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AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor.

α1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based on biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found in WT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo polymerization showing a surprising diversity of polymer topography

Native PAGE analysis of α₁-PI monomers and <i>in vitro</i> formed polymers.

<p>Polymers were formed from purified WT plasma-derived α₁-PI monomers (lane 1) by incubation at pH 4.1 (lane 2), incubation with 1.4M guanidinium chloride (lane 3) or incubation at 55°C (lane 4). Lane 5 presents purified human Z variant monomer. Quantification of the bands indicated that approximately 87% of the protein was migrating as a monomer in lane 1 (WT), markedly more than in lane 5 (69%; Z variant). M–monomer; D–dimer. Approximated molecular weights are indicated on the left. Lanes 2 to 4 were run concurrently on the same gel.</p

Comparison of α₁-PI topography generated with AFM, the AFM simulator and the crystal structure model.

<p>The AFM generated topography of the wild type α₁-PI is in a good agreement with the shape and dimensions of particles obtained with the AFM simulator and calculated from a model of the protein surface occluded by a water shell. In contrast, the waterless Van der Waals protein surface model was too rich in structural details to be efficiently compared with AFM topographs. The models were based on the crystal structure of α₁-PI WT monomer (PDB ID: 1qlp [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151902#pone.0151902.ref031" target="_blank">31</a>]). The length of a space-filled crystal structure model of the kidney-shaped antitrypsin molecule was 7–8 nm and its width up to 4–5 nm. (A) a tilted AFM image (side plot) of a typical WT monomer particle; (B) a surface model generated with the Microscope Simulator using the cone-sphere tip model of a radius 7 Å and a cone angle 25°; (C) a Van der Waals protein surface model generated with the 3V program using a probe of 10 Å radius that adds the water shell; (D) a protein surface model obtained as in (C) by applying a probe radius of 0 Å (“waterless”). Models presented in B-D are structurally aligned.</p

AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α₁-Proteinase Inhibitor - Fig 4

<p><b>Volume distributions of WT α</b>_<b>1</b><b>-PI (A) and Z variant (B) particles.</b> The distributions revealed a prevailing content of objects, which size was consistent with monomers. Surprisingly, the presence of larger molecules was also detected. Among the larger objects, particles with volume approximately twice as big as monomers were the most abundant. The content of dimers and bigger particles was more pronounced in the Z variant (B) than in the wild type (A). The volume of particles was calculated with the grain analysis of SPIP software. Relative count of particles is shown. Total of 847 and 639 particles were analyzed for the WT and Z variant, respectively. Histogram bin size was 5 nm. Fitted normal distribution curves are shown in green for monomers and in blue for dimers, whereas total frequency distribution is represented by red traces (OriginPro).</p

AFM height images of human Z mutant α₁-PI preparations obtained from a PiZ mouse liver.

<p>(A) Images of two representative fragments of fields with α₁-PI particles. The presence of long, linear, often tangled strands of polymers is striking. Arrows in the top image point at the types of particles presented in zoomed-in B to E panels on the right. The examples are marked b to e, respectively. (B)–(E) galleries of zoomed-in images of diverse polymer and oligomer types. In particular: (B) fragments of long polymer fibers with straight, smooth and compact arrangement of units; (C) fragments of long polymer fibers with grainy appearance and loose arrangement of well discernible units; (D) linear, short and thin oligomers with compact arrangement of units; (E) oligomers built from large globular units arranged as “large packed beads”.</p

<i>In vitro</i> polymerization of led to formation of structurally diverse oligomers observed with AFM.

<p>Top panels: AFM height images of representative fields of WT α₁-PI particles polymerized by: (A) incubation with 1.4M GuHCl (37.5 hrs, 25°C, pH = 7.4); (B) exposure to a low pH (pH = 4.1, 2 hrs, 25°C); (C) treatment with the elevated temperature (55°C, 4 hrs, pH = 7.4). Bottom panels: arrays of zoomed-in images of oligomers representative for each polymerization method.</p