Expression and Purification of a Recombinant Tobacco Etch Virus NIa Proteinase: Biochemical Analyses of the Full-Length and a Naturally Occurring Truncated Proteinase Form

Parks, T. Dawn; Howard, E.D.; Wolpert, T J; Arp, Daniel J.; Dougherty, William G.

doi:10.1006/viro.1995.1331

Cited by 117 publications

(102 citation statements)

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“…Autoproteolysis-The catalytic domain of TEV protease has a propensity to cleave itself in vitro between residues 218 and 219 to yield a truncated enzyme with greatly diminished activity (15,27). It is uncertain, however, whether autoinactivation of TEV protease plays any role in the physiology of viral infection.…”

Section: Resultsmentioning

confidence: 99%

Structural Basis for the Substrate Specificity of Tobacco Etch Virus Protease

Phan

Zdanov

Evdokimov³

et al. 2002

Journal of Biological Chemistry

221

243

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Because of its stringent sequence specificity, the 3C-type protease from tobacco etch virus (TEV) is frequently used to remove affinity tags from recombinant proteins. It is unclear, however, exactly how TEV protease recognizes its substrates with such high selectivity. The crystal structures of two TEV protease mutants, inactive C151A and autolysis-resistant S219D, have now been solved at 2.2-and 1.8-Å resolution as complexes with a substrate and product peptide, respectively. The enzyme does not appear to have been perturbed by the mutations in either structure, and the modes of binding of the product and substrate are virtually identical. Analysis of the protein-ligand interactions helps to delineate the structural determinants of substrate specificity and provides guidance for reengineering the enzyme to further improve its utility for biotechnological applications.The Picornaviridae are a large superfamily of (ϩ)-strand RNA viruses that are responsible for a variety of plant and animal pathologies (1). Their RNA genomes are translated into polyprotein precursors that are co-translationally cleaved by viral proteases to generate the mature proteins (2). The majority of these processing events are mediated by the picornavirus 3C-type proteases, which are structurally similar to serine proteases like trypsin and chymotrypsin, but utilize a cysteine thiol instead of a serine hydroxyl as the active-site nucleophile (1, 3). Because they play an essential role in viral replication, 3C proteases are viewed as attractive molecular targets for antiviral therapeutics (4).The stringent sequence specificity of rhinovirus 3C protease and the 3C-like nuclear inclusion protease encoded by TEV 1 has also led to their widespread application in the biotechnology sector as reagents for endoproteolytic removal of affinity tags from recombinant proteins (5). In contrast to Factor Xa, enterokinase, and thrombin, neither of these viral proteases has ever been reported to cleave genetically engineered fusion proteins at unintended locations. All 3C-type proteases exhibit a strong preference for glutamine in the P1 position of their substrates and for small aliphatic residues in the P1Ј subsite, but these are clearly not the only specificity determinants (3, 6). Studies with oligopeptide substrates have established that the P6 and P3 subsites are also important specificity determinants for TEV protease (7), whereas it is the P4 and P2Ј positions that appear to make the greatest contribution to the unique specificity of rhinovirus 3C protease (8).Despite the fact that 3C-type proteases have been the subject of considerable interest, the structural basis of their substrate specificity remains obscure. Although the crystal structures of 3C proteases from hepatitis A virus (9), rhinovirus-14 (10), and poliovirus (11, 12) have been determined, none of them have cognate peptides in the active site. Consequently, efforts to explain the substrate specificity of these enzymes have relied on modeling or, in a few cases, on the structures of e...

