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

Master'sMASTER OF SCIENC

Dynamically-Driven Inactivation of the Catalytic Machinery of the SARS 3C-Like Protease by the N214A Mutation on the Extra Domain

Despite utilizing the same chymotrypsin fold to host the catalytic machinery, coronavirus 3C-like proteases (3CLpro) noticeably differ from picornavirus 3C proteases in acquiring an extra helical domain in evolution. Previously, the extra domain was demonstrated to regulate the catalysis of the SARS-CoV 3CLpro by controlling its dimerization. Here, we studied N214A, another mutant with only a doubled dissociation constant but significantly abolished activity. Unexpectedly, N214A still adopts the dimeric structure almost identical to that of the wild-type (WT) enzyme. Thus, we conducted 30-ns molecular dynamics (MD) simulations for N214A, WT, and R298A which we previously characterized to be a monomer with the collapsed catalytic machinery. Remarkably, three proteases display distinctive dynamical behaviors. While in WT, the catalytic machinery stably retains in the activated state; in R298A it remains largely collapsed in the inactivated state, thus implying that two states are not only structurally very distinguishable but also dynamically well separated. Surprisingly, in N214A the catalytic dyad becomes dynamically unstable and many residues constituting the catalytic machinery jump to sample the conformations highly resembling those of R298A. Therefore, the N214A mutation appears to trigger the dramatic change of the enzyme dynamics in the context of the dimeric form which ultimately inactivates the catalytic machinery. The present MD simulations represent the longest reported so far for the SARS-CoV 3CLpro, unveiling that its catalysis is critically dependent on the dynamics, which can be amazingly modulated by the extra domain. Consequently, mediating the dynamics may offer a potential avenue to inhibit the SARS-CoV 3CLpro

Structural, Stability, Dynamic and Binding Properties of the ALS-Causing T46I Mutant of the hVAPB MSP Domain as Revealed by NMR and MD Simulations

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

Interaction between EphA4 and WT-/T46I-MSP domain.

<p>Superimposition of the two-dimensional ¹H-¹⁵N NMR HSQC spectra of the EphA4 ligand binding domain in the absence (blue) and presence of the WT- (a) and T46I- (c) MSP domains at molar ratios 1∶2 (green) and 1∶8 (red) (EphA4/MSP). Red letters are used to label residues with disappeared HSQC peaks while blue for residues with shifted peaks. Crystal structure of the EphA4 ligand-binding domain we previously determined (<i>37</i>) with the perturbed residues mapped back for the interaction between EphA4 and WT-MSP (b), and T46I-MSP (d). Blue is to indicate residues with unperturbed HSQC peaks, while green and red for residues with shifted and disappeared peaks respectively.</p

Trajectories of MD simulations.

<p>(a–b). Root-mean-square deviations (RMSD) of the backbone atoms for two independent MD simulations of the WT- (blue) and T46I- (red) MSP domains. (c–d). Root-mean-square fluctuations (RMSF) of the Cα atoms computed for two independent simulations (blue for simulation 1 and red for simulation 2) of the WT- (c) and T46I- (d) MSP domains. (e–f). Root-mean-square fluctuations (RMSF) of the Cα atoms computed for two independent simulations for the WT- (blue) and T46I- (red) MSP domains. The average values and standard deviations over 15 ns are computed and displayed. The green arrows are used to indicate the T46I-MSP residues with fluctuations larger than those of the WT-MSP domain.</p

Structural characterization of the T46I-MSP domain.

<p>(a). Far UV CD spectra of the WT- (blue), T46I- (red) and P56S- (black) MSP domains at protein concentrations of 20 µM. (b). Superimposition of the two-dimensional ¹H-¹⁵N NMR HSQC spectra of the WT- (blue) and T46I- (red) MSP domains at protein concentrations of 100 µM at pH 6.8. Cα (c) and Cβ (d) chemical shifts of the WT- (blue) and T46I- (red) MSP domains. The residues with the disappeared HSQC peaks are indicated by green arrows. (e). The crystal structure of the WT-MSP domain we previously determined (32) to which the T46I residues with their HSQC peaks undetected were mapped. The two mutation residues causing ALS, P56S and T46I, were displayed as spheres.</p

Interaction between the WT-/T46I-MSP domains and EphA4.

<p>(a). The ITC titration profile of the binding reaction of the WT-MSP domain to the ligand-binding domain of EphA4 (upper panel); and integrated values for reaction heats with subtraction of the corresponding blank results normalized by the amount of ligand injected versus molar ratio of MSP/EphA4 (lower panel). Superimposition of the two dimensional ¹H-¹⁵N NMR HSQC spectra of the WT- (b) and T46I- (d) MSP domains in the absence (blue) and presence of the EphA4 ligand binding domain at molar ratios 1∶2 (green) and 1∶8 (red) (MSP/EphA4). Red letters are used to label residues with disappeared HSQC peaks while blue for residues with shifted HSQC peaks. The MSP structure with the perturbed residues mapped back for the interactions between the WT-MSP and EphA4 (c), and the T46I-MSP and EphA4 (e). Blue is to indicate residues with unperturbed HSQC peaks, while green and red for residues with shifted and disappeared HSQC peaks respectively.</p

Stability of the T46I-MSP domain.

<p>(a). The urea-induced unfolding curves of the WT- (blue) and T46I- (red) MSP domains as reflected by changes of the ellipticity at 222 nm with urea concentrations ranging from 0 to 8 M. One-dimensional NMR spectra of the WT- (b) and T46I- (c) MSP domains at 15°C (green) 25°C (blue) and 45°C (red). (d). Hydrogen-deuterium (H/D) exchange results for the WT-MSP domain. Blue bars: residues with HSQC peaks detectable in the 10 mM phosphate buffer at pH 6.8. Green bars: residues with HSQC peaks detectable 15 min after re-dissolving the lyophilized protein powder in D₂O. Red bars: residues with HSQC peaks detectable 2.5 hr after re-dissolving the lyophilized protein powder in D₂O. (e). The crystal structure of the MSP domain with the H/D exchange results mapped. The yellow is to indicate the residues undetectable even in the 10 mM phosphate buffer at pH 6.8.</p