Xuewu Zhang, Ph.D. - US grants

Project Summary/Abstract Background. Plexins are transmembrane receptors for the semaphorin axon guidance molecules. Repulsive signals from semaphorin-bound plexins are critical for proper pathfinding and innervation of developing neurons. Plexin signals also play important roles in regulating cell migration, vascular patterning and immune responses. Malfunction of the plexin signaling pathways is implicated in a variety of diseases such as neurological disorders, cancer and autoimmune diseases, and plexins have emerged as new drug targets for these diseases. Essential to the signaling of plexins is their intracellular regions, which contain a R-Ras GTPase activating protein (GAP) domain. The GAP domain contributes to plexin-mediated axon guidance by inactivating R-Ras, which leads to inactivation of integrin and loss of cell adhesion. The plexin GAP domain is normally kept inactive, and its activation requires simultaneous binding of semaphorin and a RhoGTPase (Rac1, RhoD or Rnd1) to the extracellular region and the intracellular RhoGTPase binding domain (RBD) of the receptor, respectively. Objectives. The goal of this research program is to understand the molecular mechanisms of autoinhibition and activation of the plexin GAP domain. Research Design. We use X-ray crystallography in combination with biochemical and cell biological approaches to study the mechanisms. We have solved the crystal structure of the intracellular domain of plexin A3. The structure shows that the GAP domain adopts an inactive conformation, and suggests that the RBD and a N-terminal segment contribute to stabilization of this autoinhibited state. Our analyses of the structures also led to a hypothesis that the plexin intracellular domain can form a specific dimer when plexin is induced to dimerize by semaphorin, and binding of a RhoGTPase to the RBDs of this dimeric plexin allosterically induces a conformational change in the GAP domain which triggers its activation. This proposal is centered around testing this model. In Aim 1 we will perform mutational analyses of the autoinhibition mechanism using a biochemical GAP assay and a cell-based assay. We will also pursue crystal structures of other plexin family members. In Aim 2 we will use the same GAP assay and cell-based assay to test the activation mechanism involving both dimerization and RhoGTPase binding. In Aim 3 we will study the activation mechanism for the GAP domain by determining structures of the plexin intracellular domains in complex with RhoGTPases and R-Ras. These studies together will reveal the molecular basis for the autoinhibition and activation of the plexin GAP domain, and provide new routes to future drug design for diseases associated with plexin malfunction.

DESCRIPTION (provided by applicant): Plexins are the cell surface receptors of semaphorins. Plexin-mediated semaphorin signaling is essential for processes such as the development of the nervous system and the cardiovascular system and regulation of immune responses and bone homeostasis. Malfunction of plexins has been associated with neurological disorder and cancer. Understanding how plexins function will pave the way for developing targeted therapeutics for fighting the associated diseases and improving neuronal regeneration after injury. The plexin intracellular region contains a GTPase Activating Protein (GAP) domain that is essential for function. In the previous period, we have identified the small GTPase Rap as the authentic substrate for the plexin GAP domain, and have determined the structural basis for how the GAP domain is activated by semaphorin-induced dimerization and how it inactivates Rap through a non-canonical catalytic mechanism. Objectives. To study additional layers of regulation mechanisms of plexins, and mutual regulation between plexins and several of their key binding partners. Research Design. Based on a new crystal structure of ours, we will first analyze the role of the inhibitory dimer in plexin regulation, a long-standing question in the fiel. Plexin signaling requires not only its RapGAP activity, but also its ability to assemble and contro the activity of a multi-protein signaling complex at the plasma membrane. Many proteins interact with plexins, but the structural basis of their actions is largely unknown. We will focus on some of the essential binding partners, address the questions how they bind plexin and exert mutual regulation with plexins. In Aim 1 we will test an inhibitory dimer model in plexin regulation. We have determined two crystal structures of PlexinA4, which adopts a new conformation and forms a compact dimer with the GAP active site buried in the dimer interface. We propose that this dimer and the apo dimer structure of the plexin extracellular region reported previously together mediate the autoinhibited dimeric state of full-length plexin on the cell surface. Structure-based mutational analyses will be performed to test this hypothesis. In Aim 2 we will study the basis for the opposite effects of RND1/Rac and RhoD on plexin signaling. The RhoGTPases Rac1 and RND1 interact with plexin and facilitate its binding and activation by semaphorin. In contrast, RhoD inhibits plexin signaling, although it binds plexin in the same mode with similar affinity. Our structure analyses led to a hypothesis that explains this paradox, which will be tested in this aim. In Aim 3 we will analyze the interaction and regulation of FARPs by plexin. FARP1 and FARP2 are two related guanine nucleotide exchange factors (GEFs) that have been shown to interact directly with plexin and make essential contributing to its signaling. We will pursue a structure of the plexin/FARP complex to elucidate the basis for their interaction, and analyze how this interaction helps release the autoinhibition of FARPs.

