Nanobodies in Structural Biology
Written by George Biggin, Structural Biology PhD Student at The University of Manchester
Nanobodies are fast becoming a fundamental tool in many structural studies.
This article outlines their utility in:
- Stabilizing dynamic proteins
- Stabilizing transient complexes
- Streamlining protein purification
- Increasing particle size (cryo-EM)
- Preventing pseudo-two-fold symmetry (cryo-EM)
WHAT ARE NANOBODIES?
Nanobodies are the recombinant variable domain (~12-15 kDa) of heavy-chain-only camelid antibodies (fig.1a). These heavy-chain-only antibodies are raised in camelid species including camels, llamas, and alpacas. Nanobodies are now becoming a desirable alternative to conventional antibodies due to several beneficial qualities (fig.1b). Applications include diagnostic and therapeutic purposes as well as various applications in research, including use as affinity capture reagents and fluorescent imaging tools. However, an often-underappreciated application of nanobodies is their utility in structural biology.
Figure 1: (A) Domain structures of a conventional antibody, camelid heavy chain antibody, and a recombinant camelid VHH domain (nanobody). (B) Advantages of nanobodies over conventional antibodies.
(Nanobody domain figure redrawn from [1].)
NANOBODIES IN X-RAY CRYSTALLOGRAPHY
Structural biology allows us to examine proteins at a molecular level, helping us to understand how their structure at atomic resolution relates to function. X-ray crystallography is one of the most popular methods used to obtain atomic resolution structures of proteins. However, resolving the structure of some proteins can be particularly challenging. Dynamic proteins, that have variable conformations or are inherently flexible and/or unstructured, make for difficult crystallization targets as they have a reluctancy to crystallize. As such, many people are focused on developing tools to tackle these crystallization issues. One such tool which has shown success is nanobodies.
Many studies have shown how nanobodies can be raised against a target protein in order to be used as crystallization chaperones. Previously, intractable structures such as that of the transmembrane angiotensin II type 1 receptor (AT1R) have now been resolved through crystalizing with a nanobody chaperone. In a paper published in Cell by Wingler and colleagues, nanobodies were generated against AT1R using a synthetic yeast-display library. They were able to identify a nanobody which binds to the receptor’s intracellular transducer pocket, stabilizing the large conformational changes characteristic of activated GPCRs, making AT1R more susceptible to crystallization [2].
Furthermore, nanobodies have had huge success in stabilizing transient protein complexes, resulting in the high-resolution structure of many transmembrane receptors. In a landmark study reported in Nature, Rasmussen and colleagues were able to generate nanobodies against the β2 adrenergic receptor (β2AR) in complex with the Gs heterotrimer [3]. Further characterization identified a nanobody that binds and prevents dissociation of the complex by GTPγS. The nanobody bound β2AR- Gs complex was then used to obtain crystals and report the active-state structure of β2AR to 3.2 Å resolution.
Nanobodies are now becoming increasingly useful tools to facilitate structural determination of proteins in physiologically important states, as was the case for the Arp4-N-actin heterodimer [4]. Knoll and colleagues generated nanobodies against the Arp4-N-actin heterodimer. They were able to identify a nanobody which bound all 35 subunits of chromatin-associated complexes containing the Arp4-N-actin heterodimer. Subsequent crystallographic studies were able to obtain a high-resolution structure of the nanobody-bound Arp4-N-actin heterodimer, revealing the precise binding epitope. Further structural analysis was able to confirm that the nanobody recognizes the same configuration of N-actin and Arp4 as in the structure of Arp8.
Protein purification and structural studies of transmembrane proteins are particularly challenging due to their instability, flexibility, and hydrophobic nature – they require distinct detergents and other buffer components to remain stable in aqueous solutions. As such, optimizing their purification will likely lead to increased success in downstream structural studies. Nanobodies have shown to be an effective tool to aid in the purification of transmembrane receptors, including that of the Class A G protein-coupled receptor (GPCR) cannabinoid receptor CB1 [WHITEPAPER]. Nanobodies raised against protein purification tags, including GFP, can be utilized to obtain monodisperse and tag-free purified transmembrane receptors for use in structural studies [WHITEPAPER].
NANOBODIES IN CRYO-ELECTRON MICROSCOPY
In the past two decades, advancements in both detector technology and image processing have led to a rapid increase in the number of published high-resolution protein structures solved by cryo-electron microscopy (cryo-EM). This method is now preferred by many structural biologists due to lower sample requirements without the need for rigorous molecular engineering. Furthermore, the rapid freezing of protein samples in solution allows for the determination of protein structures in a more close-to-native state compared with X-ray crystallography.
However, cryo-EM can be particularly challenging for dynamic and flexible proteins as well as transiently interacting complexes, which in turn causes problems with sample stability and heterogeneity. Nanobodies were previously overlooked for cryo-EM applications due to their small size but are now being investigated as tools to overcome some of these challenges.
Specifically, nanobodies have been successful in overcoming issues with protein flexibility and in stabilizing protein complexes for cryo-EM. In a paper published in Cell Structure, researchers were able to utilize nanobodies to obtain a high-resolution structure of the fungal ortholog of the ATM kinase (Tel1), which, once activated, is responsible for the cellular response to DNA damage. Nanobodies were generated against an N-terminal fragment of Tel1. Following characterization, two of these nanobodies were utilized for an immunoaffinity pull-down to purify endogenous ctTel1 and a further nanobody was used in complex with Tel1 to stabilize the N-terminal region and obtain the final high-resolution structure.
