About author: Dr. Shanti Pal Gangwar was born and brought up in Bareilly, Uttar Pradesh. He received his M.Sc. in Biotechnology from Kumaun University, Nainital, and his Ph.D. in Biophysics and Structural Biology from Jawaharlal Nehru University, New Delhi. For his postdoctoral research, Shanti Pal moved to the USA and is currently working in the laboratory of Dr. Alexander I. Sobolevsky at Columbia University Irving Medical Center, New York. Shanti Pal’s research focuses on ionotropic glutamate receptors (iGluRs). These ligand-gated ion channels play crucial roles in communication between neuronal cells in the mammalian central nervous system. He was intrigued by the fact that plants neither have neurons nor a brain; nevertheless, plants have genes that encode iGluR-like proteins called Glutamate receptor-like channels (GLRs). While GLRs were found to play vital roles in various physiological processes in plants, including wound response, seed germination, root development, and morphogenesis, it has been unclear how they do this. What is the architecture and topology of this protein? Where and how it binds ligands? Does it form an ion channel and conducts currents, similar to iGluR? Shanti Pal’s curiosity and eagerness to answer these questions motivated him to solve the first high-resolution structure of a full-length plant GLR using cryo-electron microscopy.
How would you explain your research outcomes to the non-scientific community?
Glutamate is one of twenty amino acids representing the fundamental building blocks of proteins, which also plays a unique role as the primary neurotransmitter or messenger molecule in the mammalian brain. Glutamate establishes communication between neurons at the specialized junctions termed synapses. This communication underlies high cognitive functions such as learning and memory. This happens through the binding of presynaptically released Glutamate to different postsynaptic ionotropic glutamate receptors (iGluRs, the messenger signal receivers) and the opening of the ion channels associated with these proteins, which in turn conduct the synaptic current. Although plants do not have a nervous system, they do have long-distance signaling, which communicates the perception of environmental stimuli, such as wounds, cuts, or caterpillar bites, from one part of a plant to another by employing various mechanisms, including fast propagation of electrical signals coupled to changes in free cytosolic Ca++. It has been discovered that the plant signaling system uses proteins homologs to animal iGluRs, called Glutamate receptor-like channels (GLR), and found in various plant lineages, including moss, rice, tomato, and Arabidopsis. Amino acid sequence similarity between GLRs and iGluRs suggested possible structural and functional similarity between them. Physiological studies uncovered many GLR functions in plants, including regulation of nitrogen and carbon metabolism, water balance, ion distribution, hormone biosynthesis, and response to environmental stress. However, the molecular bases of these GLR functions have remained an enigma. It was unclear, for example, whether GLRs form a channel pore responsible for ion permeation or whether auxiliary subunits like animal iGluRs modulate them.
Similarly, it was not easy to establish the structural and functional relationship between GLRs and their mammalian counterparts iGluRs. In our research, we have solved the long-awaited puzzle of the structural organization of plant GLRs. We discovered that plant GLRs are tetrameric assemblies of four identical or similar subunits reminiscent of animal iGluRs. Similar to iGluRs, GLRs have a 3-layer architecture, which includes layers of amino-terminal domains (ATDs), ligand-binding domains (LBDs), and ion channel-forming transmembrane domains (TMDs). The binding of glutathione to the ATD and Glutamate to the LBD suggested that, unlike iGluRs, GLRs involve both types of extracellular domains in the modulation of the ion channel activity. We also discovered that the presence of the auxiliary subunit cornichon (CNIH) is critical for GLR function.
How do these findings contribute to your research area?
