Structural studies of class A and D plexins

Semaphorin signalling via the plexin receptors, important in the nervous, cardiovascular, immune and skeletal systems, requires cytoplasmic domain dimerisation but its extracellular regulation and activation mechanisms remain unclear. In the thesis I conducted structural characterisation for two particular classes of verterbrate plexins, PlxnAs and PlxnD1. Here I present crystal structures of PlxnA1, PlxnA2 and PlxnA4 full ectodomains. The first nine domains of PlxnAs forma 230 Å long ring-like stalk fromwhich the tenth C-terminal domain points away. In agreement, negative stain electron microscopy of the PlxnA1 ectodomain reveals a predominant ring-like conformation and a minor twisted-open conformation of the stalk. All PlxnA crystal structures also make intermolecular "head-to-stalk" (domain 1 to domain 4-5) interactions, which are confirmed by structure-guided biophysical assays and live cell fluorescence microscopy. Functional assays performed in COS-7 cell and dentate gyrus growth cones revealed an autoinhibitory mechanism for PlxnAs based on this head-to-stalk interface. My results reveal a two-fold role for the PlxnA ectodomains: imposing a presignalling autoinhibitory separation of the PlxnA membrane proximal domains via a head-to-stalk, intermolecular interaction and supporting PlxnA cytoplasmic domain dimerisation post semaphorin-binding. For PlxnD1, I present high-resolution crystal structures of its semaphorinbinding segment (domains 1-2) as well as an initial structural model of its complete ectodomain (domains 1-10). The sema (domain 1) of PlxnD1 has the classic fold of a plexin sema domain as well as distinct insertions potentially important for its ligand-binding specificity. The PlxnD1 ectodomain adopts a closed-ring conformation in its crystal structure, in which the tail (domain 9) interacts with the sema domain head. In-solution experiments suggest structural variability and potential weak oligomerization for the PlxnD1 ectodomain. These new structures shed light on the unique and previously uncharacterised structural basis for PlxnD1 signalling.