Molecular messenger of sight. Some of RBP3 secrets have been exposed

Scientists have for the first time looked deep into the protein structure that may determine our vision - and discovered that it is much more dynamic than previously thought. RBP3 not only changes its shape depending on its load but may also play a role in protecting the retina from diseases ranging from diabetic retinopathy to retinitis pigmentosa.

Retinol-binding protein 3 (RBP3) is a glycoprotein of about 140 kDa, found in the intercellular space of the retina. It plays an important role in the transport of retinoids - molecules essential for properly functioning the vision process. Although the existence of RBP3 has been known for years, its structure and precise mechanisms of action have remained unknown until now. The lack of this information has been a significant gap in research on eye diseases, especially those leading to irreversible vision loss.

Retinitis pigmentosa (RP) is one such disease – insidious, progressive, and still incurable. It affects millions of people worldwide, leading to the gradual loss of photoreceptors and blindness. Previous studies suggested that abnormalities in RBP3 function may be one of the contributing factors to the development of the disease, but detailed knowledge of its structure and mechanism of action was lacking. The new findings shed light on this mystery, opening the way to potential therapies that could slow or even stop retinal degeneration.

An international team of scientists, including researchers from ICTER, used modern structural analysis methods to obtain for the first time an image of the native structure of RBP3 with such high accuracy. The results were published in the journal Open Biology in a paper titled "CryoEM structure and small-angle X-ray scattering analyses of porcine retinol-binding protein 3".

For the first time, we have managed to capture the full structure of native RBP3 in pigs with a resolution of 3.67 Å. This is an important step in understanding the function of this protein, especially in the context of its role in the transport of retinoids and fatty acids in the eye, says Dr Humberto Fernandes from ICTER.

Why is RBP3 so important?

Vision is a process that begins with the conversion of light into an electrical signal by photoreceptors in the retina. A key element of this transformation is the visual cycle - a complex chain of chemical reactions in which retinoids, or derivatives of vitamin A, play an essential role. However, for them to effectively perform their function, they must be transported between different cells of the retina. This transport is the responsibility of the retinol-binding protein 3 (RBP3), which acts as a "courier" delivering retinoids to where they are needed.

RBP3 is found in the interphotoreceptor extracellular matrix (IPM), the space between the retinal pigment epithelium and the photoreceptors. This is where the transport of molecules necessary for the proper functioning of the eye takes place, including oxygen, nutrients, and retinoids. RBP3 plays a key role in delivering all-trans-retinol (at-ROL) from photoreceptors to the retinal pigment epithelium, where it is converted into 11-cis-retinal (11c-RAL), a key molecule for vision. 11c-RAL then returns to the photoreceptors, where it binds to opsins to form light-sensitive pigments that enable vision. Without RBP3, this transport cycle would be much less efficient, which could lead to retinoid deficiencies in photoreceptors and, ultimately, retinal degeneration.

Structurally, RBP3 is a large glycoprotein consisting of four retinoid-binding modules. Each of these modules has a distinctive structure that allows it to interact with a variety of molecules, including retinoids and fatty acids such as docosahexaenoic acid (DHA). However, it has not been clear yet how exactly this protein interacts with its ligands and whether its structure changes depending on the type of molecule being transported.

However, it is known that RBP3 has additional protective functions. It protects retinoids from degradation under the influence of light by limiting their oxidation and disintegration. Its presence in the IPM stabilizes the biochemical environment of the retina, which is crucial for eye health. Moreover, mutations in the gene encoding RBP3 are associated with several eye diseases, including RP and some forms of myopia.

How was the study conducted?

To capture the three-dimensional structure of RBP3, the researchers used cryo-electron microscopy (cryoEM), a technique that allows for obtaining images of biomolecules in a nearly native state at cryogenic temperature. The study also used small-angle X-ray scattering (SAXS) analysis, which allowed for determining the conformational changes of the protein in solution.

