Elimination of the native structure and solubility of the hVAPB MSP domain by the Pro56Ser mutation that causes amyotrophic lateral sclerosis.
The Pro56Ser mutation in the human VAPB MSP domain causes a familial amyotrophic lateral sclerosis. Here we present the first structural investigation of both wild-type and Pro56Ser mutant MSP domains. The results reveal that the wild-type MSP domain is well-folded at neutral pH but can undergo acid-induced unfolding reversibly. It has a thermodynamic stability energy (DeltaG degrees (N-U)) of 7.40 kcal/mol and is also active in binding to a Nir2 peptide with a K(D) of 0.65 muM. Further determination of its crystal structure reveals that it adopts a seven-strand immunoglobulin-like beta sandwich in which Pro56 adopts the unusual cis-peptide bond conformation that appears to be critical in maintaining the characteristic S-shaped loop. Markedly, the Pro56Ser mutation renders the MSP domain insoluble in buffer. Nevertheless, as facilitated by our recent discovery that "insoluble proteins" can be solubilized in salt-free water, we have successfully characterized the residue-specific conformation of the Pro56Ser mutant by CD and heteronuclear NMR spectroscopy. The Pro56Ser mutant remains lacking of the native tight packing and secondary structures under various conditions and was further characterized as having a non-native helical conformation weakly populated at pH 3.5. Intriguingly, Pro12 located in another S-shaped loop also adopts the cis-peptide bond conformation, and its mutation to Ser is able to make the MSP domain highly insoluble and unfolded like the Pro56Ser mutant. Our study thus implies that the Pro56Ser mutation might lead to ALS by eliminating the native MSP structure, which consequently leads to aggregation and loss of functions under physiological conditions.
Shi
Jiahai
J
Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore.
Lua
Shixiong
S
Tong
Justina Shihui
JS
Song
Jianxing
J
D000595:Amino Acid Sequence; D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013816:Thermodynamics; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
C496748:VAPB protein, human; D033921:Vesicular Transport Proteins