Selective Elimination of the Key Subunit Interfaces Facilitates

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Selective Elimination of the Key Subunit Interfaces Facilitates Conversion of Native 24-mer Protein Nanocage into 8-mer Nanorings Wenming Wang, Lele Wang, Hai Chen, Jiachen Zang, Xuan Zhao, Guanghua Zhao, and Hongfei Wang J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b09760 • Publication Date (Web): 17 Oct 2018 Downloaded from http://pubs.acs.org on October 19, 2018

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Journal of the American Chemical Society

Selective Elimination of the Key Subunit Interfaces Facilitates Conversion of Native 24-mer Protein Nanocage into 8-mer Nanorings Wenming Wang,† Lele Wang,† Hai Chen,‡ Jiachen Zang,‡ Xuan Zhao,† Guanghua Zhao,*,‡ and Hongfei Wang*,† †Key

Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Institute of Molecular Science, Shanxi University, Taiyuan 030006 (China) ‡College

of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083 (China)

ABSTRACT: Living systems utilize proteins as building blocks to construct a large variety of self-assembled nanoscale architectures. Yet, creating protein-based assemblies with specific geometries remains challenging. Here, we present a new approach that completely eliminates one natural intersubunit interface of multisubunit protein architecture with high symmetry, resulting in reassembly of the protein architecture into one with lower symmetry. We have applied this approach to render the conversion of the 24-mer cage-like ferritin into non-native 8-mer protein nanorings in solution. In the crystal structure, such newly formed nanorings connect with each other through hydrogen bonding in a repeating head-to-tail pattern to form nanotubes, and adjacent nanotubes stager relative to one another to create three dimensional porous protein assemblies. The above strategy has allowed the study of conversion between protein architectures with different geometries by adjusting the interactions at the intersubunit interfaces, and the fabrication of novel bionanomaterials with different geometries.

Natural proteins have acquired self-assembly properties during evolution to construct a plethora of nanoscale architectures, thereby endowing their hosts with novel functionalities.1-3 In any self-assembling protein architecture, noncovalent subunit-subunit interactions (SSIs) usually generate large, highly complementary, lowenergy intersubunit interfaces which spontaneously selfassemble and allow precise definition of the orientation of subunits relative to each other. The ability to control SSIs has produced a variety of nanoscale protein materials.1, 3, 4, 5 Despite great progress, rendering SSIs controllable is still challenging because SSIs are regulated by weak, noncovalent interactions over large surfaces. Hollow proteinaceous particles are widely distributed in nature, which are constructed from a small number of subunit building blocks by virtue of their intersubunit interfaces that self-assemble to highly cooperative, symmetrical structures. By providing a protective shell for molecular cargo, these nanocages fulfill various functions.6-11 Recently, these protein cages have attracted considerable attention from researchers in the field of nanoscience and nanotechnology, because beside their functions in vivo, their cage structures of nanometer dimensions can be explored for the preparation of nanomaterials and encapsulation of guest molecules with potential applications.12-14 One highly versatile encapsulation system is ferritin. Ferritins are ubiquitous iron storage proteins where Fe(II) sequestration prevents not only its spontaneous oxidation to Fe(III) but also production of toxic free radicals.

Ferritin is usually composed of 24 subunits which selfassemble into a hollow protein shell with octahedral symmetry.10 Ferritin subunit is composed of a four helix bundle containing two antiparallel helix pairs (A, B and C, D) and a fifth short helix (E helix) (Fig. S1a). Each ferritin molecule contains four kinds of intersubunit interfaces responsible for its shell-like assembly, namely, 24 of C3-C4 interfaces, 12 of C2 interfaces, 8 of C3 interfaces, and 6 of C4 interfaces (Figs. S1b-f). Among these four interfaces, the C3−C4 has the largest surface area, followed by C2  C3  C4. The high selectivity for cancer cells and low immunogenicity of ferritin nanocage set it apart from all other protein nanocages. Therefore, ferritin nanocage, especially recombinant human H-chain ferritin (HuHF), has emerged as a vehicle for tumor imaging, and encapsulation and delivery of drugs.15-20 However, no single protein cage architecture has all the requisite properties of size, shape, and functional-group presentation. So far, different approaches to designing self-assembling proteins have been reported in different ways, such as the use of metal-mediated interactions,21 computational interface design,4 directed evolution,22 and genetic fusion of multiple protein domains.23,24 Scheme 1. Selective removal of 49 residues from the C-terminal (which are responsible for the SSIs at the C3−C4 interface) of each ferritin subunit results in complete conversion from 24-mer protein nanocage into nanorings with D4 symmetry.

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aggregation occurs with HuHF1-125 and HuHF1-128, agreeing with their DLS results. The main band of HuHF1137 and HuHF1-143 on the native PAGE is diffuse. In contrast, HuHF1-134 is more uniform than other mutants, and exhibits a different electrophoretic behavior from native HuHF (Fig. S3b), indicative of complete conversion of native ferritin into its non-native analogue, so we focused on this mutant.

