Decrease of FU-FU interactions in the cryo-EM density maps of isomeric HdH1.

<p>Left panels: density maps of native HdH1 which are low-pass filterer to 6.8 Å from the reported 4.5 Å cryo-EM structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098766#pone.0098766-Zhang1" target="_blank">[23]</a>. Right panels: density maps of isomeric HdH1 reconstructed at 6.8 Å resolution in this study, filled with the corresponding pseudo-atomic models as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098766#pone.0098766.s003" target="_blank">Fig. S3</a>. (A) An example of loss of interaction between FUs inside one asymmetric unit, i.e., FU_B1 (red) and FU_C1 (yellow). The location of this FU-FU interaction in the didecamers is highlighted by a black box in the middle insert. Residues possibly involved in the interaction are labeled in the right panel. (B) An example of interaction loss between FUs from neighboring asymmetric units, i.e., FU_A1 (cyan) and FU_E1* (blue). A dot red line is used to indicate the boundary of two asymmetric units. The location of the interaction is highlighted by a red box in the middle insert. Residues possibly involved in the interaction are labeled in the right panel.</p

Cryo-EM Structure of Isomeric Molluscan Hemocyanin Triggered by Viral Infection

<div><p>Hemocyanins (Hcs) of arthropods and mollusks function not only as oxygen transporters, but also as phenoloxidases (POs). In invertebrates, PO is an important component in the innate immune cascade, where it functions as the initiator of melanin synthesis, a pigment involved in encapsulating and killing of pathogenic microbes. Although structures of Hc from several species of invertebrates have been reported, the structural basis for how PO activity is triggered by structural changes of Hc <i>in vivo</i> remains poorly understood. Here, we report a 6.8 Å cryo-electron microscopy (cryo-EM) structure of the isomeric form of hemocyanin, which was isolated from Abalone Shriveling syndrome-associated Virus (AbSV) infected abalone (<i>Halitotis diversicolor</i>), and build a pseudoatomic model of isomeric <i>H. diversicolor hemocyanin 1</i> (HdH1). Our results show that, compared with native form of HdH1, the architecture of isomeric HdH1 turns into a more relaxed form. The interactions between certain functional units (FUs) present in the native form of Hc either decreased or were totally abolished in the isomeric form of Hc. As a result of that, native state Hc switches to its isomeric form, enabling it to play its role in innate immune responses against invading pathogens.</p></div

The purification profile of Hcs and phenoloxidase assay from native and isomeric Hcs.

<p>(A) Flow chart for Hcs purification. (B) Eluted fractions of isomeric (blue) and native Hc (red) were tested for phenoloxidase activity by OD 280 and dot-blot experiments using 1 mM o-diphenol. (C) Spectroscopic analysis of phenoloxidase activity from isomeric (blue) and native (pink) Hcs. The control (orange) is the same buffer as used to dissolve Hcs.</p

A hypothetical diagram of PO activation in isomeric molluscan hemocyanin triggered by AbSV infection.

<p>The red and green dots represent the FU-FU interactions indicated by red and green arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098766#pone-0098766-g004" target="_blank">Fig. 4</a>, respectively.</p

The overall structure of isomeric HdH1.

<p>(A) A representative cryo-EM micrograph of isomeric HdH1. The bacilliform Hc-like particles are indicated by black arrows and the didecameric particles used in the 3D reconstruction are marked with black boxes. (B) Top view and side view of 3D reconstructed map of isomeric HdH1. An asymmetric unit is highlighted and its component Function Units (FUs) are marked with different colors. The slab area containing FU_H1 and H2 is circled by a black line in the top view panel. The upper, central and lower tiers are labeled. (C) FSC curve of isomeric HdH1 reconstruction according “gold-standard” criterion <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098766#pone.0098766-Rosenthal1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098766#pone.0098766-Scheres1" target="_blank">[27]</a>. (D) Stereo pair views of FUs in one asymmetric unit of isomeric HdH1. The FUs are named as A1 through H1, A2 through H2 and are indicated in different colors. Backview is with a rotation of 180 degree from the front view.</p

The distribution of decreased FU-FU interactions in isomeric HdH1.

