Ultrahigh and High Resolution Structures and Mutational Analysis of Monomeric Streptococcus pyogenes SpeB Reveal a Functional Role for the Glycine-rich C-terminal Loop

González-Páez, Gonzalo E.; Wolan, Dennis W.

doi:10.1074/jbc.m112.361576

Cited by 13 publications

(33 citation statements)

References 56 publications

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“…The 2477-bound SpeB structure is conserved with our previous apo and complex structures with crystallization in orthorhombic space group P2 1 2 1 2, an ordered leucine residue from the C-terminal His 6 -tag (LEH 6 ), 245 water molecules, and a bound nitrate likely introduced from the crystallization buffer. 31 The apo SpeB structure (PDB ID 4D8B) 20 served as the initial search model for molecular replacement, and the initial F o − F c electron density map exhibited unambiguous electron density for 2477 located within the SpeB active site ( Figure 2B). The final R cryst and R free for the SpeB and 2477 co-complex crystal structure were 16.1% and 18.8%, respectively, with greater than 98% of residues in the favored region of the Ramachandran plot (Table 1).…”

Section: ■ Resultsmentioning

confidence: 99%

“…21 This substrate synthesis was previously described using standard Fmoc solid phase synthesis chemistry starting with Fmoc-Lys(carbamate)-AMC Wang resin (EMD Biosciences). 20 The HTS SpeB inhibitor assay protocol was modified based on optimized fluorescence that resulted from cleavage of Ac-AIK-AMC ( Figure 1A) by 50 nM active SpeB in an activity buffer consisting of PBS at pH 7.4, 0.1 mM EDTA, 10 mM DTT, and 0.1% CHAPS. 100 nM SpeB diluted in activity buffer was incubated in low-volume, 384-well plates with a final concentration of 15 μM Maybridge HitFinder HTS compound library compounds in a volume of 10 μL (0.5% DMSO).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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Identification and Co-complex Structure of a New S. pyogenes SpeB Small Molecule Inhibitor

2015

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The secreted Streptococcus pyogenes cysteine protease SpeB is implicated in host immune system evasion and bacterial virulence. We present a small molecule inhibitor of SpeB 2477 identified from a high-throughput screen based on the hydrolysis of a fluorogenic peptide substrate Ac-AIK-AMC. 2477 inhibits other SpeB-related proteases but not human caspase-3, suggesting that the molecule targets proteases with the papain-like structural fold. A 1.59 Å X-ray crystal structure of 2477 bound to the SpeB active site reveals the mechanism of inhibition and the essential constituents of 2477 necessary for binding. An assessment against a panel of 2477 derivatives confirms our structural findings and shows that a carbamate and nitrile on 2477 are required for SpeB inhibition, as these moieties provide an extensive network of electrostatic and hydrogen-bonding interactions with SpeB active site residues. Surprisingly, despite 2477 having a reduced inhibitory potential against papain, the majority of 2477-related compounds inhibit papain to a much greater and broader extent than SpeB. These findings indicate that SpeB is more stringently selective than papain for this panel of small molecule inhibitors. On the basis of our structural and biochemical characterization, we propose modifications to 2477 for subsequent rounds of inhibitor design that will impart specificity to SpeB over other papain-like proteases, including alterations of the compound to exploit the differences in CA protease active site pocket sizes and electrostatics.

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Section: ■ Resultsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Identification and Co-complex Structure of a New S. pyogenes SpeB Small Molecule Inhibitor

2015

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“…Structural bioinformatics tools allowed us to investigate mechanisms by which zinc could mediate its inhibitory effects on SpeB, which, as a widely studied GAS virulence factor, has been crystallized by several groups who aimed to identify the enzyme's catalytic residues, substrate-binding site, protein folding, and maturation process (38)(39)(40)(41). In all those cases, SpeB was crystallized either in its mature form or in the inactive zymogen form generated by replacing the active cysteine residue with serine.…”