Here, we established a new, simple method, which is a reinterpretation of an approach we recently introduced.25, 26 In our recently reported approach, we just slightly tune the C3-C4 subunit interfaces of HuHF by selective deletion or insertion of a small number of amino acid residues (6 to 10). Consequently, such deletion or insertion can trigger the conversion of native 24-mer ferritin nanocage into a larger non-native 48-mer anlogue25 or a smaller non-native 16-mer lenticular ferritin-like nanocage.26 In this work, we completely remove one of the key subunit interfaces of multisubunit protein architecture (which usually contains several subunit interfaces) by partial truncation of the C-terminal. Consequently, other left intersubunit interfaces can drive the newly formed subunits to self-assemble into a new protein assembly with lower symmetry. By using this method, we successfully convert native 24-mer ferritin nanocages into a class of non-native 8-mer nanorings in solution (Scheme 1). In the crystal, these nanorings stack in a face-to-tail pattern through hydrogen bonds to form nanotubes. Subsequently, resulting nanotubes stagger with each other to produce 3D porous protein materials. Specifically, we applied this new method to regulate the reassembly of recombinant human H-chain ferritin (HuHF) due to its available sequence and structural information for genetic modification, and popularity as a nanocarrier in the field of nanomedicine.15−19 By analyzing the interactions at the key subunit interfaces of HuHF, we found that helix D is responsible for all interactions at the C3−C4 intersubunit interface, which is located at the Cterminal of each subunit (Fig. S1g). To eliminate the interactions at the C3−C4 interface, we genetically modified HuHF subunit by individually introducing five truncation mutants into its C-terminal. These five mutants correspond to HuHF1-125, HuHF1-128, HuHF1-134, HuHF1-137, and HuHF1-143 (Fig. S2). All these mutants were purified by GST-affinity chromatography. As expected, SDS-PAGE analyses showed that the molecular weight (MW) of these truncation mutant subunits decreases slightly with increasing the number of deletion amino acid residues (Fig. S3a). However, native PAGE and dynamic light scattering (DLS) found that the size distribution of all truncation mutants is obviously different from each other (Figs. S3b, S3c). Protein

Figure 1. Characterization of HuHF1-134 in solution. (a) The MW of HuHF1-134 was determined by SEC-MALS as 129 ± 3 kDa. Data shown is representative from three independent experiments. (b) Sedimentation coefficient distribution c(s) for HuHF1-134 (black line) and native HuHF (blue line). Conditions: [HuHF1-134] = 1.2 mg/mL, 50 mM Tris, 200 mM NaCl, pH 8.0 at 4 °C.

Figure 2. The formation of HuHF1-134 nanoring. (a) Two adjoining molecules are antiparallel, named as unit A and B, colored in magenta and blue. (b) Units A and B combine together to form a dimer. (c) Four dimers form an octameric nanoring, and the interfaces between dimers are named as C2‫׳‬. An octameric nanoring contains two walls, inner wall and outer wall. The inner wall is colored in yellow, while the outer wall is highlighted in magenta and blue. Hydrophobic interaction and salt bridge are the major interaction force in the C2‫ ׳‬interface. Hydrophobic surface were colored red and

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Journal of the American Chemical Society hydrophobic amino acids were also marked in diagram. Residues H57 and E61 in helix B are shown as colored sticks.

To gain insights into the feature of mutant HuHF1-134 in solution, we characterized this non-native protein by various methods after purification with gel filtration chromatography (Fig. S4). SDS-PAGE analyses found that the MW of HuHF1-134 subunit is about 16 kDa (Fig. S3a). The accurate MW of HuHF1-134 subunit was obtained as 16148.123 Da by MALDI-TOF-MS (Fig. S5). Size Exclusion Chromatography combined with Multi-Angle Light Scattering (SEC-MALS) was performed to determine the MW of native HuHF1-134. We found that this new protein was eluted from a Superdex 200 10/300 GL (GE Healthcare) column in a single peak at a volume about 12.6 mL (flow rate 0.4 mL/min) (Fig. 1a), giving the weight averaged molecular mass as 129±3 kDa, which is ~ 8-fold larger than the MW of the subunit (16148.123 Da), demonstrating that HuHF1-134 molecules occur as octamers in solution. Agreeing with this conclusion, analytical ultracentrifugation results showed that HuHF1-134 sediments in solution as a single discrete species with sed. coefficient as 3.41 S (128.5 kDa) as compared to native HuHF (12.10 S, 509.6 kDa) (Fig. 1b), a finding again demonstrating that this ferritin mutant mainly stays in an octameric form in solution. Further support for the above observation comes from transmission electron microscopy (TEM) analyses showing that the exterior diameter of native HuHF is around 12 nm (Fig. S6a), while HuHF1-134 exhibits a roughly circular structure with a hole at the center with the exterior diameter as ~7.0 nm (Fig. S6b). Thus, it appears that truncation of the C-terminal in native ferritin causes complete conversion form 24-mer protein nanocage into a new non-native 8-mer protein assembly. To obtain detailed structural information on this new protein assembly, we tried to crystallize HuHF1-134 and eventually obtained qualified crystals suitable for X-ray diffraction. The needle like crystals (Fig. S7) of HuHF1-134 appeared after 2 day incubation. We solved the crystal structure at 3.0 Å (I422 space group) (Supplementary Table S1). As expected, native 24-mer protein lost its shell-like structure upon the above truncation, leading to the formation of a homopolymeric protein octamer architecture where 8 of identical subunits co-assemble into a nanoring with D4 symmetry (Scheme 1). These findings are in good agreement with the above results observed in solution, demonstrating that this new octameric protein assembly is stable. To our knowledge, such protein self-assembling manner is unprecedented. The 8 subunits in the octamers can be divided into two categories with equal number, designated as units A and B which are antiparallel with each other (Fig. 2a). These two units form a dimer with a ratio of 1 to 1 (Fig. 2b), producing nearly the same interface C2 as that in native HuHF (Fig. S8). It is not surprising because we did not modify helices A and B which are responsible for the twofold interactions in native ferritin. Once the above dimers are formed, four of them assemble into a nanoring,

