The Unusual Catalytic Triad of Poliovirus Protease 3C

Sárkány, Zsuzsa; Polgár, László

doi:10.1021/bi027004w

Cited by 37 publications

(34 citation statements)

References 21 publications

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“…36 and 44). The crystal structures of the picornains of hepatitis A (45), rhinovirus (46), and poliovirus (47) show a similarity of three-dimensional structures and catalytic mechanisms to the serine proteases of the trypsin/chymotrypsin family and may be evolutionarily related (36,43,44). The pH-dependent alkylation of the active site cysteine of poliovirus protease 3C with iodoacetamide has measured its pK a at 8.86 (42), which is similar to the estimated pK a of the SrtA thiol.…”

Section: Cysmentioning

confidence: 74%

“…Moreover, our finding that the removal of the Cys 184 side chain ( C184A SrtA ⌬N59 ) has only a modest effect on the ionization state of His 120 side chain argues against the presence of an ion pair, because in the papain system the pK a of His 159 is lowered by 4.5 pH units upon the methylthiolation of the Cys 25 (25). Although the thiolate-imidazolium ion pair is a common catalytic entity of cysteine proteases, it is not universal (42,43). The absence of an ion pair in SrtA suggests its catalytic mechanism may be similar to the viral 3C proteases (picornains), a structurally and mechanistically distinct group of cysteine proteases that perform general base catalysis (reviewed in Refs.…”

Section: Cysmentioning

confidence: 92%

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Sortase from Staphylococcus aureus Does Not Contain a Thiolate-Imidazolium Ion Pair in Its Active Site

Connolly¹,

Smith

Pilpa³

et al. 2003

Journal of Biological Chemistry

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Many surface proteins are anchored to the cell wall by the action of sortase enzymes, a recently discovered family of cysteine transpeptidases. As the surface proteins of human pathogens are frequently required for virulence, the sortase-mediated anchoring reaction represents a potential target for new anti-infective agents. It has been suggested that the sortase from Staphylococcus aureus (SrtA), may use a similar catalytic strategy as the papain cysteine proteases, holding its Cys 184 side chain in an active configuration through a thiolate-imidazolium ion interaction with residue His 120 . To investigate the mechanism of transpeptidation, we have synthesized a peptidyl-vinyl sulfone substrate mimic that irreversibly inhibits SrtA. Through the study of the pH dependence of SrtA inhibition and NMR, we have estimated the pK a s of the active site thiol (Cys 184 ) and imidazole (His 120 ) to be ϳ9.4 and 7.0, respectively. These measurements are inconsistent with the existence of a thiolate-imidazolium ion pair and suggest a general base catalysis mechanism during transpeptidation.

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Section: Cysmentioning

confidence: 74%

Section: Cysmentioning

confidence: 92%

Sortase from Staphylococcus aureus Does Not Contain a Thiolate-Imidazolium Ion Pair in Its Active Site

Connolly¹,

Smith

Pilpa³

et al. 2003

Journal of Biological Chemistry

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show abstract

“…Therefore, the imidazole assistance in the hydrolysis is very likely general base catalysis, as found with serine peptidases. The acidic component of the catalytic triad is essential for activity, but its negative charge does not influence the ionization of the thiol group, as demonstrated with the E71Q variant [124]. On the other hand, in the hepatitis A virus 3C peptidase the side chains of His44 and Asp84 (corresponding to His57 and Asp102 in chymotrypsin) are not in contact.…”

Section: Further Variations In the Members Of The Catalytic Triadmentioning

confidence: 99%

The catalytic triad of serine peptidases

Polgár

2005

Cell. Mol. Life Sci.

391

311

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The catalytic action of serine peptidases depends on the interplay of a nucleophile, a general base and an acid. In the classic trypsin and subtilisin families this catalytic triad is composed of serine, histidine and aspartic acid residues and exhibits similar spatial arrangements, but the order of the residues in the amino acid sequence is different. By now several new families have been discovered, in which the nucleophile-base-acid pattern is generally conserved, but the individual components can vary. The variations illustrate how different groups and different protein structures achieve the same reaction.

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“…The shallow active site cavity utilizes Cys147 as its nucleophile, with His40 and Glu71 serving as the acid/base catalysts (44).…”

mentioning

confidence: 99%

Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-Dependent RNA Polymerase

Marcotte

Wass

Gohara

et al. 2007

J Virol

109

123

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Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3C pro and the viral polymerase 3D pol and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4-Å resolution and the G64S fidelity mutant of 3D pol at a 3.0-Å resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active site is intact in both the 3CD and the 3D pol G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain's N terminus to the VPg binding site suggest how 3D pol makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.Poliovirus (PV), a member of the Picornaviridae family of RNA viruses, must simultaneously perform many tasks inside a host cell for efficient and successful viral replication, and only a small number of gene products are responsible for these processes. The virus produces a single polyprotein that is cleaved by virally encoded proteases. Many of the viral proteins, including precursor proteins, play multiple roles in viral replication.The viral protein 3CD is a multifunctional precursor to the poliovirus protease 3Cpro and the RNA-dependent RNA polymerase 3Dpol . The 3CD molecule retains its ability to function as a protease but lacks polymerase activity (20). Its proteolytic functions include cleavages, resulting in the production of structural proteins VP0, VP1, and VP3 and nonstructural proteins 3AB, 3CD, 3C pro , and 3D pol . In many of these functions, 3CD serves as a better protease than 3C pro , suggesting that the 3D region of 3CD contributes to that activity (34).3CD is also a crucial component of the viral replication complex. The 5Ј-terminal region of the poliovirus genome adopts a cloverleaf structure to which 3CD can bind in the presence of the viral protein 3AB or the cellular protein PCBP2 (2, 35, 49). Both forms of this ribonucleoprotein complex appear to play important roles in inducing RNA synthesis (49). The initiation of RNA synthesis is primed by the addition of two uridines to the 22-amino-acid protein primer VPg (3B) at the side-chain hydroxyl of Tyr3. Positive-strand synthesis is thought to use an RNA hairpin structure as its template within the PV genome (37,42). One model suggests that this cisacting replication signal in the 2C-noncoding region of the PV genome, cre(2C), binds to 3CD and forms a mu...

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The Unusual Catalytic Triad of Poliovirus Protease 3C

Cited by 37 publications

References 21 publications

Sortase from Staphylococcus aureus Does Not Contain a Thiolate-Imidazolium Ion Pair in Its Active Site

Sortase from Staphylococcus aureus Does Not Contain a Thiolate-Imidazolium Ion Pair in Its Active Site

The catalytic triad of serine peptidases

Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-Dependent RNA Polymerase

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