Periodic Reporting for period 1 - ComplexSignal (Deciphering the combinatorial signalling patterns in brain development by structural studies of axon guidance receptors and adhesive GPCRs)
Berichtszeitraum: 2018-05-01 bis 2020-04-30
Brain development relies on the coordinated migration of neuronal cells and their cellular extensions (axons, dendrites). Correct navigation during these cell migration events is essential and requires the combined actions of 'cell guidance receptors' and their ligands. These cell membrane receptors typically trigger repulsive or attractive/adhesive cell responses depending on the receptor type(s) and ligands involved. In addition to their role in neuronal cell guidance, membrane receptor-ligand interactions have important roles in adult brain such as synapse formation and ability of synapses to change their activity level. As a result, malfunction of these proteins has been associated with many neurological and neurodevelopmental disorders. Therefore, deciphering the complete function of these ligands and the molecular mechanism of their interactions with the receptors is a major task towards understanding the biology of brain, pathology of neuronal disorders as well as their clinical applications.
Research in the last four decades has led to the identification of major guidance protein families. However, our understating so far is limited to the effect of individual receptor-ligand interactions. How multiple ligands and receptors work together to generate the complexity found in brain tissues is poorly understood. Multifunctional ligands and receptors are thought to interact in context-dependent combinations to increase their functional versatility, but the mechanistic details underpinning these 'combinatorial' interactions remain largely elusive. Even less understood is the role of other extracellular molecules, for example, sugars of the heparan sulphate family. Recent studies have shown that many receptors bind these sugars, adding a further layer of functional complexity and versatility.
Recently, our group has made a pioneering progress and elucidated the structure of a “super-complex” fragment consisting of three types of cell receptors: FLRT proteins, adhesion GPCRs Latrophilin (Lphn) and guidance receptors Unc5. This has opened up a series of questions that need to be addressed to fully exploit the initial findings. In this project, our goal is to investigate how super-complex formation impacts on the downstream soluble and transmembrane signalling domains to direct cell behaviour. We specifically aim at elucidating the full length structure of the super-complex to reveal the molecular details of the interactions, understanding how complex formation impacts on the functions of these receptors, and exploring the influence of heparan sulphate sugars on the super-complex structure and function.
Research in the last four decades has led to the identification of major guidance protein families. However, our understating so far is limited to the effect of individual receptor-ligand interactions. How multiple ligands and receptors work together to generate the complexity found in brain tissues is poorly understood. Multifunctional ligands and receptors are thought to interact in context-dependent combinations to increase their functional versatility, but the mechanistic details underpinning these 'combinatorial' interactions remain largely elusive. Even less understood is the role of other extracellular molecules, for example, sugars of the heparan sulphate family. Recent studies have shown that many receptors bind these sugars, adding a further layer of functional complexity and versatility.
Recently, our group has made a pioneering progress and elucidated the structure of a “super-complex” fragment consisting of three types of cell receptors: FLRT proteins, adhesion GPCRs Latrophilin (Lphn) and guidance receptors Unc5. This has opened up a series of questions that need to be addressed to fully exploit the initial findings. In this project, our goal is to investigate how super-complex formation impacts on the downstream soluble and transmembrane signalling domains to direct cell behaviour. We specifically aim at elucidating the full length structure of the super-complex to reveal the molecular details of the interactions, understanding how complex formation impacts on the functions of these receptors, and exploring the influence of heparan sulphate sugars on the super-complex structure and function.
Truncated domains of FLRT2, Lphn3 and Unc5D had been reported to form an octameric super-complex with an unusual combination, four Lphn3 molecules interacting with 2 copies of FLRT2 and Unc5D (see figure). Biochemical and biophysical analyses using the full length proteins, rather than the truncations, revealed that these receptors did not form the reported octameric complex but a tetrameric one, with 2 copies of Lphn3 binding to 1 copy of FLRT2 and Unc5D. While this could suggest a potential mechanism of action of the receptors (e.g. forming multiple complexes), it might also indicate an experimental fault of the earlier findings. We also identified that this complex was quite unstable, falling apart to its constituents rather than staying as a single entity in solution. We created mutants of these receptors which had residues exchanged to cysteines on special positions. Organisms use disulphide bridges between two cysteine residues to stabilize the structure of proteins. We exploited this mechanism to link receptors together and increase the stability of the receptor complex. This allowed us to obtain the sample quality needed for cryo-electron microscopy (cryo-EM) experiments.
