The structure of the neurotoxin palytoxin determined by MicroED

Palytoxin (PTX) is a potent neurotoxin found in marine animals that can cause serious symptoms such as muscle contractions, haemolysis of red blood cells and potassium leakage. Despite years of research, very little is known about the mechanism of PTX. However, recent advances in the field of cryoEM, specifically the use of microcrystal electron diffraction (MicroED), have allowed us to determine the structure of PTX. It was discovered that PTX folds into a hairpin motif and is able to bind to the extracellular gate of Na,K-ATPase, which is responsible for maintaining the electrochemical gradient across the plasma membrane. These findings, along with molecular docking simulations, have provided important insights into the mechanism of PTX and can potentially aid in the development of molecular agents for treating cases of PTX exposure.


Introduction 27
Na,K-ATPase is an essential protein for maintaining proper cell function and is targeted by the 28 potent marine toxin palytoxin (PTX) (Habermann, 1989; Christian Skou & Esmann, 1992;Tubaro 29 et al., 2012). PTX is a non-proteinaceous natural product that was first isolated from tropical 30 marine corals and later found in dinoflagellates (Usami et al., 1995 Rhodes et al., 2002). Aquarium hobbyists may also be 34 exposed to PTX when mishandling Palythoa coral or inhaling aerosolized PTX (Hoffmann et al.,35 2008; Rumore & Houst, 2014). Inhaling PTX from blooming events of Ostreopsis has also caused 36 severe illness and hospitalization (Ciminiello et al., 2006). Understanding the structure of PTX 37 and how it binds to Na,K-ATPase is crucial for developing molecular agents that can treat cases 38 of PTX exposure and protect against its toxic effects. 39 PTX's binding to Na,K-ATPase with high affinity and its ability to convert it into a passive cation 40 pore has serious implications for cellular function and can lead to a range of health effects, 41 including skeletal muscle contractions, heart failure, hemolysis, and platelet aggregation 42 (Böttinger et al., 1986;Riobó & Franco, 2011;Artigas & Gadsby, 2003;Wang & Horisberger, 43 1997). The irreversible depolarization of the membrane caused by PTX can also contribute to 44 bone resorption and tumorigenesis (Lazzaro et al., 1987;Aligizaki et al., 2011). The extremely 45 low lethal dose for humans highlights the severity of PTX poisoning (Tubaro et al., 2011;Wiles et 46 al., 1974). 47 The development of anti-PTX molecules that can inhibit the binding of PTX on Na,K-ATPase is 48 crucial for the treatment of PTX exposure. The structure of PTX when bound to an antibody 49 fragment (scFv) was determined using microcrystal electron diffraction (MicroED) (Shi et al.,50 2013; Nannenga et al., 2014)at 3.2 Å resolution. This provided valuable information on the binding 51 mode of PTX, which was then used to perform docking simulations to determine the potential 52 binding mode of PTX on Na,K-ATPase. These findings pave the way for the development of 53 molecular agents that can treat cases of PTX exposure by inhibiting the binding of PTX on Na,K-54 ATPase, and can potentially save many lives. 55 56 57 Results 58

Characterization of scFv-PTX complex and crystallization 59
To date, very little has been uncovered about the three-dimensional structure of PTX. Many 60 studies have utilized anti-PTX antibodies to investigate PTX (Lau et al., 1995;Taniyama et al., 61 2003; Levine et al., 1987). The scFv antibody used in this study is a 26 kDa protein developed,

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expressed, and purified by Zabbio (San Diego, CA). The binding of PTX to scFv was confirmed 63 using size exclusion chromatography (SEC). The shift in the SEC trace of free scFv and PTX-64 bound scFv was compared to confirm binding ( Figure 1A). Furthermore, the binding affinity of 65 PTX to scFv was determined using microscale thermophoresis (MST). PTX binds to scFv at a 66 KD of 2.1 μM ( Figure 1B). 67 SEC fractions corresponding to the stable scFv-PTX complex were collected, concentrated to 10 68 mg/mL, and subjected to sparse matrix crystallization screening to identify crystallization condition 69 hits. The scFv-PTX complex was crystallized by the hanging drop vapor diffusion technique. The 70 well solution contained 27 % Jeffamine ED-2001 pH 7.0 and 100 mM sodium citrate tribasic 71 dihydrate pH 5.6. The scFv-PTX complex was combined with the well solution at 2: The crystals were thin rods that formed in dense bundles ( Figure 1C). The average size of each 73 crystal was 5 μm x 500 μm. The crystals in the drop were then transferred to an electron 74 microscopy (EM) sample grid, blotted to remove surrounding crystallization media, and vitrified 75 by plunge freezing into liquid ethane. Crystals were stored in liquid nitrogen prior to use.

