Unlocking Vision’s Secrets: Researchers Reveal 3D Structure of Key Eye Protein

Researchers have uncovered the 3D structure of RBP3, a key protein in vision, revealing how it transports retinoids and fatty acids and how its dysfunction may lead to retinal diseases.

Proteins play a critical role in the human body, acting as essential structural and functional components of cells, tissues, and organs. They are involved in a wide range of biological processes, from fundamental cellular functions such as DNA replication to more complex physiological activities, including those that enable vision.

Within the visual system, proteins are indispensable for detecting light, synthesizing photopigments in photoreceptor cells, and transmitting signals within these cells. Any disruption, whether through genetic mutation or protein malfunction, can impair normal vision and lead to a range of visual disorders.

Recently, scientists at the Institute of Physical Chemistry, Polish Academy of Sciences in collaboration with the International Centre for Translational Eye Research (ICTER) provided new structural insights into the RBP3 protein. Their findings have advanced our understanding of the visual cycle and its link to retinal diseases.

A Natural Optical Detector

The human eye, our natural optical sensor, is a remarkably complex organ that enables us to perceive the world. Its function depends on the coordinated activity of numerous molecules. Vision begins in the retina, a thin layer of tissue lining the back of the eye, where light-sensitive cells known as photoreceptors (rods and cones) are located.

These photoreceptors detect light and convert it into electrical signals which are then transmitted to the brain via the optic nerve, allowing us to form visual images. A key molecule in this process is 11-cis-retinal (11cRAL), a light-sensitive compound that binds to opsin proteins such as rhodopsin. This interaction triggers the conversion of light into an electrical signal, initiating the visual process.

When photons are absorbed, a cascade of chemical reactions, including the isomerization of 11-cis-retinal (11cRAL) to all-trans-retinal, initiates vision. To enable continued vision, the 11cRAL must be continuously regenerated through a process called the visual cycle. Here the story begins…

Enter RBP3: The Retinoid Transporter

This is where another molecule enters the picture. That is Retinol-binding protein 3 (RBP3), a special protein located in the intercellular matrix that maintains the proper functioning of the visual cycle. RBP3 works as a transporter of retinoids between photoreceptors and retinal pigment epithelium cells and is also known to bind some important fatty acids. It shuttles crucial molecules back and forth from the photoreceptors making the visual pigments ready for the multiple reactions under the photons triggering.

The severity of diabetic retinopathy, an eye disease associated with diabetes, is associated with decreased levels of RBP3, and leads to progressive vision loss.

As RBP3 interacts with receptors like the glucose transporter 1 (GLUT1) and vascular endothelial growth factor (VEGF), been involved in blood vessel growth and cellular signaling in the eye. Disrupted RBP3 causes accumulation of retinal “waste products”, such as lipofuscin, which may cause oxidative damage to the RPE and photoreceptor cells. Besides diabetic retinopathy, RBP3 level disruption can also lead to retinitis pigmentosa, pan-retinal degeneration, and myopia.

Uncovering RBP3’s Structure

Although the RBP3 connection with these diseases is well known, the mechanisms of the binding to retinoids to transport them are still not satisfactorily described. This mystery intrigued the international team of researchers led by Dr. Humberto Fernandes from the Institute of Physical Chemistry, Polish Academy of Sciences – International Centre for Translational Eye Research (ICTER) to solve that mystery. They focused on the insight into the detailed structure of the RBP3 when it binds different retinoids and fatty acids.

The main aim of their investigations was to overcome the lack of an experimental structural model for the native form of RBP3. To achieve this, the authors purified the porcine RBP3 (pRBP3) and analyzed its structure using cryo-electron microscopy (cryoEM), where data was collected under cryogenic conditions, and after that data was refined by multiple steps and software to get the final 3D structure/model of the protein.

Additionally, small-angle X-ray scattering (SAXS) was used to provide data on the conformation changes depending on the cargo molecules. Interestingly, the structure of the RBP3 can be elongated, or bent, suggesting the dynamic changes in the structure while docking its cargo.

“Based on previous knowledge of RBP3 properties and straightforward methods for isolation of the porcine variant of RBP3, we purified porcine RBP3, and obtained a protein with Förster resonance energy transfer behaviour analogous to other RBP3s. Through analysis of cryoEM data, we determined a structure at 3.67 Å resolution of the porcine RBP3 protein and observed conformational changes upon ligand binding,” says Dr. Humberto Fernandes

Insights into RBP3 Function

Experimental results enabled the determination of the 3D structure and revealed conformational changes upon binding to its ligand as a step forward in the insight into the RBP3 functional mechanisms during the visual cycle.

RBP3 as a large molecule consisting of four retinoid-binding modules, has long lost its original catalytic functionality, and it evolved to be a cargo transporter interacting with a variety of molecules and delivering retinoids and fatty acids in the eye.

Research findings show the protein changes employing its shape during the binding of different molecules, which relates to the effectiveness of the interaction with the other molecules in the cargo and signaling. As a result, the conformational changes may play a significant role in the regulation of the light conversion into the visual signals.

Dr. Fernandes remarks, “In all measured parameters, the porcine variant mimics the more completely characterized bovine variant. The capacity of RBP3 to load different retinoids and fatty acids, the ability of the latter to displace the former, and the conformational changes dependent on ligand identity might be the basis for the loading and unloading of retinoids (and potentially DHA) to the intended cell types bordering the IPM intercellular matrix. Thus, RBP3 complexes merit further investigation.”

Understanding the proteins, including genetic mutations that affect the protein’s behaviour, like RBP3, is crucial to describe the mechanisms of the processes that appear in retinal diseases. Revealing the detailed structure of this bioactive molecule is a milestone in the studies on the interactions with different proteins.

The presented findings bring the bright light into potentially more effective and faster diagnostics, where the RBP3 molecule would work as an early-stage retinal disease development biomarker. What is more, it can help in the regulation of the RBP3 activity to develop treatments for the disruption of the visual process.

Reference: “CryoEM structure and small-angle X-ray scattering analyses of porcine retinol-binding protein 3” by Vineeta Kaushik, Luca Gessa, Nelam Kumar, Matyáš Pinkas, Mariusz Czarnocki-Cieciura, Krzysztof Palczewski, Jiří Nováček and Humberto Fernandes, 1 January 2025, Open Biology.
DOI: 10.1098/rsob.240180

The work was supported Foundation for Polish Science co-financed by the European Union under European Funds for Smart Economy (FENG.02.01-IP.05-T005/23), and (MAB/2019/12) project within the International Research Agendas programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund. It was also supported by the National Institutes of Health R01EY009339. The authors also acknowledge support to the Department of Ophthalmology Gavin Herbert Eye Institute at the University of California, Irvine from an unrestricted Research to Prevent Blindness award, from NIH core grant P30EY034070, support by MEYS CR (LM2023042) and European Regional Development Fund-Project “Innovation of Czech Infrastructure for Integrative Structural Biology” (No. CZ.02.01.01/00/23_015/0008175) and iNEXT-Discovery, project number 871037, funded by the Horizon 2020 program of the European Commission, the PASIFIC postdoctoral fellowship programme (Agreement No PAN.BFB.S.BDN.315.022.2022; Project No. DPE/2023/00007), this project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 847639 and from the Ministry of Science and Higher Education, and cryoEM training through the Wellcome/MRC-funded cryoEM training program (218785/Z/19/Z).

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