Despite the availability of genetic information obtained during GLR cloning and the well-established importance of GLRs for long-distance signaling, their molecular architecture and structural mechanisms of activation and regulation have remained a puzzle. It was also unclear how similar GLRs and iGluRs are and how GLRs promote Ca++ influx into the cytosol in response to mechanical injury or wounding of a plant by insect bites or drought. Our research allowed us to solve the first structure of a plant GLR, revealing its 3-dimensional architecture and binding sites of amino acids ligands, Glu/Ser/Met, and glutathione to LBD and ATD, respectively. We identified residues contributing to ligand-binding sites and demonstrated the role of GLRs as ligand-gated channels permeable to Ca++ ions. Our study improves understanding of the general mechanisms of functional regulation of GLR, such as ligand binding, gating, and allosteric regulation. It also helps the researchers apply genome editing strategies in plants. It provides critical information regarding the functional role of GLRs in amino acid homeostasis and long-distance signaling. Our structural information provides a perspective on the evolution of bacterial and animal proteins and the plant lineage. It represents a tool to engineer plant GLRs to reach a more profound understanding of their basic physiology. Overall, our cryo-EM structure of the full-length GLR and crystal structures of its isolated ligand-binding domains allow a much better understanding of the molecular mechanisms of GLR function. Structural studies of plant GLRs are now entering a new exciting era of research that promises many exciting practical outcomes in the near future.
What was the exciting moment during your research?
In our research of GLRs, there was not a single Eureka moment of Archimedes. The progress on the project was slow and gradual. Initially, we were struggling to find the right conditions for protein preparation, then for cryo-EM sample preparation, and finally for high-resolution GLR structure determination. Not a single but a series of exciting moments have happened on the way, starting from protein expression to building a 3D model of GLR. Since our primary tool, structural biology, is like an “all or none phenomenon,” the realization of a successful outcome of the project does not come unless we have a density map and 3D structure. Therefore, I consider the most significant Eureka moment of my research was when, after many ups and downs, we finally solved the first high-resolution structure of the full-length GLR. Those feelings were soothing. Another exciting moment was discovering glutathione binding to the amino-terminal domain and its effect on the ion channel gating mechanism, previously never identified for iGluR-like proteins.
What do you hope to do next?
Comprehensive structural information is an arbiter between models of biological processes and serves as an inspiration for new hypotheses. I am fascinated with structural biology and studies of macromolecules, including plant GLRs and animal iGluRs, exploration of structural bases of signaling in plants, and neuronal communication in the animal brain. Both targets of my research, iGluRs, and GLRs, are similarly complicated and require comprehensive approaches for their studies. The exploration of one type of molecule complements the other. I plan to start my research group and contribute to the mystery of biological phenomena using the cutting-edge tools of structural biology, focusing on ion channels.
Where do you seek scientific inspiration from?
Inspiration is everywhere and is incredibly sporadic and comes in diverse ways – one just needs to open the eyes and breathe it in. For example, we do not have a memory chip in our head, yet we store and recall our memories instantly. Thus, curiosity to understand where and how our memory is stored in the brain might lead someone to be a neuroscientist. I believe that scientist’s inspiration is derived from the straightforward joy of thinking and understanding, the need to gain more insightful information, find a cure or a new purpose, and understand what is happening around us and how to make it better. My teachers, mentors, friends, family, and everyone around has inspired me. I also read books describing life stories of great scientists, telling about their ups and downs and encouraging me in my endeavors.
How do you intend to help Indian science improve?
Higher education in India has expanded a lot over the past few decades. However, with the growth in quantity, keeping a tab on quality has become a primary concern. Therefore, my primary goal will be to emphasize quality and not quantity in scientific research and focus my efforts on achieving better quality Ph.D. education by implementing interdisciplinary research and teaching our young talents at the highest science and technology standards. In addition, I will focus on exchanging and amalgamating ideas and concepts, interaction with people from various educational backgrounds, improving communication skills, presentation, public speaking, academic writing, and publishing high-quality research articles. As we all know, India is a developing country. We do not have sufficient facilities in research institutions. Therefore, it is critical to emphasize the importance of collaboration among the Indian research community and work together to produce the best out of their hard work. Moreover, there is an urgent need to scale up the investment in Science and Technology and encourage cooperation between academia and industry. I will also give lectures, discuss scientific topics in university knowledge exchange programs, and mentor young research graduates/undergraduates in scientifically interactive and creative ways.
Green, M.N*., Gangwar, S.P*., Michard, E., Simon, A.A., Portes, M.T., Barbosa-Caro, J., Wudick, M.M., Lizzio, M.A., Klykov, O., Yelshanskaya, M.V., et al. (2021). Structure of the Arabidopsis thaliana glutamate receptor-like channel GLR3.4. Mol Cell 81, 3216-3226 e3218. (Equal contribution)
Edited by: Vikramsingh Gujar