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Section: Resultsmentioning

confidence: 99%

Structural Basis for the Substrate Specificity of Tobacco Etch Virus Protease

Phan

Zdanov

Evdokimov³

et al. 2002

Journal of Biological Chemistry

221

243

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“…Expression and purification of TEV protease was performed as described previously [26]. TEV protease was concentrated with a Centriprep 10 concentrator (Amicon), shock-frozen and stored in multiple aliquots at 280 8C.…”

Section: Expression and Purification Of Tev Proteasementioning

confidence: 99%

Characterization of the ternary complex between Rab7, REP‐1 and Rab geranylgeranyl transferase

Alexandrov

Simón²,

Yurchenko³

et al. 1999

European Journal of Biochemistry

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Geranylgeranylation is a post-translational modification of Rab GTPases that enables them to associate reversibly with intracellular membranes. Geranylgeranylation of Rab proteins is critical for their activity in controlling intracellular membrane transport. According to the currently accepted model for their action, newly synthesized Rab proteins are recruited by Rab escort protein (REP) and are presented to the Rab geranylgeranyl transferase (RabGGTase) which covalentely modifies the Rab protein with two geranylgeranyl moieties. After prenylation, the Rab protein remains in complex with REP and is delivered to the target membrane by the latter. In this work, we show that RabGGTase can form a stable complex with Rab7±REP in the absence of its lipid substrate geranylgeranyl pyrophosphate. In order to characterize this interaction, we developed three fluorescence assays reporting on the interaction of RabGGTase with the Rab7±REP complex. For this interaction we determined a K d value of about 120 nm.Association of RabGGTase with the Rab7±REP complex occurs with a rate constant of < 10 8 m 21´s21 . We demonstrate that the state of the nucleotide bound to Rab7 does not influence the affinity of RabGGTase for the Rab7±REP-1 complex. Finally, we address the issue of substrate specificity of RabGGTase. Titration experiments demonstrate that, in contrast with farnesyl transferase, RabGGTase does not recognize a defined C-terminal sequence motif. Experiments using Rab7 mutants in which the last 16 amino acids were either mutated or truncated revealed that the distal part of the C-terminus makes only a limited contribution to the binding affinity between RabGGTase and the Rab7±REP-1 complex. This demonstrates the functional dissimilarity between RabGGTase and geranylgeranyl transferase I and farnesyl transferase, which interact specifically with the C-terminus of their substrates. Based on these experiments, we propose that RabGGTase recognizes the overall structure arising from the association of Rab and REP and then`scans' the flexible C-terminus to position the proximal cysteines into the active site.Keywords: geranylgeranylation; prenyltransferase; Rab7; Rab geranylgeranyl transferase; REP.Rab proteins are members of the Ras superfamily of small GTP-binding proteins. Many of them were shown to be implicated in control of intracellular vesicular traffic [1,2]. For their function Rab proteins require modification by geranylgeranyl isoprenoids. This allows them to associate reversibly with a membrane in a tightly controlled fashion [3,4]. Covalent attachment of geranylgeranyl groups to two C-terminal cysteines is catalyzed by Rab geranylgeranyl transferase (RabGGTase).Mammalian RabGGTase (GGTase II) is a heterodimer composed of tightly associated a and b subunits of 60 and 38 kDa, respectively [5,6], and belongs to the family of protein prenyl transferases together with farnesyl transferase and geranylgeranyl transferase I (GGTase I) [7]. Substrates of farnesyl transferase include members of the Ras family, lamin b and...

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“…This process has been extensively studied for many picornavirus superfamily members including poliovirus (Jore et al, 1988 ;Ypma-Wong et al, 1988 a ;Baum et al, 1991 ;Ha$ mmerle et al, 1991 ;Kean et al, 1991), potyviruses (Carrington et al, 1989Dougherty & Parks, 1991 ;Parks et al, 1995 ;Verchot et al, 1991), comoviruses (Dessens & Lomonossoff, 1991 ;Peters et al, b, 1995 and, to a lesser extent, nepoviruses (Demangeat et al, 1992 ;Margis et al, 1994 ; Author for correspondence : He! le ' ne Sanfaçon (at the Pacific AgriFood Research Centre).…”

Section: Introductionmentioning

confidence: 99%