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Preparaing crystal lamellae and collection of MicroED data 78 The crystals that were obtained for this study were too thick for MicroED and needed to be thinned 79 to a thickness that would allow for the transmission of electrons (Martynowycz et al., 2021). To 80 achieve this, thin crystal lamellae were produced using a cryogenic focused ion beam scanning 81 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 1, 2023. ; https://doi.org/10.1101/2023.03.31.535166 doi: bioRxiv preprint electron microscope (FIB/SEM) milling instrument (Martynowycz et al., 2019). The process began 82 by loading the EM grid with crystals into the FIB/SEM at cryogenic temperature, followed by 83 imaging using the SEM ( Figure 1D). Potential milling sites were then observed in the FIB view of 84 the specimen ( Figure 1E), and the targeted crystal and surrounding media were milled into a thin 85 lamella using the gallium ion beam. The final product was a lamella that measured 7 μm wide and 86 300 nm thick. 87 After the crystal lamellae were produced, they were transferred to a Titan Krios transmission 88 electron microscope that was operating at 300 kV and cooled cryogenically. The sites of the 89 lamellae were identified using low magnification imaging and adjusted to eucentric height. To 90 ensure high-resolution diffraction, a preview of the lamella was taken ( Figure 1F and 2A one PTX bound to each scFv monomer. The density map contoured at 1.5 σ had continuous 105 density for the backbone of the scFv and the side chains of the amino acids were also well 106 resolved ( Figure 2B). Continuous density was obtained for PTX after multiple rounds of refinement 107 ( Figure 2C). The Rwork and Rfree of the refinement were 28% and 32%, respectively. 108 The MicroED structure of scFv-PTX complex. 109 The scFv creates a binding pocket into which an internal segment of the PTX chain is inserted, 110 forming a hairpin motif (depicted in Figure 3). The deepest part of the pocket consists of a double 111 ring with two cyclic ethers, which is flanked by two hydrocarbon chains running antiparallel to 112 each other. Hydrogen bonds are formed between a cyclic ether of the double ring and residues 113 Y106, E108, and Y169 of the scFv. Hydrophobic amino acid side chains, including F59, W224, 114 V231, and V165, sequester the hydrophobic segments of PTX flanking the double ring from the 115 external aqueous environment. At the entrance to the binding pocket, a network of intramolecular 116 hydrogen bonds is formed by several hydroxyl groups. Outside the binding pocket, the two tail 117 ends of PTX are observed traveling in opposite directions in the solvent channel of the crystal. 118 119 Molecular docking. 120 The scFv-PTX complex structure obtained by MicroED was used to investigate the potential 121 binding of PTX to the Na,K-ATPase protein using molecular docking. The hairpin-like motif of PTX 122 from the scFv-PTX complex was used as the ligand, and the human Na,K-ATPase structure in 123 E1 state was used as the receptor protein molecule (Guo et al., 2022). Rigid docking simulations 124 were performed using the Patchdock server (Schneidman-Duhovny et al., 2005), which 125 suggested that the hairpin motif of PTX binds to the extracellular gate of the Na,K-ATPase protein.

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The cyclic ether forms hydrogen bonds, while the hydrocarbon chains are protected by 127 hydrophobic transmembrane alpha-helices. The two tails of PTX are between the alpha and beta 128 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made and block the channel, leading to a possible explanation for the cytotoxic effects of PTX. 135 136

Discussion 137
The findings of this study provide significant insights into the molecular mechanism of PTX binding 138 to Na,K-ATPase. The hairpin motif formed by the hydrophobic region of PTX when bound to scFv 139 was found to also fit into the extracellular gate of Na,K-ATPase like a plug, blocking the ion 140 channel and rendering the pump inactive. This corroborates earlier reports that this region of PTX 141 is chiefly responsible for its interaction with biomembranes and may be important in the 142 conversion of Na,K-ATPase from a pump to a passive cation pore ( binding and its effects on Na,K-ATPase. 148 The use of scFv in this study allowed for the determination of the 3D structure of the scFv-PTX 149 complex using MicroED. The scFv-PTX complex was crystallized using the hanging drop vapor 150 diffusion method, and the crystals were thinned using cryogenic FIB milling prior to MicroED 151 diffraction. The needle-shaped crystals of the scFv-PTX complex that were obtained for this study 152 were typically thin (3-5 m) and long (several hundred microns) and they formed in bundles. Such 153 morphologies are extremely challenging for analyses by x-ray crystallography, often leading to 154 multiple lattices and weak scattering. Using MicroED and FIB milling was advantageous in this 155 case because the entire crystal bundle could be transferred to the EM grid and crystal sites were 156 accessed by using a FIB mill to generate crystal lamellae and ultimately a MicroED structure. 157 The structure showed that PTX binds to the scFv in a highly specific manner, forming several key 158 interactions with amino acid residues in the complementarity-determining regions (CDRs) of the 159 scFv. In particular, PTX binds to the CDR H3 loop of the scFv, which is known to be a critical 160 region for antigen binding ( Figure 2B). The 3D binding mode of PTX to scFv was used to perform 161 docking simulations to predict the binding mode of PTX to Na,K-ATPase. The docking simulations 162 suggested that PTX binds to Na,K-ATPase in a similar manner to scFv, with the key interactions 163 occurring in the extracellular ion gate of Na,K-ATPase (Figure 3). This provides important insights 164 into the mechanism of PTX binding to Na,K-ATPase and can aid in the development of anti-PTX 165 molecules that prevent the binding of PTX to Na,K-ATPase. 166 This research paves the way for the development of possible treatments for PTX exposure. The 167 detailed structural information obtained from our MicroED study can aid in the creation of new 168 inhibitors that can block the binding of PTX to Na,K-ATPase, thus preventing its toxic effects. 169 Additionally, the knowledge gained from this study can be applied to develop methods for 170 identifying and monitoring the accumulation of PTX and its analogues in the environment, 171 potentially preventing harmful exposure to both humans and marine life. In summary, this 172 research not only elucidates the mechanism of action of PTX, but also offers valuable insights 173 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 1, 2023. ; https://doi.org/10.1101/2023.03.31.535166 doi: bioRxiv preprint into the development of potential therapeutics and environmental monitoring techniques. 174 Significantly, this study reinforces the utility of MicroED as a powerful tool for revealing the 175 structures of important biomolecules, such as the long-awaited structure of palytoxin, which has 176 been elusive to x-ray crystallography. 177 178

