Staphylococcal SplB Serine Protease Utilizes a Novel Molecular Mechanism of Activation

Pustelny, Katarzyna; Zdzalik, Michal; Stach, Natalia; Steć-Niemczyk, Justyna; Cichoń, Paweł; Czarna, Anna; Popowicz, Grzegorz M.; Mak, Paweł; Drąg, Marcin; Salvesen, Guy S.; Władyka, Benedykt; Potempa, Jan; Dubin, Adam; Dubin, Grzegorz

doi:10.1074/jbc.m113.507616

Cited by 15 publications

(27 citation statements)

References 33 publications

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“…6a). This was similar to the zymogens of the staphylococcal SplB protease (Pustelny et al, 2014) and bovine trypsin (Fehlhammer et al, 1977), in which the spatial arrangement of the catalytic residues (Ser214, Asp102, His57 and Ser195; chymotrypsin numbering) in the zymogens was almost similar, but with slightly distorted or inadequately formed S1 pockets compared with the mature enzymes. Such an arrangement in the zymogen could also explain the complete absence of activity against the specific substrate (Fig.…”

Section: A Structural Perspective On Pro-esp Activationsupporting

confidence: 63%

Structural insights into the role of the N-terminus in the activation and function of extracellular serine protease fromStaphylococcus epidermidis

Manne

Narayana

2020

Acta Cryst Sect D Struct Biol

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Extracellular serine protease (Esp) from Staphylococcus epidermidis is a glutamyl endopeptidase that inhibits the growth and formation of S. aureus biofilms. Previously, crystal structures of the matured and active Esp have been determined. Interestingly, many of the staphylococcal glutamyl endopeptidase zymogens, including V8 from Staphylococcus aureus and Esp from S. epidermidis, contain unusually long pro-peptide segments; however, their function is not known. With the aim of elucidating the function of these pro-peptide segments, crystal structures of the Esp zymogen (Pro-Esp) and its variants were determined. It was observed that the N-terminus of the Pro-Esp crystal structure is flexible and is not associated with the main body of the enzyme, unlike in the known active Esp structure. In addition, the loops that border the putative substrate-binding pocket of Pro-Esp are flexible and disordered; the structural components that are responsible for enzyme specificity and efficiency in serine proteases are disordered in Pro-Esp. However, the N-terminal locked Pro-Esp variants exhibit a rigid substrate-binding pocket similar to the active Esp structure and regain activity. These structural studies highlight the role of the N-terminus in stabilizing the structural components responsible for the activity and specificity of staphylococcal glutamyl endopeptidases.

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Section: A Structural Perspective On Pro-esp Activationsupporting

confidence: 63%

Structural insights into the role of the N-terminus in the activation and function of extracellular serine protease fromStaphylococcus epidermidis

Manne

Narayana

2020

Acta Cryst Sect D Struct Biol

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“…[26,27] Other MRSA biofilm gene clusters with changes in expression profiles due to HP-14 include: spl (serine proteases; [28] activated), opp (oligopeptide transporters; [29] activated), gap (glycolysis; [30] inhibited), arc (arginine deiminase; [31] inhibited), ure (urease; [32] inhibited), hem (heme biosynthesis; [33] inhibited), and an uncharacterized gene cluster containing dnaC (DNA synthesis and repair; [34] inhibited; see Figures 1 and 2 in the Supporting Information). Several gene clusters with unknown functions or lower levels of activation/inhibition in response to HP-14 are presented in the Supporting Information (for WoPPER details, see Tables 6 and 7).…”

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confidence: 99%

Transcript Profiling of MRSA Biofilms Treated with a Halogenated Phenazine Eradicating Agent: A Platform for Defining Cellular Targets and Pathways Critical to Biofilm Survival

et al. 2018

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Bacterial biofilms are surface-attached communities of non-replicating bacteria innately tolerant to antibiotics. Biofilms display differential gene expression profiles and physiologies as compared to their planktonic counterparts; however, their biology remains largely unknown. In this study, we used a halogenated phenazine (HP) biofilm eradicator in transcript profiling experiments (RNA-seq) to define cellular targets and pathways critical to biofilm viability. WoPPER analysis with time-course validation (RT-qPCR) revealed that HP-14 induces rapid iron starvation in MRSA biofilms, as evident by the activation of iron-acquisition gene clusters in 1 hour. Serine proteases and oligopeptide transporters were also found to be up-regulated, whereas glycolysis, arginine deiminase, and urease gene clusters were down-regulated. KEGG analysis revealed that HP-14 impacts metabolic and ABC transporter functional pathways. These findings suggest that MRSA biofilm viability relies on iron homeostasis.

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“…Proteolitic activity of SplE was routinely verified with zymography using b-casein as a substrate (Arvidson et al, 1973). SplB was expressed and purified as described previously (Pustelny et al, 2014). Mutants were generated by site directed mutagenesis, verified by sequencing and purified similarly to the wild type SplB.…”

Section: Star+methodsmentioning

confidence: 99%

“…It is unlikely that the observed conformation is induced directly by crystal-specific interactions (tight packing), since the closest adjacent molecule is more than 6 Å away from the discussed region and there is no indication of ordered solvent in the water channel between the two molecules. At the same time, however, the N terminus of SplE is involved in multiple interactions with an adjacent molecule, while it has been previously demonstrated that precise positioning of the N terminus influences the activity of SplB (Pustelny et al, 2014). Although this might suggest indirect influence of crystal packaging on the conformation of loop 1, we rather believe that the observed conformation constitutes a native arrangement and is related to a substrate-induced activation mechanism, as previously suggested for SplC (Popowicz et al, 2006) and analyzed in more detail in the discussion section.…”

Section: Substrate Preference Of Sple Extends Beyond P1 Residuementioning

confidence: 99%

Unique Substrate Specificity of SplE Serine Protease from Staphylococcus aureus

et al. 2018

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Staphylococcus aureus is a dangerous human pathogen characterized by alarmingly increasing antibiotic resistance. Accumulating evidence suggests the role of Spl proteases in staphylococcal virulence. Spl proteases have restricted, non-overlapping substrate specificity, suggesting that they may constitute a first example of a proteolytic system in bacteria. SplA, SplB, and SplD were previously characterized in terms of substrate specificity and structural determinants thereof. Here we analyze the substrate specificity of SplE documenting its unique P1 preference among Spl proteases and, in fact, among all chymotrypsin-like (family S1) proteases characterized to date. This is interesting since our understanding of the general aspects of proteolysis is based on seminal studies of S1 family members. To better understand the molecular determinants of the unusual specificity of SplE, the crystal structure of the protein is determined here. Conclusions from structural analysis are evaluated by successful grafting of SplE specificity on the scaffold of SplB protease.

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Staphylococcal SplB Serine Protease Utilizes a Novel Molecular Mechanism of Activation

Cited by 15 publications

References 33 publications

Structural insights into the role of the N-terminus in the activation and function of extracellular serine protease fromStaphylococcus epidermidis

Structural insights into the role of the N-terminus in the activation and function of extracellular serine protease fromStaphylococcus epidermidis

Transcript Profiling of MRSA Biofilms Treated with a Halogenated Phenazine Eradicating Agent: A Platform for Defining Cellular Targets and Pathways Critical to Biofilm Survival

Unique Substrate Specificity of SplE Serine Protease from Staphylococcus aureus

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