Periodic Reporting for period 3 - FLEXINGPLEXIN (A structure-function analysis to discover how receptor conformations and interactions determine semaphorin-neuropilin-plexin signalling outputs.)
Berichtszeitraum: 2023-10-01 bis 2025-03-31
During the development of a multicellular organism the members of a few distinctive families of extracellular proteins interact with specific cell surface receptors to guide cells to their correct location. Cell guidance functions continue to be essential throughout life to maintain healthy tissues. These functions depend on a number of cell guidance family members working in unison to provide exquisitely detailed instructions in an appropriately timely and location dependent manner. Such orchestration requires multiple, switchable, levels of control over the signalling outputs. Semaphorins use plexins as their main receptors and the semaphorin and plexin families together constitute one of the largest and functionally diverse of the cell guidance systems. Co-receptors, such as the neuropilins, have been implicated in context dependent switching of semaphorin-plexin signalling. To date we know very little about the molecular mechanisms that act at the cell surface to determine divergent signalling outputs despite their vital role in controlling timely, location-dependent functions. To address this lack of knowledge we are investigating the mechanisms that control switches in signalling output for the semaphorin-neuropilin-plexin system.
Although there have been enormous advances revealing semaphorin-neuropilin-plexin function at the level of genetic and cellular experiments, our knowledge of the molecular level mechanisms which deliver and discriminate between the numerous biological outcomes is still sparse. The myriad physiological functions of the semaphorin-neuropilin-plexin system are now known to span angiogenesis, bone maintenance, heart development, and regulation of immune responses, as well as their plethora of roles in the nervous system. Conversely, these proteins have been found to be involved in a broad range of pathologies, including cancer, and neurogenetic disorders. For this reason, semaphorins, plexins and neuropilins have emerged as potential points for clinical intervention. Already insights into the determinants of ligand-receptor binding, generated by us and others, are being applied in the design of novel therapeutic agents. To better guide such efforts, we need to be able to pinpoint the factors that control signalling outcomes. Our aim is to provide the understanding and tools required to manipulate semaphorin-neuropilin-plexin signalling in more tightly targeted and functionally specific modes than currently available.
Some of the most fundamental gaps in our knowledge concern the mechanisms controlling the signalling outputs of the secreted class 3 semaphorins (Sema3s), their PlexinA1-4 and PlexinD1 receptors and their co-receptors, Neuropilin 1 and 2 (Nrp1, Nrp2). The overarching objective of FLEXINGPLEXIN is to discover the mechanisms by which Sema3 variants, plexin ectodomain conformation and interactions with neuropilin result in multiple different signalling outcomes. We have subdivided this objective into three aims:
AIM 1. The structural determinants and mechanisms of action by which class 3 semaphorins exert differing effects on signalling.
AIM 2. The conformational state of the plexin ectodomain in different contexts and its contribution to signal outcome.
AIM 3. The mechanisms by which neuropilin binding can switch the outcomes of plexin signalling.
Although there have been enormous advances revealing semaphorin-neuropilin-plexin function at the level of genetic and cellular experiments, our knowledge of the molecular level mechanisms which deliver and discriminate between the numerous biological outcomes is still sparse. The myriad physiological functions of the semaphorin-neuropilin-plexin system are now known to span angiogenesis, bone maintenance, heart development, and regulation of immune responses, as well as their plethora of roles in the nervous system. Conversely, these proteins have been found to be involved in a broad range of pathologies, including cancer, and neurogenetic disorders. For this reason, semaphorins, plexins and neuropilins have emerged as potential points for clinical intervention. Already insights into the determinants of ligand-receptor binding, generated by us and others, are being applied in the design of novel therapeutic agents. To better guide such efforts, we need to be able to pinpoint the factors that control signalling outcomes. Our aim is to provide the understanding and tools required to manipulate semaphorin-neuropilin-plexin signalling in more tightly targeted and functionally specific modes than currently available.
Some of the most fundamental gaps in our knowledge concern the mechanisms controlling the signalling outputs of the secreted class 3 semaphorins (Sema3s), their PlexinA1-4 and PlexinD1 receptors and their co-receptors, Neuropilin 1 and 2 (Nrp1, Nrp2). The overarching objective of FLEXINGPLEXIN is to discover the mechanisms by which Sema3 variants, plexin ectodomain conformation and interactions with neuropilin result in multiple different signalling outcomes. We have subdivided this objective into three aims:
AIM 1. The structural determinants and mechanisms of action by which class 3 semaphorins exert differing effects on signalling.
AIM 2. The conformational state of the plexin ectodomain in different contexts and its contribution to signal outcome.
