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Conformational switching in an aspartic proteinase
A Y Lee1, S V Gulnik, J W Erickson
1Structural Biochemistry Program, SAIC Frederick, National Cancer Institute, Maryland 21702-1201, USA.
Abstract:
The crystal structure of a catalytically inactive form of cathepsin D (CatDhi) has been obtained at pH 7.5. The N-terminal strand relocates by 30 A from its position in the interdomain beta-sheet and inserts into the active site cleft, effectively blocking substrate access. CatDhi has a five-stranded interdomain beta-sheet and resembles Intermediate 3, a hypothetical structure proposed to be transiently formed during proteolytic activation of the proenzyme precursor. Interconversion between active and inactive forms of CatD is reversible and may be regulated by an ionizable switch involving the carboxylate side chains of Glu 5, Glu 180, and Asp 187. Our findings provide a structural basis for the pH-dependent regulation of aspartic proteinase activity and suggest a novel mechanism for pH-dependent modulation of substrate specificity.
Insights
The crystal structure of inactive Cathepsin D (CatDhi) reveals an N-terminal strand blocking the active site. This reversible structural change explains pH-dependent enzyme activity and substrate specificity.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Cathepsin D (CatD) is an aspartic proteinase involved in various cellular processes.
- Understanding CatD's regulation is crucial for its role in physiology and disease.
Purpose of the Study:
- To elucidate the structural basis of catalytically inactive Cathepsin D (CatDhi).
- To investigate the mechanism of pH-dependent regulation of CatD activity and substrate specificity.
Main Methods:
- X-ray crystallography was used to determine the structure of CatDhi at pH 7.5.
- Structural analysis focused on the N-terminal region and active site cleft.
Main Results:
- The crystal structure of CatDhi shows the N-terminal strand repositioned to block the active site cleft.
- CatDhi shares structural similarity with Intermediate 3, a proposed transient form during enzyme activation.
- A reversible interconversion between active and inactive CatD forms is suggested, regulated by an ionizable switch involving Glu 5, Glu 180, and Asp 187.
Conclusions:
- The findings provide a structural explanation for the pH-dependent regulation of aspartic proteinase activity.
- A novel mechanism for pH-dependent modulation of substrate specificity in CatD is proposed.