The first step was to obtain the protein in its native form. To do this, the researchers isolated RBP3 from pig retinas obtained from local slaughterhouses. The tissues were stored in conditions that minimized protein degradation - on ice, in the dark. RBP3 was then purified using advanced chromatographic techniques, including affinity chromatography, ion exchange chromatography, and gel filtration. Each step of the procedure was aimed at obtaining a stable and functional form of the protein, which was crucial for further studies.

After obtaining pure samples of the protein, the researchers began experiments using cryo-electron microscopy. RBP3 samples were cooled to extremely low temperatures and placed in an electron beam, which allowed them to obtain hundreds of thousands of images of single molecules. Assembling these images into a three-dimensional model allowed the reconstruction of the protein structure at an unprecedented resolution of 3.67 Å.

In parallel, SAXS analysis was used, which provided additional information about the conformational plasticity of RBP3 in solution. These experiments allowed them to observe how the protein changes its shape when bound to different molecules, including retinoids and fatty acids. Thanks to this, the researchers discovered that RBP3 adopts different conformations depending on the type of cargo it transports, which may be crucial for its function as a dynamic retinoid carrier.

This is one of the most detailed structural studies of RBP3 ever performed. By combining cryoEM and SAXS, we have gained unique insight into how this protein works, explains Dr Humberto Fernandes from ICTER.

What was found?

One of the key findings of this study is the ability of RBP3 to change shape depending on the type of molecule being bound. Experiments have shown that after binding 11-cis-retinal (11c-RAL) and all-trans-retinol (at-ROL), the protein assumes different conformations - from compact to open. SAXS analyses have shown that at higher concentrations of these retinoids, RBP3 is elongated, which may suggest the mechanism of its action as a dynamic retinoid transporter.

This finding is particularly interesting because it suggests that RBP3 may act as a flexible retinoid carrier, changing shape to optimize the transport of these molecules between the retinal pigment epithelium and photoreceptors - says Dr Vineeta Kaushik from ICTER.

Additional information on the plasticity of the protein was provided by molecular docking analyses, which indicated the presence of two main ligand binding sites. Interestingly, SAXS analysis showed that binding fatty acids such as DHA (docosahexaenoic acid) did not lead to significant changes in the structure of RBP3, indicating that its role in transporting these molecules may differ from that of retinoids.

This groundbreaking discovery changes our understanding of the role of RBP3 in the eye. Rather than being a passive transporter of retinoids, we are beginning to see it as an active, adaptive mechanism that can precisely regulate the delivery of key molecules to photoreceptors. Understanding these dynamics opens new avenues for research into the visual cycle and potential therapies for retinal degenerative diseases.

What's next?

The discovery of the full structure of RBP3 and its conformational plasticity opens a new chapter in research on the functioning of the visual cycle and the mechanisms leading to retinal degeneration. The results may have key implications for the diagnosis and treatment of eye diseases, including diabetic retinopathy, retinitis pigmentosa, and high myopia.

Previous studies have suggested that reduced levels of RBP3 in the retina are associated with the progression of diabetic retinopathy, and its stabilization could have a protective effect. Now, thanks to precise structural data, scientists can focus on developing new therapeutic strategies that would modulate RBP3 activity and could slow down the progression of this disease. It is also possible to use RBP3 as a diagnostic biomarker, which could help identify patients at risk of vision loss at an early stage of the disease. The team plans to continue research on the dynamics of RBP3 function in both physiological and pathological conditions.

This is just the beginning. Now that we have a 3D model of RBP3, we can study how exactly it interacts with other retinal proteins and how we can use this information to develop new treatments, concludes Dr Humberto Fernandes.

Authors of the paper "CryoEM structure and small-angle X-ray scattering analyses of porcine retinol-binding protein 3": Vineeta Kaushik, Luca Gessa, Nelam Kumar, Matyáš Pinkas, Mariusz Czarnocki-Cieciura, Krzysztof Palczewski, Jiří Nováček and Humberto Fernandes.

Author: scientific editor Marcin Powęska