<p>The decreased FU-FU interactions inside one asymmetric unit are indicated with green arrows, while the decreased FU-FU interactions between asymmetric units are indicated with red arrows. (A) The side view of isomeric HdH1. The boundaries of one asymmetric unit is marked with red dashed lines. All FUs in the asymmetric unit are colored as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098766#pone-0098766-g001" target="_blank">Fig. 1</a>. (B) Longitudinal section view of the isomeric HdH1 with a rotation of 180 degree from (A).</p

Northwestern blotting showing Pns6 binding to ssRNA and dsRNA.

<p>Lanes 1 and 2: Coomassie Brilliant Blue staining. “Mark” indicates the protein molecular standards, the arrowheads indicate 66 kDa and 43 kDa bands. The black triangle indicates the His-Pns6 band. Lane 3: His-Pns6 was detected by western blotting (WB) with anti-His antibody. Lane 6: after incubation and UV crosslinking, the digoxin-labeled single-stranded RNA probe ssA-Dig bound to His-Pns6 on the membrane was detected with anti-digoxin (AP-conjugated) antibody. Lanes 4 & 5: negative controls. Lane 7: the radiolabeled dsRNA probe dsR bound to His-Pns6 was detected after 8 hours of exposure.</p

EMSA confirmation of the RNA binding ability of Pns6 and the influence of salt concentrations on the binding.

<p>(A) and (B) EMSA (electrophoresis mobility shift assays) showing Pns6 binding to dsRNA and ssRNA, respectively. And the RNA binding activities of Pns6 was affected by salt concentration. Free and bound RNA probes are indicated by ‘F’ and ‘B’, respectively. Free probes were quantified with ImageJ software version 1.4. The relative values of free probe are presented below each of the lanes. In (A) and (B) 30 nanogram (ng) of BSA was added in lanes labeled ‘−’ as a negative control. In (A), the random dsRNA (dsR) and conserved dsRNA (S3-5) were used at 5 ng, and lanes 1, 2, 3, and 4 represent His-Pns6 used at 10, 20, 30 or 40 ng, respectively. In (B), Pns6 was added at 30 ng in lanes labeled ‘+’. The random ssRNA (ssR) and conserved RDV ssRNA (S3-5s, S3-5a, S3-3s and S3-3a) were used at 2.5 ng. (C) and (D) Effects of salt concentrations on Pns6-RNA interaction. In (C) and (D), no protein was added in lanes labeled ‘−’. BSA and His-P9 served as negative controls. Proteins were added at 50 ng in each lane. In (C), the dsRNA (S3-5) was used at 2.5 ng. In (D), the ssRNA (S3-5a) was used at 2.5 ng.</p

Sequences of dsRNA and ssRNA probes.

<p>Sequences of dsRNA and ssRNA probes.</p

N-terminal region of Pns6 is responsible for RNA binding.

<p>(A)–(D) Northwestern blotting of mutant proteins showed that mutant M3 and all mutants containing M1 had the ability to bind ssRNA. Lane 1: BSA (negative control); 2: His-P7 (positive control); 3: His-Pns6; 4: His-GKS; 5: His-M12; 6: His-M3; 7: GST; 8: GST::M1; 9: GST::M13; 10: GST::M2; 11: GST::M23. (A) Coomassie Brilliant Blue staining; (B) western blot with anti-His antibody; (C) western blot with anti-GST antibody; (D) Northwestern blot detecting digoxin-labeled probe ssA-Dig. (E) EMSA showing dsRNA-binding activity of HisM1 and lack of such activity by HisM3. Labeled S3-5 probe was used at 5 ng in each lane. Lanes 0, 2 and 4 represent the proteins used at 0, 20 and 40 ng. Relative values of free labeled probes are presented below each lane.</p