding of two R-helices and activation of the catalytic machinery, which results in formation of infectious virions. Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) is unique among the herpesvirus proteases in possessing an autolysis site in the dimer interface, which removes the carboxyl-terminal 27 amino acids comprising an R-helix adjacent to the active site. Truncation results in the irreversible loss of dimerization and concomitant inactivation. We characterized the conformational and functional differences between the active dimer, inactive monomer, and inactive truncated protease to determine the different protease regulatory mechanisms that control the KSHV lytic cycle. Circular dichroism revealed a loss of 31% R-helicity upon dimer dissociation. Comparison of the full-length and truncated monomers by NMR showed differences in 21% of the protein structure, mainly located adjacent to the dimer interface, with little perturbation of the overall protein upon truncation. Fluorescence polarization and active site labeling, with a transition state mimetic, characterized the functional effects of these conformational changes. Substrate turnover is abolished in both the full-length and truncated monomers; however, substrate binding remained intact. Disruption of the helix 6 interaction with the active site oxyanion loop is therefore used in two independent regulatory mechanisms of proteolytic activity.The nine known human herpesviruses (HHVs) 1 are responsible for diseases ranging from herpes labialis, caused by Herpes simplex virus-1, to Kaposi's sarcoma (KS), caused by Kaposi's sarcoma-associated herpesvirus (KSHV, HHV8) (1-3). These nine HHVs are grouped into three families, based on phenotypic and genomic similarities. Although the R-, the -, and the γ-herpesviruses (including KSHV) vary in tissue distribution and disease states, all herpesviruses possess homologous structural and enzymatic proteins involved in their lytic cycles. Among these proteins are the protease (Pr) and assembly protein (AP), which are necessary for infectious virion formation (4-8). Pr is expressed as a Pr/AP fusion (9-13), AP binds the major capsid protein, and the complex translocates to the nucleus where the immature capsid is assembled around the AP scaffold (14). Pr cleaves the Pr/AP fusion at the release site (R-site) and the maturation site (M-site) (10,11,13,15,16), allowing DNA packaging, formation of the infectious virion, and completion of the lytic cycle. Additional internal cleavage sites have been identified in multiple herpesvirus proteases; however, KSHV Pr is unique in that autolysis at the D-site (dimer d...

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Functional Consequences of the Kaposi's Sarcoma-Associated Herpesvirus Protease Structure: Regulation of Activity and Dimerization by Conserved Structural Elements^,

Cited by 38 publications

References 30 publications

Herpesvirus Protease Inhibition by Dimer Disruption

Herpesvirus Protease Inhibition by Dimer Disruption

Displacements of Prohead Protease Genes in the Late Operons of Double-Stranded-DNA Bacteriophages

One Functional Switch Mediates Reversible and Irreversible Inactivation of a Herpesvirus Protease

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Functional Consequences of the Kaposi's Sarcoma-Associated Herpesvirus Protease Structure: Regulation of Activity and Dimerization by Conserved Structural Elements,

Cited by 38 publications

References 30 publications

Herpesvirus Protease Inhibition by Dimer Disruption

Herpesvirus Protease Inhibition by Dimer Disruption

Displacements of Prohead Protease Genes in the Late Operons of Double-Stranded-DNA Bacteriophages

One Functional Switch Mediates Reversible and Irreversible Inactivation of a Herpesvirus Protease

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Functional Consequences of the Kaposi's Sarcoma-Associated Herpesvirus Protease Structure: Regulation of Activity and Dimerization by Conserved Structural Elements^,