Characterization of the structure and function of the Rabphilin 3A

Abstract

This PhD thesis deals with both the structural and functional characterization of the C2 domains of Rabphilin 3A. This protein contains two C2 domains, C2A and C2B, located in tandem. There are some precedents that show the participation of this protein in several processes such as exocytosis or vesicle fusion due to its ability to interact with the SNARE complex. Rabphilin 3A controls the process of recaption of new vesicles, being the interaction with SNAP25 the key regulation step of the overall process. Several biochemical and biophysical studies have demonstrated that each of the C2 domains shows a different mode to bind to calcium, as well as different affinities for phosphoinositides, what could imply a difference in the regulation of the synaptic transmission and the processes related to vesicle secretion. The tandem domain C2AB has a characteristic behaviour markedly different form that of the independent C2A and C2B domains, which has been studied through biophysical, biochemical, and molecular biology techniques. The comparison of the behaviour of the wild type protein with several mutant constructs have served to characterize the binding mode of this protein to the membrane and the SNARE complex. The isothermal titration calorimetry (ITC) technique has been used to determine that the C2A domain of the Rabphilin 3A binds to the membranes formed by POPC/POPS/PI(4,5)P2 mainly through its lysine-rich cluster. The presence of calcium in the media allows the anchoring of the domain to the lipid membrane through a different region, also called the calcium-binding region. The binding areas of the C2B domain remain blocked due to the relative tandem orientation of the two domains in the C2AB structure, being accessible the H2 helix, which in turn becomes the main area able to interact with the SNARE complex. The C2AB domain of Rabphilin 3A is not able to form aggregates on its own, but the presence of vesicles containing phopholipids allows that the domains of the proteins bind to two membranes in a calcium-dependant manner, hence bringing them together and provoking the enlargement of the size of the aggregate particles. This experimental fact can easily observed by analyzing the sample with the dynamic light scattering (DLS) technique. The use of transmission electronic microscopy has corroborated the size of these aggregates, visualizing the potential ability of this protein to fuse vesicles. The generation of mutant constructs have served to determine that the linker among both domains could hamper the aggregation ability, since a mutation on the aminoacid residues of the linker provoked a significant enhancement of the binding capability. Another checkpoint of the ability to bind membrane was found in the loops of the calcium binding region of the C2A domain, acting as a positive control checkpoint of the binding of the domain to the membrane. The determination of the crystal structure of the C2B domain of Rabphilin 3A interacting with SNAP25 has allowed the identification of the key residues involved in this interaction. A combination of several biophysical and biochemical techniques have been used to propose that the tandem C2AB domains are able to coordinate calcium and PI(4,5)P2 through the calcium-binding region and the lysine rich cluster, while the binding to the SNAP25 takes place through the H2 helix of the C2B domain. In addition, it has been determined that the interaction observed of the C2AB of Rabphilin 3A and SNAP25 also occurs in the complexes SNAP25/STX1A and SNAP25/STX1A/VAMP2. This binding is calcium-dependant and needs the whole domain to happen. With all these results in mind we have proposed a model to explain how the Rabphilin 3A participates in the vesicle fusion process along with the cooperation of calcium and PI(4,5)P2.