Structural plasticity broadens the specificity of an engineered protease

Bone, Roger; Silen, Joy L.; Agard, David A.

doi:10.1038/339191a0

Cited by 162 publications

(123 citation statements)

References 25 publications

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“…Similar flexible binding sites were also observed when mutations were made in the ␣-lytic protease S 1 subsite. Crystallographic data of M192A and M213A ␣-lytic protease mutants, with and without substrate analogues bound, demonstrated that there were local changes in conformation to accommodate the various P 1 residues; consequently, the mutant appeared to have relaxed specificity (43). Furthermore, crystal structures of Savinase with a Gly substitution residue, Ile 107 , which is analogous to Glu 255 in Kex2, demonstrated that the Savinase S 4 subsite was structurally flexible.…”

Section: Figmentioning

confidence: 93%

Plasticity of Extended Subsites Facilitates Divergent Substrate Recognition by Kex2 and Furin

Rozan

Krysan

Rockwell

et al. 2004

Journal of Biological Chemistry

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Yeast Kex2 and human furin are subtilisin-related proprotein convertases that function in the late secretory pathway and exhibit similar though distinguishable patterns of substrate recognition. Although both enzymes prefer Arg at P 1 and basic residues at P 2 , the two differ in recognition of P 4 and P 6 residues. To probe P 4 and P 6 recognition by Kex2p, furin-like substitutions were made in the putative S 4 and S 6 subsites of Kex2. T252D and Q283E mutations were introduced to increase the preference for Arg at P 4 and P 6 , respectively. Glu 255 was replaced with Ile to limit recognition of P 4 Arg. The effects of putative S 4 and S 6 mutations were determined by examining the cleavage by purified mutant enzymes of a series of fluorogenic substrates with systematic changes in P 4 and/or P 6 . Whereas wild type Kex2 exhibited little preference for Arg at P 6 , the T252D mutant and T252D/Q283E double mutant exhibited clear interactions with P 6 Arg. Moreover, the T252D and T252D/Q283E substitutions altered the influence of the P 6 residue on P 4 recognition. We infer that cross-talk between S 4 and S 6 , not seen in furin, allows wild type and mutant forms of Kex2 to adapt their subsites for altered modes of recognition. This apparent plasticity may allow the subsites to rearrange their local environment to interact with different substrates in a productive manner. E255I-Kex2 exhibited significantly decreased recognition of P 4 Arg in a tetrapeptide substrate with Lys at P 1 , although the general pattern of selectivity for aliphatic residues at P 4 remained unchanged.The subtilisin superfamily includes a subfamily of related processing proteases, the proprotein convertases that function in the late secretory pathway of diverse eukaryotic organisms including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and mammals (1-4). Unlike the degradative subtilisins, which display a broad substrate specificity for hydrophobic residues (5), the proprotein convertases are post-translational modifying enzymes that process secretory proteins in a sequence-specific manner. In general, these proteases cleave C-terminal to clusters of basic residues, but their exact sequence specificity differs among the members of this family, even though they are Ն45% identical within their subtilisin-related domains. Similarities and differences in substrate recognition were illustrated by the enzymatic characterization of two members of this family, the S. cerevisiae protease, Kex2, and the human homologue, furin.A detailed understanding of substrate recognition by Kex2 and furin has emerged from extensive analysis of the purified secreted, soluble enzymes using model peptide substrates. Based on these studies, the consensus cleavage site for Kex2 was determined to be (Ali/Arg)-Xaa-(Lys/Arg)-Arg2 (where Ali indicates an aliphatic amino acid), with the principal determinants being a basic residue at P 2 and Arg at P 1 (6 -9).

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Section: Figmentioning

confidence: 93%

Plasticity of Extended Subsites Facilitates Divergent Substrate Recognition by Kex2 and Furin

Rozan

Krysan

Rockwell

et al. 2004

Journal of Biological Chemistry

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“…Some proteases exhibit no promiscuity, whereas others are as promiscuous as the detoxification enzymes. Structural plasticity has been correlated with increased promiscuity in mutants of native proteases (48,49). Given that proteases share a highly conserved catalytic mechanism and yet display widely variable promiscuous behavior, they may serve as a useful system to study the structural and dynamic determinants of promiscuity.…”

