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Probing catalytic rate enhancement during intramembrane proteolysis

  • Elena Arutyunova , Cameron C. Smithers , Valentina Corradi , Adam C. Espiritu , Howard S. Young , D. Peter Tieleman and M. Joanne Lemieux EMAIL logo
Published/Copyright: April 12, 2016
Biological Chemistry
From the journal Biological Chemistry Volume 397 Issue 9

Abstract

Rhomboids are ubiquitous intramembrane serine proteases involved in various signaling pathways. While the high-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed an active site comprised of a serine-histidine dyad and an extensive oxyanion hole, the molecular details of rhomboid catalysis were unclear because substrates are unknown for most of the family members. Here we used the only known physiological pair of AarA rhomboid with its psTatA substrate to decipher the contribution of catalytically important residues to the reaction rate enhancement. An MD-refined homology model of AarA was used to identify residues important for catalysis. We demonstrated that the AarA active site geometry is strict and intolerant to alterations. We probed the roles of H83 and N87 oxyanion hole residues and determined that substitution of H83 either abolished AarA activity or reduced the transition state stabilization energy (ΔΔG‡) by 3.1 kcal/mol; substitution of N87 decreased ΔΔG‡ by 1.6–3.9 kcal/mol. Substitution M154, a residue conserved in most rhomboids that stabilizes the catalytic general base, to tyrosine, provided insight into the mechanism of nucleophile generation for the catalytic dyad. This study provides a quantitative evaluation of the role of several residues important for hydrolytic efficiency and oxyanion stabilization during intramembrane proteolysis.

Keywords: catalytic dyad; FRET; GlpG; intramembrane protease; oxyanion hole; rhomboid protease; serine protease

Acknowledgments

We thank Dr. Matthew Freeman, Oxford University, for the TatA clone. We also thank Dr. Tracy Raivio, University of Alberta for the GlpG knockout strain, and Dr. Robert Campbell, University of Alberta, for the CyPet and YPet vector. We thank Dr. Pankaj Panwar and Ms. Pauline Skiba for early discussions.

  1. Funding information: This study was supported in part by funding from Alberta Innovates Health Solutions and the Parkinson’s Society of Canada. Work in M.J.L.’s group is also supported by grant MOP-93557 from the Canadian Institutes of Health Research. M.J.L. is an Alberta Innovates Health Solutions Scientist and Parkinson Society of Canada New Investigator. Work in D.P.T.’s group was supported by grant MOP-62690 from the Canadian Institutes of Health Research. D.P.T. is an Alberta Innovates Health Solutions Scientist and Alberta Innovates Technology Futures Strategic Chair in (Bio)Molecular Simulation. Simulations used West-Grid/Compute Canada facilities.

  2. Conflict of interest statement: The authors declare that they have no conflicts of interest regarding the contents of this article.

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Supplemental Material:

The online version of this article (DOI: 10.1515/hsz-2016-0124) offers supplementary material, available to authorized users.

Received: 2016-1-27
Accepted: 2016-4-6
Published Online: 2016-4-12
Published in Print: 2016-9-1

©2016 Walter de Gruyter GmbH, Berlin/Boston

Articles in the same Issue

  1. Frontmatter
  2. Guest Editorial
  3. Highlight: proteolytic networks across cellular boundaries
  4. HIGHLIGHT: IPS 2015 – 9TH GENERAL MEETING OF THE INTERNATIONAL PROTEOLYSIS SOCIETY
  5. A personal journey with matrix metalloproteinases
  6. Type II transmembrane serine proteases as potential targets for cancer therapy
  7. Membrane trafficking and proteolytic activity of γ-secretase in Alzheimer’s disease
  8. Dipeptidyl peptidase 9 substrates and their discovery: current progress and the application of mass spectrometry-based approaches
  9. Tetraspanin 8 is an interactor of the metalloprotease meprin β within tetraspanin-enriched microdomains
  10. Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors
  11. Granzyme B inhibits keratinocyte migration by disrupting epidermal growth factor receptor (EGFR)-mediated signaling
  12. Myeloid conditional deletion and transgenic models reveal a threshold for the neutrophil survival factor Serpinb1
  13. Probing catalytic rate enhancement during intramembrane proteolysis
  14. Human 20S proteasome activity towards fluorogenic peptides of various chain lengths
  15. Research Articles/Short Communications
  16. Protein Structure and Function
  17. Biophysical analysis of three novel profilin-1 variants associated with amyotrophic lateral sclerosis indicates a correlation between their aggregation propensity and the structural features of their globular state
  18. The potential of the Galleria mellonella innate immune system is maximized by the co-presentation of diverse antimicrobial peptides
https://doi.org/10.1515/hsz-2016-0124
Keywords for this article
catalytic dyad; FRET; GlpG; intramembrane protease; oxyanion hole; rhomboid protease; serine protease

Articles in the same Issue

  1. Frontmatter
  2. Guest Editorial
  3. Highlight: proteolytic networks across cellular boundaries
  4. HIGHLIGHT: IPS 2015 – 9TH GENERAL MEETING OF THE INTERNATIONAL PROTEOLYSIS SOCIETY
  5. A personal journey with matrix metalloproteinases
  6. Type II transmembrane serine proteases as potential targets for cancer therapy
  7. Membrane trafficking and proteolytic activity of γ-secretase in Alzheimer’s disease
  8. Dipeptidyl peptidase 9 substrates and their discovery: current progress and the application of mass spectrometry-based approaches
  9. Tetraspanin 8 is an interactor of the metalloprotease meprin β within tetraspanin-enriched microdomains
  10. Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors
  11. Granzyme B inhibits keratinocyte migration by disrupting epidermal growth factor receptor (EGFR)-mediated signaling
  12. Myeloid conditional deletion and transgenic models reveal a threshold for the neutrophil survival factor Serpinb1
  13. Probing catalytic rate enhancement during intramembrane proteolysis
  14. Human 20S proteasome activity towards fluorogenic peptides of various chain lengths
  15. Research Articles/Short Communications
  16. Protein Structure and Function
  17. Biophysical analysis of three novel profilin-1 variants associated with amyotrophic lateral sclerosis indicates a correlation between their aggregation propensity and the structural features of their globular state
  18. The potential of the Galleria mellonella innate immune system is maximized by the co-presentation of diverse antimicrobial peptides
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