AIM 3. The mechanisms by which neuropilin binding can switch the outcomes of plexin signalling.
To provide information on molecular mechanism we are integrating structural biology (x-ray crystallography and cryo electron microscopy, cryoEM), in vitro interaction assay and in cellulo molecular imaging-based approaches. This integrated structural biology approach underpins our work on all three aims. The combination of techniques allows us to span from the atomic to cellular scale and we are using them, alongside functional assays (in-house for in cellulo, and in collaboration for further cellular as well as in vivo studies), to evaluate molecular-level models for mechanism in their biological context. We are now at the mid-point of our studies and have undertaken a number of structural and biophysical analyses of Sema3s, their plexin receptors and co-receptors by x-ray crystallography and/or single particle cryoEM. Highlights of these studies to date include:
An atomic resolution level structure-function analysis of class 3 semaphorins Sema3A, Sema3E and Sema3G providing the insights to engineer bespoke interactions with plexin and neuropilin receptors.
Analyses of the extent to which the extracellular regions (ecodomains) of Plexin A, Plexin B and Plexin D receptors can flex between ring-like and more open shapes (conformations), the changes to these properties in engineered plexin variants and, in collaboration, the impact on biological function.
An atomic resolution level structure-function analysis of class 3 semaphorins Sema3A, Sema3E and Sema3G providing the insights to engineer bespoke interactions with plexin and neuropilin receptors.
Analyses of the extent to which the extracellular regions (ecodomains) of Plexin A, Plexin B and Plexin D receptors can flex between ring-like and more open shapes (conformations), the changes to these properties in engineered plexin variants and, in collaboration, the impact on biological function.
Detailed structural information on class 3 semaphorins has been limited to Sema3A. Prior to the start of this project we used our knowledge of the structure-function of Sema3A to guide the design of a truncated Sema3A (T-sema3A) stabilised by an engineered dimerization site that introduced a disulphide bridge. Subsequent work by our collaborators points to the potential utility of T-sema3A as a novel agent that could help restore balance to the cellular immune system in autoimmune diseases (see Eiza et al. Front Pharmacol. 2023 DOI: 10.3389/fphar.2022.1085892). During the initial period of FLEXINGPLEXIN we have advanced our knowledge of the structural determinants of receptor binding and signal complex assembly for class 3 semaphorins (AIM 1). We have now been able to shed light on distinctive structure-function features of Sema3E and Sema3G and are using these insights to engineer novel Sema3s to manipulate signalling outcomes. We anticipate that by the end of the project we will have engineered multiple novel semaphorin ligands, characterised their structure-function properties in cellulo and, with collaborators, used them to probe mechanisms of action and determinants of signalling outcome in multiple biomedically relevant biological contexts.
At the start of the project our knowledge of plexin structure-function was sufficient to allow us to engineer a disulphide bridge to lock the PlexinD1 ring-like conformation (see Figure, adapted from Mehta et al. Nature 2020 DOI: 10.1038/s41586-020-1979-4). This locked ring conformation still allows the receptor to trigger repulsive cellular responses on binding Sema3E but prevents it from acting as a mechanosensor of sheer stress. In FLEXINGPLEXIN we have advanced these studies with structural and biophysical analyses of the determinants of plexin ectodomain conformation (AIM 2). We have engineered variant plexin ectodomains and in collaborations are combining our molecular level analyses with in cellulo and in vivo functional analyses to probe the role of plexin ectodomain conformation and flexion in biological function and dysfunction. For the remainder of the project we anticipate we will bring to bear the tools and insights we have generated through work on AIM 1 and 2 to dissect the interplay of semaphorin, plexin and the co-receptor neuropilin in determining signalling outcome in a range of biological contexts (AIM 3).
At the start of the project our knowledge of plexin structure-function was sufficient to allow us to engineer a disulphide bridge to lock the PlexinD1 ring-like conformation (see Figure, adapted from Mehta et al. Nature 2020 DOI: 10.1038/s41586-020-1979-4). This locked ring conformation still allows the receptor to trigger repulsive cellular responses on binding Sema3E but prevents it from acting as a mechanosensor of sheer stress. In FLEXINGPLEXIN we have advanced these studies with structural and biophysical analyses of the determinants of plexin ectodomain conformation (AIM 2). We have engineered variant plexin ectodomains and in collaborations are combining our molecular level analyses with in cellulo and in vivo functional analyses to probe the role of plexin ectodomain conformation and flexion in biological function and dysfunction. For the remainder of the project we anticipate we will bring to bear the tools and insights we have generated through work on AIM 1 and 2 to dissect the interplay of semaphorin, plexin and the co-receptor neuropilin in determining signalling outcome in a range of biological contexts (AIM 3).