generating four new interfaces named as C2‫׳‬. This represents the first major difference in the structure between native ferritin and HuHF1-134. The interactions at the C2‫׳‬, interfaces consist of hydrophobic interactions and two salt bridges as shown in Fig. 2c. In the native HuHF, there are two hydrophobic cores at the two ends of the four helical bundle (Fig S9). Elimination of helix D made these two inherent hydrophobic cores exposed to water, which is unstable. In the structure of HuHF1-134, hydrophobic core 1 from one molecule and hydrophobic core 2 from another molecule were combined into a bigger hydrophobic core, thereby forming the new interface C2'. The second interface difference between native ferritin and its mutant corresponds to the four-fold and three-fold interactions. Four short helices E lie roughly parallel in native ferritin (Fig. S1c), forming the four-fold interactions and three helix D forming the three-fold interactions, whereas such C4 and C3 interactions in HuHF1-134 lack due to the elimination of helices D and E.

Figure 3. The formation of HuHF1-134 nanotube arrays through different interactions in the crystal. (a) Hydrogen bonds are formed by Q14 and E94 from different layer molecules. (b) Salt bridges between R79 and D84 are the main forces of adjacent tubes. The residues contribute to the interaction are shown as stick.

The inner and outer diameters of the newly formed nanorings are 3.2 nm and 7 nm, respectively, and their height is 5.1 nm (Scheme 1). The inner wall of the nanorings is composed of only helix B, while its outer wall

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comprises helix A, helix C and BC loops (Fig. 2c). Interestingly, in the crystal, octameric nanorings pack layer-by-layer in uniform orientation, creating the tubelike arrangement (Fig. S10). Hydrogen bonding between Q14 and E94 from different layers mediates the contacts (Fig. 3a). Adjacent nanotubes stagger with each other and are in contact by interactions of salt bridge between D79 and D84 (Fig. 3b). This assembly behavior is reminiscent of α keratin protofilaments which are formed from two staggered rows of head-to-tail associated coiled coils.27 Nanotube arrays extend through the crystal, resulting in the formation of two kinds of pores. One is the round pore which exists inside the nanotube with crosssectional area around 8.04 Å2, and another four angle star shaped pore is left between the outer walls of the individual nanotubes with a little bigger crosssectional area of 10.54 Å2 (Scheme 1). By using DLS, we found that the newly fabricated nanorings are stable only over the pH range of 8.0 to 9.0 at temperature from 25 to 35 C (Figs. S11 and S12). In closing, protein nanotubes or nanorings hold the promise of ease of functionalization, intrinsic biocompatibility, and modular molecular recognition. Such properties have been difficult to reach with carbon or inorganically derived nanotubes. Therefore, it is of great importance to explore new methods to fabricate such protein assemblies. Herein, we built a new protein engineering method by selectively removal of one kind of the key subunit interfaces through truncation of the inherent C-terminal, resulting in the formation of new protein nanorings in solution and nanotubes in crystals. The present study represents an alternative approach to engineering nanoring and nanotube assemblies.

ASSOCIATED CONTENT Supporting Information. Crystallographic data for HuHF1-134 (PDB and mmCIF). Supplementary methods, figures and Table (PDF).

AUTHOR INFORMATION Corresponding Author E-mail address: [email protected] E-mail address: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The work was supported by the National Natural Science Foundation of China (Nos. 21601112, 31730069, and 21671125). We thank the staffs from BL17B/BL18U1/19U1 beamline of National Center for Protein Sciences Shanghai (NCPSS) at Shanghai Synchrotron Radiation Facility for assistance during data collection. Crystallographic coordinates of HuHF1-134 deposited into the Protein Data Bank with PDB code 5ZND.

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