Section: Discussionmentioning

confidence: 99%

A Quantitative Index of Substrate Promiscuity

2007

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Catalytic promiscuity is a widespread, but poorly understood, phenomenon among enzymes with particular relevance to the evolution of new functions, drug metabolism, and in vitro biocatalyst engineering. However, there is at present no way to quantitatively measure or compare this important parameter of enzyme function. Here we define a quantitative index of promiscuity (I) that can be calculated from the catalytic efficiencies of an enzyme toward a defined set of substrates. A weighted promiscuity index (J) that accounts for patterns of similarity and dissimilarity among the substrates in the set is also defined. Promiscuity indices were calculated for three different enzyme classes: eight serine and cysteine proteases, two glutathione S-transferase (GST) isoforms, and three cytochrome P450 (CYP) isoforms. The proteases ranged from completely specific (granzyme B, J ) 0.00) to highly promiscuous (cruzain, J ) 0.83). The four drug-metabolizing enzymes studied (GST A1-1 and the CYP isoforms) were highly promiscuous, with J values between 0.72 and 0.92; GST A4-4, involved in the clearance of lipid peroxidation products, is moderately promiscuous (J ) 0.37). Promiscuity indices also allowed for studies of correlation between substrate promiscuity and an enzyme's activity toward its most-favored substrate, for each of the three enzyme classes.

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“…The suppressor alleles could serve to relax some of the constraints imposed on the identity of splice-site residues, thereby allowing mutant as well as wild-type sequences to be utilized. In well-studied systems of protein-substrate recognition, mutations that permit a wider range of interactions have been observed to increase the flexibility of the binding interface or pocket (Bone et al 1989;Morton and Matthews 1995). Such effects are not limited to mutations at the site of binding, and, in fact, may occur at considerable distances (Mace et al 1995;Gutierrez et al 1998).…”

Section: A Role For Prp8 At the Spliceosomal Catalytic Corementioning

confidence: 99%

Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome

Collins¹,

Guthrie²

1999

Genes & Development

111

122

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The highly conserved spliceosomal protein Prp8 is known to cross-link the critical sequences at both the 5 (GU) and 3 (YAG) ends of the intron. We have identified prp8 mutants with the remarkable property of suppressing exon ligation defects due to mutations in position 2 of the 5 GU, and all positions of the 3 YAG. The prp8 mutants also suppress mutations in position A51 of the critical ACAGAG motif in U6 snRNA, which has been observed previously to cross-link position 2 of the 5 GU. Other mutations in the 5 splice site, branchpoint, and neighboring residues of the U6 ACAGAG motif are not suppressed. Notably, the suppressed residues are specifically conserved from yeast to man, and from U2-to U12-dependent spliceosomes. We propose that Prp8 participates in a previously unrecognized tertiary interaction between U6 snRNA and both the 5 and 3 ends of the intron. This model suggests a mechanism for positioning the 3 splice site for catalysis, and assigns a fundamental role for Prp8 in pre-mRNA splicing. The removal of introns from eukaryotic genes to generate mRNA is catalyzed by the spliceosome, a complex RNA-protein machine composed of small nuclear RNAs (snRNAs), and at least 60 proteins. The pre-mRNA splicing reaction consists of two sequential transesterification steps: The first step results in cleavage of the 5Ј splice site (5ЈSS) and formation of a branched (lariat) intermediate; the second step results in ligation of the 5Ј and 3Ј exons. The existence of self-splicing group-II introns, which utilize a similar two-step transesterification mechanism, has led to the hypothesis that premRNA splicing is catalyzed by RNA (Sharp 1985;Cech 1986). Indeed, mutational and cross-linking analyses have revealed a network of RNA-RNA interactions, which have been proposed to form the catalytic core of the spliceosome (Newman 1994;Nilsen 1994).Whether any of the spliceosomal proteins make direct structural or chemical contributions to the active site remains unknown. Numerous cross-linking studies have placed one spliceosomal protein, Prp8, at or near the catalytic core. As summarized in Figure 1, Prp8 has been observed to cross-link to RNA residues within each critical sequence component of the intron: The consensus sequences that define the 5ЈSS, the branch-point (BP), and the 3Ј splice site (3ЈSS) (MacMillan et

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Structural plasticity broadens the specificity of an engineered protease

Cited by 162 publications

References 25 publications

Plasticity of Extended Subsites Facilitates Divergent Substrate Recognition by Kex2 and Furin

Plasticity of Extended Subsites Facilitates Divergent Substrate Recognition by Kex2 and Furin

A Quantitative Index of Substrate Promiscuity

Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome

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