Abstract
T46I is the second mutation on the hVAPB MSP domain which was recently identified from non-Brazilian kindred to cause a familial amyotrophic lateral sclerosis (ALS). Here using CD, NMR and molecular dynamics (MD) simulations, we characterized the structure, stability, dynamics and binding capacity of the T46I-MSP domain. The results reveal: 1) unlike P56S which we previously showed to completely eliminate the native MSP structure, T46I leads to no significant disruption of the native secondary and tertiary structures, as evidenced from its far-UV CD spectrum, as well as Cα and Cβ NMR chemical shifts. 2) Nevertheless, T46I does result in a reduced thermodynamic stability and loss of the cooperative urea-unfolding transition. As such, the T46I-MSP domain is more prone to aggregation than WT at high protein concentrations and temperatures in vitro, which may become more severe in the crowded cellular environments. 3) T46I only causes a 3-fold affinity reduction to the Nir2 peptide, but a significant elimination of its binding to EphA4. 4) EphA4 and Nir2 peptide appear to have overlapped binding interfaces on the MSP domain, which strongly implies that two signaling networks may have a functional interplay in vivo. 5) As explored by both H/D exchange and MD simulations, the MSP domain is very dynamic, with most loop residues and many residues on secondary structures highly fluctuated or/and exposed to bulk solvent. Although T46I does not alter overall dynamics, it does trigger increased dynamics of several local regions of the MSP domain which are implicated in binding to EphA4 and Nir2 peptide. Our study provides the structural and dynamic understanding of the T46I-causing ALS; and strongly highlights the possibility that the interplay of two signaling networks mediated by the FFAT-containing proteins and Eph receptors may play a key role in ALS pathogenesis.
Highlights
Amyotrophic lateral sclerosis (ALS) is the most prevalent fatal motor neuron disease, which affects people of all race and ethnic background
By extensive CD, NMR characterization and molecular dynamics (MD) simulations, we found that the T46I-major sperm protein (MSP) domain still retains the native MSP structure with overall dynamics largely unaltered
A large portion of the T46I protein existed in inclusion body, there was a small portion of the soluble T46I form in supernatant
Summary
Amyotrophic lateral sclerosis (ALS) is the most prevalent fatal motor neuron disease, which affects people of all race and ethnic background. ALS8 was identified from a large Brazilian family with dominant motor neuron diseases, which encodes a mutated VAPB (vesicle-associated membrane protein-associated protein B). The missense P56S point mutation results in a typical ALS phenotype with rapid progression or late onset spinal muscular atrophy [2,3,4,5]. Very recently, another mutation T46I on VAPB has been unraveled to cause ALS in non-Brazilian kindred [6]. The human VAP family proteins were originally identified as homologues of vesicle-associated membrane protein (VAMP)associated protein (VAP) in Aplysia californica (aVAP33) [7,8], which include VAPA, VAPB, and VAPC. The VAP proteins interact with intracellular proteins [19,20] including Nir, Nir, and Nir via the FFAT motif which differentially affects the organization of the ER [11]
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