FAM46C (Family with sequence similarity 46, C) is one of the most frequently mutated genes in multiple myeloma (found in over 20% of the patients). Mutations of other FAM46 family members (A, B and D) are also associated with various human diseases. Despite the strong connections with diseases, the functions of FAM46 in either physiological or pathological settings are unknown. The goal of the project is to fill the knowledge gap on the functions of the FAM46 proteins and the underlying mechanisms, paving the way for developing therapeutic strategies for the associated diseases. The project is based on our preliminary work showing the direct physical interactions of FAM46 with polo-like kinase 4 (Plk4) and BRCA2 and CDKN1A(p21) interacting protein (BCCIP). Plk4 is the master regulator of centrosome duplication and ciliogenesis. BCCIP has been shown to be involved in regulating DNA repair and centrosome duplication. These together suggest that the FAM46 proteins regulate centrosome duplication, ciliogenesis and DNA repair. Centrosomes organize the bi-polar spindle formation and proper chromosome segregation in the cell cycle. In non-dividing cell, centrosomes mediate the formation of primary cilia, specialized signaling organelles critical for organogenesis and tissue homeostasis. A role in regulating these processes are consistent with the strong connection between FAM46 mutations and cancer. These hypotheses will be tested in three aims by combining X-ray crystallographic, biophysical, biochemical and cell-based functional approaches. Aim 1 will be focused on biophysical and structural analyses of the FAM46/Plk4 interaction. These studies will reveal the binding mode between FAM46 and Plk4, and identify key residues that can be tested by mutations in functional assays. In addition, potential mutual regulation of the enzymatic activities between FAM46 and Plk4 will be analyzed. Aim 2 will be focused on biophysical and structural analyses of the FAM46/BCCIP interaction. The crystal structure of the FAM46 and BCCIP complex will be determined to elucidate their interaction in atomic detail. Structural analyses will also be directed at the potential FAM46, Plk4 and BCCIP? tripartite complex. Mutational analyses will follow to test the binding mode and interface residues. The goal of Aim 3 is to analyze the roles of the interactions among FAM46, Plk4 and BCCIP? in regulating centrosome duplication, ciliogenesis and DNA repair. Fluorescence microscopy will be used to probe the localization of FAM46, Plk4 and BCCIP?, and their colocalization in and out of centrosomes in various stages of the cell cycle. The regulation of centrosome duplication, ciliogenesis and DNA repair by FAM46 through the interactions with Plk4 and BCCIP will also be analyzed by fluorescence microscopy.

Plexins are single-pass transmembrane receptors that serve as the primary receptors for the guidance molecules semaphorins. Some semaphorins also require the neuropilin co-receptor for binding and activating plexin. Semaphorin/plexin/neuropilin-mediated signaling is essential for many processes, including the development of the nervous system and the cardiovascular system. Malfunction of this signal pathway is associated with diseases such as neurological disorder and cancer. A better understanding of the mechanisms of this pathway will provide a foundation for developing targeted therapies for these diseases and improving neuronal regeneration after injury. Plexin and neuropilin are both large proteins, and use their N-terminal membrane distal domains to bind semaphorin. Semaphorin are dimeric molecules, and activate plexin by inducing the formation of the active dimer. One major remaining mechanistic question is how the membrane proximal and transmembrane regions in plexin and neuropilin couple the semaphorin binding at the N-terminal domains to the activation of the plexin cytoplasmic domain, which relays the signal to downstream pathways. There is evidence suggesting that the membrane proximal and transmembrane regions play active roles in plexin activation. Another outstanding question concerns how many of the newly identified binding partners of plexin contribute to the signaling. The proposed research will be focused on addressing these mechanistic questions. In Aim 1, we will analyze how the interactions mediated by the transmembrane region of plexin regulate the formation of the plexin active dimer. We will design plexin constructs that contain the transmembrane region and cytoplasmic region but not the N-terminal autoinhibitory domains. These constructs are expected to form the active dimer spontaneously in the absence of semaphorin binding. We will determine the structure of this active dimer through either X-ray crystallography or cryo-electron microscopy to visualize how the transmembrane region interacts and promotes the formation of the plexin active dimer. In Aim 2, we will pursue the structure of full-length plexin and neuropilin in complex with semaphorin by using cryo-electron microscopy. These structures will provide a direct view of the entire receptor/ligand complex, and reveal how the membrane proximal and transmembrane regions couple the binding of semaphorin at the N-terminal domains to the cytoplasmic domains. In Aim 3, we will investigate the interactions and regulation of plexin by regulatory proteins. Some of these proteins may only exert their regulatory function in the context of the lipid bilayer, which we will investigate by using plexin reconstituted in lipid discs. The structural studies will be correlated by in vitro biophysical and cell-based functional assays. In addition, new approaches developed for the plexin system will be used to study other transmembrane signaling proteins in order to gain a general understanding of the principles of transmembrane signaling.

This application is to request fund for purchasing of a high-end computation and data storage system for the cryo-electron microscopic (cryo-EM) work under the parent project that is focused on cryo-EM structural and mechanistic analyses of the transmembrane receptor plexin. In the past few years, the parent project has generated a number of high-impact cryo-EM structures of plexin and related transmembrane receptors, which substantially advanced the understanding of their signaling mechanisms. During this period, the infrastructure for computation and data storage have become a major bottleneck. The throughput of cryo-EM data collection has been increasing at a very fast rate, leading to vast amounts of data that enable the determination of high-resolution structures of proteins that was previously not possible. Accordingly, fast computers are required for processing these data and take advantage of new methods that are improving on a daily basis but also more resource demanding. Large storage servers are needed for secure storage and easy access of the data. The lab has been relying on a single workstation with a 12-TB hard-disk. With the data collection rate at ~2 TB per day and ~5 TB per trial of a protein, the available disk space is severely limiting the progress of the project. The calculation of each structure on the GPU workstation takes days or weeks for difficult cases, which leads to delays as datasets waiting in the queue. In addition, the current GPUs have 12 GB memory, limiting the size of the protein complex structures that could be processed. This proposal therefore requests for fund to purchase a computational system built for cryo-EM, with 16 GPUs with 48 GB memory and 1 PB RAID hard-disk unit. This is an invest that will greatly benefit the parent project and the research field for years to come.