Stabilizing the subtilisin BPN' pro‐domain by phage display selection: How restrictive is the amino acid code for maximum protein stability?

Ruan, Biao; Hoskins, Joel R.; Wang, Lan; Bryan, Philip N.

doi:10.1002/pro.5560071111

Cited by 40 publications

(42 citation statements)

References 39 publications

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“…The bacterial prodomain is predominantly unfolded when the catalytic subtilisin domain is not present, but adopts the α/β‐plait structure in the subtilisin‐bound state [Fig. 2(B)] 21–23. In contrast, the mammalian PC1 prodomain is independently folded [Fig.…”

Section: Resultsmentioning

confidence: 99%

Solution NMR structure of a sheddase inhibitor prodomain from the malarial parasite Plasmodium falciparum

Chen

Oganesyan

et al. 2012

Proteins

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Plasmodium subtilisin 2 (Sub2) is a multidomain protein that plays an important role in malaria infection. Here, we describe the solution NMR structure of a conserved region of the inhibitory prodomain of Sub2 from Plasmodium falciparum, termed prosub2. Despite the absence of any detectable sequence homology, the protozoan prosub2 has structural similarity to bacterial and mammalian subtilisin-like prodomains. Comparison with the three-dimensional structures of these other prodomains suggests a likely binding interface with the catalytic domain of Sub2 and provides insights into the locations of primary and secondary processing sites in Plasmodium prodomains.

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Section: Resultsmentioning

confidence: 99%

Solution NMR structure of a sheddase inhibitor prodomain from the malarial parasite Plasmodium falciparum

Chen

Oganesyan

et al. 2012

Proteins

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“…Interestingly, these two different protease regulatory mechanisms appear combined in the chaperoning capabilities of IMC-dependent systems. Not surprisingly, the chaperoning ability and inhibitory functions of IMC domains correlate well, and IMC variants with diminished binding affinity are often weaker chaperones (73, 79, 80). However, this correlation is not always true, as the propeptide of aqualysin (a closely related thermostable homologue of subtilisin) (81) and a computationally designed synthetic peptide are potent slow-binding inhibitors (72), yet much weaker chaperones of subtilisin.…”

Section: The Concept Of Intramolecular Chaperonesmentioning

confidence: 99%

Insights from Bacterial Subtilases into the Mechanisms of Intramolecular Chaperone-Mediated Activation of Furin

Shinde

Thomas

2011

Methods in Molecular Biology

111

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Prokaryotic subtilisins and eukaryotic proprotein convertases (PCs) are two homologous protease subfamilies that belong to the larger ubiquitous super-family called subtilases. Members of the subtilase super-family are produced as zymogens wherein their propeptide domains function as dedicated intramolecular chaperones (IMCs) that facilitate correct folding and regulate precise activation of their cognate catalytic domains. The molecular and cellular determinants that modulate IMC-dependent folding and activation of PCs are poorly understood. In this chapter we review what we have learned from the folding and activation of prokaryotic subtilisin, discuss how this has molded our understanding of furin maturation, and foray into the concept of pH sensors, which may represent a paradigm that PCs (and possibly other IMC-dependent eukaryotic proteins) follow for regulating their biological functions using the pH gradient in the secretory pathway.

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“…This involved four steps: (1) engineering of highly stable and refoldable subtilisin (11,12), (2) modifications of binding pockets resulting in a strong preference for sequences of the form P4−P1 = YRAL (13−17), (3) selection of an independently stable prodomain with high affinity for subtilisin (18,19), and (4) engineering a tunable active site by means of a D32A1 mutation which renders activity dependent on the presence of certain small anions such as fluoride and azide (20−22). The resulting switchable subtilisin, whose activity against cognate sequences (e.g., YRAL-) can be accelerated more than 3000-fold by 100 mM azide, is denoted S189.1

A shorthand for denoting amino acid substitutions employs the single-letter amino acid code as follows: N218S denotes the change of asparagine 218 to serine, and Δ75−83 denotes the deletion of residues 75−83 from the amino acid sequence.

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confidence: 99%

Structure of a Switchable Subtilisin Complexed with a Substrate and with the Activator Azide

et al. 2009

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An engineered variant of the protease subtilisin from Bacillus amyloliquefaciens, in which the D32A mutation renders the enzyme’s activity dependent on the presence of certain small anions such as fluoride or azide, has been produced. This modified enzyme has applications as an azide or fluoride-triggered expression−purification tool. We report activity measurements showing that the enzyme is activated more than 3000-fold by azide and describe the 1.8 Å resolution structure of an inactive form (by replacing the catalytic nucleophile Ser 221 with alanine) of the protease, in complex with azide and with a substrate that spans the active site. Both enzyme and substrate have been engineered to increase their stability and the affinity of their interaction. The substrate is based on a stabilized subtilisin prodomain, extended across the active site by the addition of four residues at its C-terminus. In the crystal structure, the substrate is well-ordered across the active site, and the azide anion is observed bound adjacent to Ala 32. The structures of the substrate complex in three different crystals (anion-free, fluoride-soaked, and azide-soaked) are compared. These structures provide extensive information for understanding subtilisin’s substrate binding and catalytic mechanism, and for the development of biotechnology tools based on anion-activated proteolysis. The mechanism of anion-dependent proteolysis appears to be a slight modification of the accepted charge-relay mechanism for serine proteases.

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Stabilizing the subtilisin BPN' pro‐domain by phage display selection: How restrictive is the amino acid code for maximum protein stability?

Cited by 40 publications

References 39 publications

Solution NMR structure of a sheddase inhibitor prodomain from the malarial parasite Plasmodium falciparum

Solution NMR structure of a sheddase inhibitor prodomain from the malarial parasite Plasmodium falciparum

Insights from Bacterial Subtilases into the Mechanisms of Intramolecular Chaperone-Mediated Activation of Furin

Structure of a Switchable Subtilisin Complexed with a Substrate and with the Activator Azide

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