Section: Discussionmentioning

confidence: 99%

Metal-Mediated Modulation of Streptococcal Cysteine Protease Activity and Its Biological Implications

et al. 2014

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dStreptococcal cysteine protease (SpeB), the major secreted protease produced by group A streptococcus (GAS), cleaves both host and bacterial proteins and contributes importantly to the pathogenesis of invasive GAS infections. Modulation of SpeB expression and/or its activity during invasive GAS infections has been shown to affect bacterial virulence and infection severity. Expression of SpeB is regulated by the GAS CovR-CovS two-component regulatory system, and we demonstrated that bacteria with mutations in the CovR-CovS two-component regulatory system are selected for during localized GAS infections and that these bacteria lack SpeB expression and exhibit a hypervirulent phenotype. Additionally, in a separate study, we showed that expression of SpeB can also be modulated by human transferrin-and/or lactoferrin-mediated iron chelation. Accordingly, the goal of this study was to investigate the possible roles of iron and other metals in modulating SpeB expression and/or activity in a manner that would potentiate bacterial virulence. Here, we report that the divalent metals zinc and copper inhibit SpeB activity at the posttranslational level. Utilizing online metal-binding site prediction servers, we identified two putative metal-binding sites in SpeB, one of which involves the catalytic-dyad residues 47 Cys and 195 His. Based on our findings, we propose that zinc and/or copper availability in the bacterial microenvironment can modulate the proteolytic activity of SpeB in a manner that preserves the integrity of several other virulence factors essential for bacterial survival and dissemination within the host and thereby may exacerbate the severity of invasive GAS infections.

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“…Recombinant production of streptopain variants in Escherichia coli was pursued for convenient exploration of point mutations [12]. These protein variants were purified by combinations of ion exchange chromatography [13-17], dyeligand chromatography [13, 15], size-exclusion chromatography [14, 15, 17], or Ni 2+ -chelating chromatography [12, 16, 18-20]. …”

Section: Introductionmentioning

confidence: 99%

“…This cleavage can be performed by mature streptopain or by exogenous proteases [22]. Most previously published recombinant purifications yielded the zymogen, which was subsequently activated by incubation with mature streptopain [12, 18-20], although some cases of streptopain self-activation during expression and purification were also reported [17, 18]. …”

Section: Introductionmentioning

confidence: 99%

Highly efficient recombinant production and purification of streptococcal cysteine protease streptopain with increased enzymatic activity

Lane

Seelig

2016

Protein Expression and Purification

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Streptococcus pyogenes produces the cysteine protease streptopain (SpeB) as a critical virulence factor for pathogenesis. Despite having first been described seventy years ago, this protease still holds mysteries which are being investigated today. Streptopain can cleave a wide range of human proteins, including immunoglobulins, the complement activation system, chemokines, and structural proteins. Due to the broad activity of streptopain, it has been challenging to elucidate the functional results of its action and precise mechanisms for its contribution to S. pyogenes pathogenesis. To better study streptopain, several expression and purification schemes have been developed. These methods originally involved isolation from S. pyogenes culture but were more recently expanded to include recombinant E. coli expression systems. While substantially easier to implement, the latter recombinant approach can prove challenging to reproduce, often resulting in mostly insoluble protein and poor purification yields. After extensive optimization of a wide range of expression and purification conditions, we applied the autoinduction method of protein expression and developed a two-step column purification scheme that reliably produces large amounts of purified soluble and highly active streptopain. This method reproducibly yielded 3 mg of streptopain from 50 mL of expression culture at >95% purity, with an activity of 5,306 +/− 315 U/mg, and no remaining affinity tags or artifacts from recombinant expression. This improved method therefore enables the facile production of the important virulence factor streptopain at higher yields, with no purification scars that might bias functional studies, and with an 8.1-fold increased enzymatic activity compared to previously described procedures.

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Ultrahigh and High Resolution Structures and Mutational Analysis of Monomeric Streptococcus pyogenes SpeB Reveal a Functional Role for the Glycine-rich C-terminal Loop

Cited by 13 publications

References 56 publications

Identification and Co-complex Structure of a New S. pyogenes SpeB Small Molecule Inhibitor

Identification and Co-complex Structure of a New S. pyogenes SpeB Small Molecule Inhibitor

Metal-Mediated Modulation of Streptococcal Cysteine Protease Activity and Its Biological Implications

Highly efficient recombinant production and purification of streptococcal cysteine protease streptopain with increased enzymatic activity

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