Mechanism and function of Vav1 localisation in TCR signalling.

The antigen-specific binding of T cells to antigen presenting cells results in recruitment of signalling proteins to microclusters at the cell-cell interface known as the immunological synapse (IS). The Vav1 guanine nucleotide exchange factor plays a critical role in T cell antigen receptor (TCR) signalling, leading to the activation of multiple pathways. We now show that it is recruited to microclusters and to the IS in primary CD4(+) and CD8(+) T cells. Furthermore, we show that this recruitment depends on the SH2 and C-terminal SH3 (SH3(B)) domains of Vav1, and on phosphotyrosines 112 and 128 of the SLP76 adaptor protein. Biophysical measurements show that Vav1 binds directly to these residues on SLP76 and that efficient binding depends on the SH2 and SH3(B) domains of Vav1. Finally, we show that the same two domains are critical for the phosphorylation of Vav1 and its signalling function in TCR-induced calcium flux. We propose that Vav1 is recruited to the IS by binding to SLP76 and that this interaction is critical for the transduction of signals leading to calcium flux.

Crucial structural role for the PH and C1 domains of the Vav1 exchange factor.

The Vav family of proteins are guanine nucleotide exchange factors (GEFs) for the Rho family of GTPases, which regulate various cellular functions, including T-cell activation. They contain a catalytic Dbl homology (DH) domain that is invariably followed by a pleckstrin homology (PH) domain, which is often required for catalytic activity. Vav proteins are the first GEFs for which an additional C1 domain is required for full biological activity. Here, we present the structure of a Vav1 fragment comprising the DH-PH-C1 domains bound to Rac1. This structure shows that the PH and C1 domains form a single structural unit that packs against the carboxy-terminal helix of the DH domain to stabilize its conformation and to promote nucleotide exchange. In contrast to previous reports, this structure shows that there are no direct contacts between the GTPase and C1 domain but instead suggests new mechanisms for the regulation of Vav1 activity.

Cross-interaction chromatography as a rapid screening technique to identify the stability of new antibody therapeutics.

Protein aggregation can be a major problem in the manufacturing of new biopharmaceuticals and there is a desirability for development of techniques that can predict the behaviour of new biopharmaceuticals early on in the development process. A technique that can be used to predict aggregation is self-interaction chromatography that is used to determine the second virial coefficient, B22, but one of the limitations includes the need to immobilise every protein of interest. In this study a related technique, cross interaction chromatography (CIC), is evaluated which overcomes this limitation. Three antibodies were studied across a range of NaCl concentrations with each antibody being studied as both a mobile phase and as the stationary phase - in total 6 different stationary-mobile phase combinations. The B22 values obtained for all three proteins correlated strongly with the B23 results obtained for the same protein in the mobile phase, and were significantly independent of the protein immobilised on the stationary phase. This observation allows the use of pre-prepared columns with known immobilised model proteins such as a polyclonal antibody or mAb, with other unknown monoclonal antibodies in the mobile phase. Preliminary experiments using a series of known immobilised mAbs columns with an unknown mAb in the mobile phase resulted in at least a 50 fold reduction in the amount of unknown protein needed and a rapid semi-quantitative assessment of aggregation propensity. CIC can speed up the screening process with minimum preparation time and therefore more rapidly be able to identify the aggregation stability of new antibody formulations.

Function of the nucleotide exchange activity of vav1 in T cell development and activation.

The guanine nucleotide exchange factor (GEF) Vav1 is essential for transducing T cell antigen receptor (TCR) signals and therefore plays a critical role in the development and activation of T cells. It has been presumed that the GEF activity of Vav1 is important for its function; however, there has been no direct demonstration of this. Here, we generated mice expressing enzymatically inactive, but normally folded, Vav1 protein. Analysis of these mice showed that the GEF activity of Vav1 was necessary for the selection of thymocytes and for the optimal activation of T cells, including signal transduction to Rac1, Akt, and integrins. In contrast, the GEF activity of Vav1 was not required for TCR-induced calcium flux, activation of extracellular signal-regulated kinase and protein kinase D1, and cell polarization. Thus, in T cells, the GEF activity of Vav1 is essential for some, but not all, of its functions.

Small-angle X-ray scattering and NMR studies of the conformation of the PDZ region of SAP97 and its interactions with Kir2.1.

The functional localization of potassium inward rectifiers is regulated by SAP97, a PDZ membrane-associated guanylate kinase protein. We describe here an investigation of the conformation of the PDZ domain region of SAP97 PDZ1-3. The NMR and SAXS data reveal conformational dynamics. The NMR data show minimal interdomain contacts, with the U3 linker region between PDZ2 and PDZ3 being largely unstructured. Shape analysis of the SAXS profiles revealed a dumbbell for the PDZ12 double domain. An overall elongated, asymmetric shape comprised of two to three distinct components characterizes the triple domain PDZ1-3. In addition, rigid body modeling shows that the representative average shape does not provide the full picture and that the data for the triple domain are consistent with large variations, suggesting significant conformational flexibility. However, the dynamics appears to be restricted as PDZ3 is located essentially within approximately 40 A from PDZ12. We also show that the Kir2.1 cytoplasmic domain interacts with all three PDZ domains but with a clear preference for PDZ2 even in the presence of the U3 region. We speculate that the restricted dynamics and preferential Kir2.1 binding to PDZ2 are features that enable SAP97 to function as a scaffold protein, allowing other proteins each to bind to the other two PDZ domains in sufficient proximity to yield productive channelosomes.