Structure Function Studies of Vesicle-associated membrane protein-associated protein B (VAPB) associated with Amyotrophic lateral Sclerosis (ALS)

LUA SHIXIONG

Publication

Structure Function Studies of Vesicle-associated membrane protein-associated protein B (VAPB) associated with Amyotrophic lateral Sclerosis (ALS)

LUA SHIXIONG

Abstract

The process of protein folding is remarkably efficient, but sometimes it can go wrong. This can have harmful consequences, as the incorrect folding of proteins is thought to be the cause of diseases. Amyotrophic lateral sclerosis 8 (ALS8) caused by the missense Thr46Ile and Pro56Ser mutation in the MSP domain of Vesicle-associated membrane protein-associated protein B (VAPB) is one example of such ? misfolding diseases?, and also the main focus of my research. In this thesis, the first structural investigation on both wild-type, Thr46Ile and Pro56Ser mutated MSP domains is presented. The results revealed that the wild-type MSP domain is well-folded at neutral pH but can undergo acid-induced unfolding reversibly. It has thermodynamic stability energy of 7.40kcal/mol and is also active in binding to a Nir2 peptide with a Kd of 0.65?M. Further determination of its crystal structure reveals that it adopts a seven-stranded immunoglobulin-like ? sandwich. By contrast, the Pro56Ser mutation renders the MSP domain to be insoluble in buffer. Nevertheless, as facilitated by the discovery that ?insoluble proteins? can be solubilized in salt-free water (Li et al., 2006), we have successfully characterized the residue-specific conformation of the Pro56Ser mutant by CD and heteronuclear NMR spectroscopy. Surprisingly, the Pro56Ser mutant remains highly-unstructured under various conditions, lacking of tight tertiary packing and well-formed secondary structure, only with non-native helical conformation weakly-populated over the sequence. As such, the abolishment of native MSP structure consequently leads to aggregation and loss of functions under the physiological condition. Unexpectedly, unlike the Pro56Ser MSP domain mutant, the Thr46Ile mutation did not eliminate the native secondary and tertiary structures, as demonstrated by its far-UV CD spectrum, as well as Ca and C? NMR chemical shifts. However, the Thr46Ile mutation did result in a reduced thermodynamic stability and loss of the cooperative urea-unfolding transition which consequently causes it to be prone to aggregation at high protein concentrations and temperatures in vitro. The same mutation also causes a 3 fold reduction in its ability to bind to the Nir2 peptide and significantly eliminate its ability to bind to EphA4. We have also provided evidence that the EphA4 and Nir2 peptide appear to have overlapped binding interfaces on the MSP domain, which strongly implies that two signalling networks may have a functional interplay in vivo. Our study provides the first molecular basis for understanding the Pro56Ser and Thr46Ile ALS-causing mutations. We have also shown that by introducing additional Proline residues in the right context, the MSP domain could gain resistant to the Pro56Ser mutation. Lastly, we hypothesized that the interplay of two signalling networks mediated by the FFAT-containing proteins and Eph receptors respectively may play a key role in ALS pathogenesis.