The role of the L2 loop in the regulation and maintaining the proteolytic activity of HtrA (DegP) protein from Escherichia coli

Sobiecka-Szkatula, Anna; Giełdoń, Artur; Scirè, Andrea; Tanfani, Fabio; Figaj, Donata; Koper, Tomasz; Ciarkowski, Jerzy; Lipińska, Barbara; Skórko-Glonek, Joanna

doi:10.1016/j.abb.2010.05.028

Cited by 5 publications

(8 citation statements)

References 29 publications

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“…CtHtrA activity for these substrates was previously published by Huston et al [2011]. The reduced activity observed in the mutated proteins is consistent with a previous study on EcHtrA, where all mutations in the active site affected ␤ -casein hydrolysis activity except I228N [Sobiecka-Szkatula et al, 2010]. All mutated proteins were able to hydrolyse ␤ -casein when assessed by conducting the assay and subsequently monitoring the remaining amount of ␤ -casein using Coomassie-stained PAGE.…”

Section: Identified L2 Loop Residues Are Important For Protease Activsupporting

confidence: 76%

“…1 c). Therefore, there can be some confidence that these residues are important for substrate specificity as the structural loops of EcHtrA, in particular the L2 loop, have been shown to be involved in substrate binding by forming the S1 specificity pocket [Krojer et al, 2002Sobiecka-Szkatula et al, 2010]. A microbial protein BLAST and alignment of the results for CtHtrA loop 1 and 2 revealed that from 250 returned matches, I242 and I265 were conserved in 99% of sequences.…”

Section: V266 Is Predicted To Be Involved In Forming the S1-binding Pmentioning

confidence: 99%

“…During oligomerization, the L1 and L2 loops break their interactions with the protruding LA loop from the neighbouring monomer, and arrange into an ordered conformation with resolved subsite pockets to facilitate proteolysis [Krojer et al, 2010]. Studies on the role of the L2 loop in Escherichia coli HtrA/DegP (EcHtrA) have shown that the L2 loop, in particular the apical region, is necessary for correct active site conformation [Sobiecka-Szkatula et al, 2010]. Key residues in EcHtrA were identified which are involved in the stabilization of the L2 loop, in particular residue L229 which when mutated completely eliminated the proteolytic activity [SobieckaSzkatula et al, 2010].…”

Section: Introductionmentioning

confidence: 99%

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The Active Site Residue V266 of Chlamydial HtrA Is Critical for Substrate Binding during both in vitro and in vivo Conditions

Gloeckl

Tyndall²,

Stansfield³

et al. 2012

Microb Physiol

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HtrA is a complex, multimeric chaperone and serine protease important for the virulence and survival of many bacteria. Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for severe disease pathology. C. trachomatis HtrA (CtHtrA) has been shown to be highly expressed in laboratory models of disease. In this study, molecular modelling of CtHtrA protein active site structure identified putative S1–S3 subsite residues I242, I265, and V266. These residues were altered by site-directed mutagenesis, and these changes were shown to considerably reduce protease activity on known substrates and resulted in a narrower and distinct range of substrates compared to wild type. Bacterial two-hybrid analysis revealed that CtHtrA is able to interact in vivo with a broad range of protein sequences with high affinity. Notably, however, the interaction was significantly altered in 35 out of 69 clones when residue V266 was mutated, indicating that this residue has an important function during substrate binding.

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Section: Identified L2 Loop Residues Are Important For Protease Activsupporting

confidence: 76%

Section: V266 Is Predicted To Be Involved In Forming the S1-binding Pmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Active Site Residue V266 of Chlamydial HtrA Is Critical for Substrate Binding during both in vitro and in vivo Conditions

Gloeckl

Tyndall²,

Stansfield³

et al. 2012

Microb Physiol

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“…Among interactions involving the LAЈ and L2 loops, the hydrophobic interaction of Ile-236 (L2) and Phe-68 (LAЈ) appears to be particularly interesting. As reported previously (30), the substitution I236N resulted in increased proteolytic activity of HtrA at low temperatures (up to 35°C). This effect was ascribed to the disturbance of the hydrophobic contact with Ile-228, which is expected to stabilize the inactive conformation.…”

Section: Discussionmentioning

confidence: 48%

“…Analysis of Proteolytic Activity-HtrA proteolytic activity was analyzed using ␤-casein as substrate at temperatures of 20, 35, and 45°C as described in Sobiecka-Szkatula et al (30). The proteolytically active variants of HtrA (0.17 M) were incubated with ␤-casein (17 M) in 25 mM Tris-HCl, pH 8.0.…”

Section: Methodsmentioning

confidence: 99%

The LA Loop as an Important Regulatory Element of the HtrA (DegP) Protease from Escherichia coli

Figaj

Giełdoń

Polit

et al. 2014

Journal of Biological Chemistry

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Background: An understanding of the HtrA protease activation mechanism is incomplete with respect to its LA regulatory loop.Results: A theoretical model of the LA structure is provided and experimentally verified. Conclusion: LA intersubunit contacts strongly contribute to the stabilization of the inactive HtrA. Significance: This is the first report that simultaneously offers a theoretical three-dimensional structure of LA and its biophysical and functional properties.

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Temperature dependent dynamics of DegP-trimer: A molecular dynamics study

Rai

Amutha

2015

Computational and Structural Biotechnology Journal

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DegP is a heat shock protein from high temperature requirement protease A family, which reacts to the environmental stress conditions in an ATP independent way. The objective of the present analysis emerged from the temperature dependent functional diversity of DegP between chaperonic and protease activities at temperatures below and above 28 °C, respectively. DegP is a multimeric protein and the minimal functional unit, DegP-trimer, is of great importance in understanding the DegP pathway. The structural aspects of DegP-trimer with respect to temperature variation have been studied using molecular dynamics simulations (for 100 ns) and principal component analysis to highlight the temperature dependent dynamics facilitating its functional diversity. The DegP-trimer revealed a pronounced dynamics at both 280 and 320 K, when compared to the dynamics observed at 300 K. The LA loop is identified as the highly flexible region during dynamics and at extreme temperatures, the residues 46–80 of LA loop express a flip towards right (at 280) and left ( at 320 K) with respect to the fixed β-sheet connecting the LA loop of protease for which Phe46 acts as one of the key residues. Such dynamics of LA loop facilitates inter-monomeric interaction with the PDZ1 domain of the neighbouring monomer and explains its active participation when DegP exists as trimer. Hence, the LA loop mediated dynamics of DegP-trimer is expected to provide further insight into the temperature dependent dynamics of DegP towards the understanding of its assembly and functional diversity in the presence of substrate.

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The role of the L2 loop in the regulation and maintaining the proteolytic activity of HtrA (DegP) protein from Escherichia coli

Cited by 5 publications

References 29 publications

The Active Site Residue V266 of Chlamydial HtrA Is Critical for Substrate Binding during both in vitro and in vivo Conditions

The Active Site Residue V266 of Chlamydial HtrA Is Critical for Substrate Binding during both in vitro and in vivo Conditions

The LA Loop as an Important Regulatory Element of the HtrA (DegP) Protease from Escherichia coli

Temperature dependent dynamics of DegP-trimer: A molecular dynamics study

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