Reactive Center Loop Insertion in α-1-Antitrypsin Captured by Accelerated Molecular Dynamics Simulation

Area of Science:

• Biochemistry
• Structural Biology
• Computational Biology

Background:

• Protease inhibition by serine protease inhibitors (serpins) involves large conformational changes.
• Complex formation triggers reactive center loop (RCL) cleavage and insertion into β-sheet A, trapping the protease.

Purpose of the Study:

• To perform the first detailed accelerated molecular dynamics simulation of RCL insertion in alpha-1-antitrypsin (α₁AT).
• To investigate the role of structural elements and mutations in serpin inhibition and stability.

Main Methods:

• Accelerated molecular dynamics simulations of cleaved RCL insertion in α₁AT.
• Analysis of internal water pathways and structural plasticity.
• Generation and analysis of α₁AT mutants (K168E, E346K, K168E/E346K) for inhibitory activity, stability, and polymerization.

Main Results:

• Simulations revealed internal water pathways facilitating RCL residue incorporation.
• Observed plasticity in helix F (hF) and a novel chaperone role for hF and thFs3A in RCL insertion.
• Transient electrostatic interactions modulate inhibitory activity.
• The E346K mutation enhanced inhibitory activity but increased polymerization rates.

Conclusions:

• The study elucidates the mechanism of RCL insertion in serpins, highlighting the roles of water, hF, and thFs3A.
• Mutation E346K suggests a gatekeeping role for residue E346 in maintaining the metastable native state of α₁AT.
• Findings provide insights into serpin function and potential therapeutic strategies.