Structural Investigations of Human A2M Identify a Hollow Native Conformation That Underlies Its Distinctive Protease-Trapping Mechanism.

Harwood SL, Lyngsø J, Zarantonello A, Kjøge K, Nielsen PK, Andersen GR, Pedersen JS, Enghild JJ, Mol Cell Proteomics 20:100090 (2021) Europe PMC

SASDJQ3 – Trypsin-treated alpha-2-macroglobulin, deglycosylated

Cationic trypsin
Alpha-2-macroglobulin
MWI(0) 665 kDa
MWexpected 695 kDa
log I(s) 8.08×10-1 8.08×10-2 8.08×10-3 8.08×10-4
Cationic trypsin Alpha-2-macroglobulin small angle scattering data  s, nm-1
ln I(s)
Cationic trypsin Alpha-2-macroglobulin Guinier plot ln 8.08×10-1 Rg: 6.6 nm 0 (6.6 nm)-2 s2
(sRg)2I(s)/I(0)
Cationic trypsin Alpha-2-macroglobulin Kratky plot 1.104 0 3 sRg
p(r)
Cationic trypsin Alpha-2-macroglobulin pair distance distribution function Rg: 6.4 nm 0 Dmax: 20.7 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Cationic trypsin Alpha-2-macroglobulin CUSTOM IN-HOUSE model

SAXS data were collected from a solution of human deglycosylated alpha-2-macroglobulin treated with bovine pancreatic trypsin in a 20 mM HEPES, 150 mM NaCl, pH 7.4, buffer (I(s) vs s, where s = 4π sin θ/λ; 2θ is the scattering angle; λ = 0.134 nm). Each alpha-2-macroglobulin tetramer contains two bovine trypsin molecules, which results in a total molecular mass of 689 kDa for the alpha-2-macroglobulin tetramer of 161 kDa and two trypsins of 23 kDa. The data were collected at the in-house Bruker AXS NanoSTAR instrument with an Excillum Ga Metal Jet X-ray source at Aarhus University. The instrument has a homebuilt scatterless pinhole in front of the sample and is equipped with a Bruker AXS Vantec2000 detector. Data were collected for 1800 s for the sample and buffer, respectively, and the data were normalized to absolute scale using the scattering from pure water. The structure was modelled using home-written software, which performs rigid body refinement on the structure with P222 symmetry for alpha-2-macroglobulin and no symmetry for the two trypsins. The program takes into account a hydration layer.

Cationic trypsin
Mol. type   Protein
Organism   Bos taurus
Olig. state   Dimer
Mon. MW   25.8 kDa
 
UniProt   P00760 (1-246)
Sequence   FASTA
 
Alpha-2-macroglobulin (A2M)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Tetramer
Mon. MW   160.8 kDa
 
UniProt   P01023 (24-1474)
Sequence   FASTA