Acknowledgments 179
The authors would like to thank Zabbio (San Diego, CA) for development and generation of ScFv. 180 This study was supported by the National Institutes of Health P41GM136508 and the Department 181 of Defense HDTRA1-21-1-0004. The Gonen laboratory is supported by funds from the Howard 182 Hughes Medical Institute. Coordinates and maps were deposited in the protein data bank 183 (Accession code XXXX) and the EM Data bank (Accession code YYYY).    Capillaries, NanoTemper Technologies). Thermophoresis was measured at 21°C for 15 sec with 230 50% LED power and 100% (auto-detect) power. 231 Crystallization. The complex was purified by size-exclusion chromatography and the elution 232 fractions were concentrated to 10 mg/mL. Palytoxin was incubated with scFv at a 2:1 molar ratio 233 for 30 minutes at room temperature. A sparse matrix screening hit was identified in the PEGRxHT 234 well condition C01 (Hampton Research) by sitting drop vapor diffusion using a Mosquito 235 crystallization robot. This condition was optimized for robust crystallization using hanging drop 236 vapor diffusion. In the final condition, the complex was crystallized by mixing with 27 % Jeffamine 237 ED-2001 pH 7.0, and 100 mM sodium citrate tribasic dihydrate pH 5.6 in 1.5 uL drops with a 2:1 238 sample-to-mother liquor ratio. 239 Cryo-preservation. The cover slip with crystal drop was removed from the screening tray and 240 the drop was gently applied to a Cu200 R2/2 holey carbon EM grid (quantifoil). The EM grid was 241 negatively glow-discharged prior to sample application. The grid was blotted in a Leica GP2 set 242 to 95% humidity and 12°C and plunge-frozen into liquid ethane. The sample was stored in liquid 243 nitrogen until further use. 244

Machining crystal lamellae using the cryo-FIB/SEM. The vitrified EM grid was loaded into a 245
Thermo Fisher Aquilos dual-beam FIB/SEM operating at cryogenic temperature following 246 established procedures (Martynowycz et al., 2019). The sample was sputter coated with a thin 247 layer of platinum to preserve the sample during imaging and ion beam milling. A whole-grid atlas 248 of the drop was acquired by the SEM and potential milling sites were selected. The targeted 249 crystal and surrounding media were milled into a thin lamella using the gallium ion beam. The first 250 stage of milling used a beam current of 0.5 nA and gradually decreased to a minimum of 10 pA 251 as the lamella became thinner at later stages of milling. The final lamellae were 7 μm wide and 252 200 nm thick. 253

MicroED Data Collection.
Grids with milled lamellae were transferred to a cryogenically cooled 254 Thermo Fisher Scientific Titan Krios G3i TEM operating at an accelerating voltage of 300 kV. The 255 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 1, 2023. ; https://doi.org/10.1101/2023.03.31.535166 doi: bioRxiv preprint Krios was equipped with a field emission gun and a Falcon4 direct electron detector. A low 256 magnification atlas of the grid was acquired using EPUD (Thermo Fisher) to locate milled 257 lamellae. The stage was translated to the lamellae position and the eucentric height was set. The 258 100 μm selected area aperture was inserted and centered on the crystal to block background 259 reflections. In diffraction mode, the beam was defined using a 50 μm C2 aperture, a spotsize of 260 11, and a beam diameter of 20 μm. MicroED data were collected by continuously rotating the 261 stage at 0.2 ° / s. MicroED data from three different crystal lamellae were selected for downstream 262 data processing. was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 1, 2023. ; https://doi.org/10.1101/2023.03.31.535166 doi: bioRxiv preprint was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 1, 2023. was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 1, 2023. ; https://doi.org/10.1101/2023.03.31.535166 doi: bioRxiv preprint