Structure and function of aspartic proteinases

Abstract

Crystallographic and Computational Studies on Aspartic Proteinases: Structural and Functional Insights

Abstract and Synopsis Chapter 1 - Introduction Aspartic proteinases are a superfamily of enzymes involved in diverse biological processes. Examples include:

Renin, which regulates blood pressure via the renin-angiotensin system.

Retroviral proteinases (e.g., HIV proteinase), essential for viral maturation and targeted in AIDS therapy.

Cathepsin D, implicated in tumorigenesis.

Pepsin, crucial for protein hydrolysis in the stomach but also responsible for tissue damage in peptic ulcer disease.

Aspartic proteinases are also linked to amyloid diseases, malaria, and fungal infections such as candidiasis, making them important therapeutic targets. Their catalytic mechanism involves active-site aspartate residues and a water molecule that attacks the scissile bond carbonyl, forming a tetrahedral gem-diol intermediate. This thesis reports crystallographic and computational analyses to understand their structure-function relationships.

Chapter 2 - Methods Provides an overview of protein crystallography methods:

Data collection and processing.

Refinement using single-crystal oscillation photography.

Use of software packages such as DENZO, CNS, O, PROCHECK, MSP, ALIGN for structure validation and analysis.

Chapter 3 - Comparative Analysis of Pepsin-like Proteinases

Multiple structure alignment of 10 native aspartic proteinases using STAMP.

Identification of 17 conserved water molecules.

Roles attributed to stabilization of active site geometry, flap region conformation, and overall structural integrity.

Chapter 4 - pH Effects on Rhizopuspepsin

Crystal structures determined at pH 4.6, 7.0, and 8.0, compared with pH 6.0.

Data collected using MAR imaging plate and Rigaku RU-200 generator.

Refinement with CNS and validation with standard packages.

Identified a pH-sensitive region influencing protein stability and activity.

Chapter 5 - pH Effects on Porcine Pepsin

Structures determined at pH 1.0, 2.0, 3.5, and 4.0.

Data collected using MARXDS and refined with CNS.

Hexagonal crystals diffracted to 2.4 Å, but data collected to 2.94 Å.

Limited resolution hindered detailed solvent-protein interaction analysis.

Chapter 6 - Anti-malarial Peptide Inhibitors

Crystal structures of two peptide inhibitors (INI & IN2) of plasmepsin I determined using CAD4 and Bruker SMART APEX instruments.

INI forms ribbon-like blocks stabilized by hydrogen bonds.

IN2 forms columns with intra-column hydrogen bonds, resembling peptide-like inhibitors.

Binding modes analyzed, with INI showing distinct conformations requiring further study.

Conclusions Conserved water molecules play critical roles in stabilizing aspartic proteinases.

pH strongly influences protein conformation and activity.

Structural studies of peptide inhibitors provide insights into anti-malarial drug design.