Nanobodies have also been used in cryo-EM to resolve issues with preferred protein orientation within the ice as well as particle alignment during single particle analysis (SPA). Ideally, proteins are trapped within the ice in random orientations, which is crucial to obtain a high-resolution 3D reconstruction. However, due to the surface properties of some proteins, specific regions of a protein can preferentially adhere to the air-water interface. This was the case for the GABA receptor, where Laverty and colleagues generated a nanobody against the α1 subunit and fused this nanobody to a protein scaffold to generate a “megabody”[5]. Using this megabody, they were able to overcome these issues in cryo-EM data collection and processing, reporting a 3.2 Å structure of the GABA receptor.
Nanobodies, in these megabody configurations, have since been used to increase particle size and overcome preferential orientation at the water-air interface. Nanobodies have further been used in cryo-EM to break the pseudo-twofold symmetry of proteins, including that of the ABC exporter, which improved single particle analysis, resulting in a higher-resolution reconstruction [6].
NANOBODIES IN CLEM
Correlative Light and Electron Microscopy (CLEM) is becoming an increasingly widespread approach combining both fluorescence light microscopy and electron microscopy. CLEM utilizes fluorescent imaging of cells to determine the location of specific proteins at high spatial and temporal resolutions and, when coupled with electron microscopy, can reveal protein structure at an ultrastructural level. Nanobody-based tools have been used in CLEM for the detection of less abundant proteins and, more recently, to localize specific protein interactions. Through the development of a novel enzymatic tag named “APEX,” Martell and researchers were able to show that when APEX is fused to a protein of interest and expressed within cells. Cells can be fixed and overlaid with a solution of DAB and hydrogen peroxide. APEX then catalyzes the polymerization and local deposition of DAB, which subsequently recruits electron-dense osmium, giving contrast in electron microscopy [7]. Building on this further, Ariotti and colleagues genetically fused a conditionally stable GFP nanobody to APEX, which, when expressed in the presence of any GFP-tagged protein of interest, its localization can be determined using CLEM with high signal-to-noise. These APEX nanobody tools are now being developed for bimolecular fluorescence complementation, allowing for the detection and localization of intracellular protein interactions at an ultrastructural scale [8].
NANOBODIES FOR STRUCTURAL BIOLOGY AT THE UNIVERSITY OF MANCHESTER
Ongoing research at The University of Manchester is seeking to understand the role of fibrillin 1 in regulating the bioavailability and activation of TGFβ. Fibrillin 1 is an extracellular matrix protein which is incorporated into microfibrils. These microfibrils are crucial in the formation of elastic fibers, which provide our tissues with flexibility and extensibility, which is crucial for the functioning of many tissues. When fibrillin 1 is mutated, as seen in Marfan syndrome, this results in abnormal fibrillin 1 synthesis and or secretion and leads to dysregulated TGFβ signaling, which causes tissue degradation.
Determining the structure of the fibrillin-1-latent TGFβ complex using cryo-EM will give insight into the molecular mechanisms that regulate latent TGFβ activation. However, determining the structure of fibrillin 1 is challenging due to its beads-on-a-string-like structure and inherent flexibility [9]. Ongoing work is aiming to co-express two fibrillin 1 nanobodies joined by a linker. It is hypothesized that when bound to fibrillin 1, these nanobodies could act in a clamp-like mechanism to reduce the flexibility of fibrillin 1 and make structural determination by cryo-EM more attainable (fig.2).
Figure 2. Schematic representation of how Nanobody reagents can be used as tools to stabilize the flexible fibrillin 1 for downstream structural analysis.
CONCLUSION
In conclusion, the emergence of nanobodies has revolutionized the field of structural biology, offering a new perspective for advancing our understanding of complex biological systems. Targeting challenging protein structures, membrane proteins, and dynamic complexes, these small, stable, and versatile reagents have displayed an array of utility in various structural techniques: X-ray crystallography, cryo-electron microscopy, Correlative Light and Electron Microscopy (CLEM), and more. By harnessing the power of nanobodies, researchers can unlock frontiers in structural biology, biotechnology, and beyond.
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References
- Bannas, P., J. Hambach, and F. Koch-Nolte, Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics. Front Immunol, 2017. 8: p. 1603.
- Wingler, L.M., et al., Distinctive Activation Mechanism for Angiotensin Receptor Revealed by a Synthetic Nanobody. Cell, 2019. 176(3): p. 479-490.e12.
- Rasmussen, S.G.F., et al., Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature, 2011. 477(7366): p. 549-555.
- Knoll, K.R., et al., The nuclear actin-containing Arp8 module is a linker DNA sensor driving INO80 chromatin remodeling. Nature Structural & Molecular Biology, 2018. 25(9): p. 823-832.
- Laverty, D., et al., Cryo-EM structure of the human α1β3γ2 GABAA receptor in a lipid bilayer. Nature, 2019. 565(7740): p. 516-520.
- Hofmann, S., et al., Conformation space of a heterodimeric ABC exporter under turnover conditions. Nature, 2019. 571(7766): p. 580-583.
- Martell, J.D., et al., Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat Biotechnol, 2012. 30(11): p. 1143-8.
- Ariotti, N., et al., Ultrastructural localisation of protein interactions using conditionally stable nanobodies. PLoS Biol, 2018. 16(4): p. e2005473.
- McMahon, C., et al., Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nat Struct Mol Biol, 2018. 25(3): p. 289-296.
