Structural studies of a2-macroglobulin from the horseshoe crab Limulus polyphemus
This work is focused on structural studies of the innate immune protein a2-macroglobulin from the horseshoe crab, Limulus Polyphemus, using crystallography and structure prediction software to reveal clues about the structure and function of this key immune mediator.
The a2-macroglobulin superfamily of proteins, characterised by the presence of an internal thiol-ester bond, is seen in humans as a2-macroglobulin, pregnancy zone protein (PZP), and Complement components C3, C4 and C5. a2-Macroglobulin (a2m) is a multifunctional serum protein, whose primary function is serving as a protease inhibitor. Rather than a traditional active-site inhibition, a2m immobilises target proteases via proteolytic cleavage of its bait region resulting in structural reorganisation of a2m and molecular entrapment of the protease. The nature of the bait region sequence allows for cleavage by a wide number of proteases which thus become entrapped. Small amines such as methylamine can also induce a2m activation resulting in the same structural reorganisation seen in proteolytic activation.
The crystal structure of Limulus a2m was not determined during this study, however this work represents the first reports of protein crystals of Limulus a2m activated with methylamine. Crystals were tested at the Diamond Light Source and diffraction was detected to 6Å with a predicted orthorhombic space group of P222 and unit cell dimensions of a = 115Å, b = 141Å, and c = 338Å.
In addition to crystallographic analysis the Limulus Polyphemus a2m sequence was submitted to the I-TASSER server for structure prediction. I-TASSER predicted general structural homology with the human analogue although differences arise from the human model representing the activated form and I-TASSER building a native , non-activated structure for the Limulus homologue. The bioinfomatic analysis and structure prediction presented here provides convincing structural models coupled with novel insights into the activation mechanism of Limulus a2m and how this might relate to its functions downstream.
|Dec 1, 2016