Meanwhile, we crystallized FLRT2 (LRR domain) in the presence of a heparan sulphate sugar analog (SOS) and determined the sugar-bound structure. We revealed a dimeric FLRT2 structure, two copies aligning face to face, with the sugar binding to a cleft between the two FLRT2 molecules (see figure). Further analysis of the structure revealed that the interface that FLRT2 was using to bind the other copy in the structure with sugar overlapped with the interface it used to bind Lphn3s in the described super-complex. This indicated that these two complexes would be mutually exclusive. In addition, the sugar analog was binding the same region as the second copy of Lphn3 (B copy in the super-complex) binds to FLRT2. Overall these observations suggested that the heparan sulfate sugars at the extracellular matrix could regulate the super-complex either by controlling the amount of FLRT2 molecules available for super-complex formation or by blocking the surface on FLRT2 for second Lphn3 binding and therefore influencing the number of Lphn3s in the complex. By using a Lphn homolog (Lphn2) we were able to create a trimeric complex with just one copy of Lphn2, FLRT2 and Unc5D. These two complexes –tetrameric and trimeric– could reflect different functional states of these receptor complexes. We are at the moment elucidating the structures of these to understand if there is any structural changes when different number of receptors are present in the complexes.
Finally, we wanted to investigate how super-complex formation effects Lphn3 activity and performed GPCR activity assays. Remarkable, we observed that neither FLRT2 nor FLRT2-Unc5D presence did not change the Lphn3 activity. This indicated that the super-complex may not act via Lphn3 but maybe via Unc5D or FLRT2, which needs further investigation.
Meanwhile, we crystallized FLRT2 (LRR domain) in the presence of a heparan sulphate sugar analog (SOS) and determined the sugar-bound structure. We revealed a dimeric FLRT2 structure, two copies aligning face to face, with the sugar binding to a cleft between the two FLRT2 molecules (see figure). Further analysis of the structure revealed that the interface that FLRT2 was using to bind the other copy in the structure with sugar overlapped with the interface it used to bind Lphn3s in the described super-complex. This indicated that these two complexes would be mutually exclusive. In addition, the sugar analog was binding the same region as the second copy of Lphn3 (B copy in the super-complex) binds to FLRT2. Overall these observations suggested that the heparan sulfate sugars at the extracellular matrix could regulate the super-complex either by controlling the amount of FLRT2 molecules available for super-complex formation or by blocking the surface on FLRT2 for second Lphn3 binding and therefore influencing the number of Lphn3s in the complex. By using a Lphn homolog (Lphn2) we were able to create a trimeric complex with just one copy of Lphn2, FLRT2 and Unc5D. These two complexes –tetrameric and trimeric– could reflect different functional states of these receptor complexes. We are at the moment elucidating the structures of these to understand if there is any structural changes when different number of receptors are present in the complexes.
Finally, we wanted to investigate how super-complex formation effects Lphn3 activity and performed GPCR activity assays. Remarkable, we observed that neither FLRT2 nor FLRT2-Unc5D presence did not change the Lphn3 activity. This indicated that the super-complex may not act via Lphn3 but maybe via Unc5D or FLRT2, which needs further investigation.
In this project, we have also developed so-called nanobodies against FLRT2, Lphn3 and Unc5D. Nanobodies are fragments of unusual antibodies from camels and related species. Antibodies are indispensable tools in biological and medical research, with a wide range of applications from determining the location of a molecule inside a cell to targeting drugs to desired tissues in an organism. Nanobodies function similar to regular antibodies and they even outperform them in many applications; therefore, they can be seen as good alternatives to regular antibodies. Moreover, nanobodies can be produced by any microorganism. Hence, they could replace the regular antibodies and reduce the number of animals used in antibody production.
Receptor nanobodies we have developed are highly versatile and function as good as commercially available antibodies in various applications, such as fluorescence microscopy and affinity purification. We expect that these nanobodies will replace the conventional antibodies currently in use. In addition, the nanobodies have the potential to bind the receptors and block their activity by preventing their interaction with other proteins, and therefore acting like an inhibitor. We plan to use these nanobodies as inhibitors to investigate the role of the receptors in neuronal cells as well as in developing organisms.
Finally, malfunctioning of FLRTs, Lphns and Unc5s had been reported in several neurological disorders. Some of these had even been identified overabundantly in certain cancer cells. The developed nanobodies could in theory be used to target such malfunctioning receptors or to block the activity of excess proteins in patients. Therefore, these nanobodies could have applications in medical research as well.
Receptor nanobodies we have developed are highly versatile and function as good as commercially available antibodies in various applications, such as fluorescence microscopy and affinity purification. We expect that these nanobodies will replace the conventional antibodies currently in use. In addition, the nanobodies have the potential to bind the receptors and block their activity by preventing their interaction with other proteins, and therefore acting like an inhibitor. We plan to use these nanobodies as inhibitors to investigate the role of the receptors in neuronal cells as well as in developing organisms.
Finally, malfunctioning of FLRTs, Lphns and Unc5s had been reported in several neurological disorders. Some of these had even been identified overabundantly in certain cancer cells. The developed nanobodies could in theory be used to target such malfunctioning receptors or to block the activity of excess proteins in patients. Therefore, these nanobodies could have applications in medical research as well.