Molecular Mechanism Underlying the Thermal Stability and pH-induced Unfolding of CHABII

Zheng, Wei; Song, Jianxing

doi:10.1016/j.jmb.2005.02.028

Cited by 40 publications

(47 citation statements)

References 78 publications

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“…However, it seems that if at pH 4.0, the NS2B-NS3pro complex exists as an intermediate which already has substantial secondary structures and tertiary packing. However, the tight tertiary packing is not completely achieved and consequently the complex undergoes μs-ms conformational exchanges, very similar to what we previously observed on a molten globule formed by a small protein at pH 4.0 [39,40]. The intermediate of the NS2B-NS3pro complex at pH 4.0 is enzymatically inactive, but nevertheless, once it is transferred into the buffer at pH 7.5, the tightly-packed structure will immediately form and the complex can reach the fully active state.…”

Section: Structure and Activity Of The Refolded Ns2b-ns3pro Complexessupporting

confidence: 81%

NMR and MD Studies Reveal That the Isolated Dengue NS3 Protease Is an Intrinsically Disordered Chymotrypsin Fold Which Absolutely Requests NS2B for Correct Folding and Functional Dynamics

2015

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Dengue genome encodes a two component protease complex (NS2B-NS3pro) essential for the viral maturation/infectivity, thus representing a key drug target. Previously, due to its “complete insolubility”, the isolated NS3pro could not be experimentally studied and it remains elusive what structure it adopts without NS2B and why NS2B is indispensable. Here as facilitated by our previous discovery, the isolated NS3pro has been surprisingly deciphered by NMR to be the first intrinsically-disordered chymotrypsin-like fold, which exists in a loosely-packed state with non-native long-range interactions as revealed by paramagnetic relaxation enhancement (PRE). The disordered NS3pro appears to be needed for binding a human host factor to trigger the membrane remodeling. Moreover, we have in vitro refolded the NS3pro in complex with either NS2B (48–100) or the full-length NS2B (1–130) anchored into the LMPC micelle, and the two complexes have similar activities but different dynamics. We also performed molecular dynamics (MD) simulations and the results revealed that NS2B shows the highest structural fluctuations in the complex, thus providing the dynamic basis for the observation on its conformational exchange between open and closed states. Remarkably, the NS2B cofactor plays a central role in maintaining the correlated motion network required for the catalysis as we previously decoded for the SARS 3CL protease. Indeed, a truncated NS2B (48–100;Δ77–84) with the flexible loop deleted is able to trap the NS2B-NS3pro complex in a highly dynamic and catalytically-impotent state. Taken together, our study implies potential strategies to perturb the NS2B-NS3pro interface for design of inhibitors for treating dengue infection.

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Section: Structure and Activity Of The Refolded Ns2b-ns3pro Complexessupporting

confidence: 81%

NMR and MD Studies Reveal That the Isolated Dengue NS3 Protease Is an Intrinsically Disordered Chymotrypsin Fold Which Absolutely Requests NS2B for Correct Folding and Functional Dynamics

2015

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“…The loss of a cooperative unfolding transition of T46I implies that its tight tertiary packing is disrupted to some degree [49], [50] and consequently the T46I molecule may be more accessible to partially- or highly unfolded states prone to aggregation. Indeed, we observed that aggregation occurred for T46I at high protein concentrations and temperatures.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2011

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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.

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“…The pH ranges at which these transitions occur suggest that the protonation of His side chains may be involved. Indeed, it has been shown that His residues may function as pH sensors for many molecular mechanisms involving protein-membrane interaction (11,12) and/or MG formation (13,14).…”

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confidence: 99%

Concerted Protonation of Key Histidines Triggers Membrane Interaction of the Diphtheria Toxin T Domain

Perier

Chassaing

Raffestin

et al. 2007

Journal of Biological Chemistry

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The translocation domain (T domain) of the diphtheria toxin contributes to the transfer of the catalytic domain from the cell endosome to the cytosol, where it blocks protein synthesis. Translocation is initiated when endosome acidification induces the interaction of the T domain with the membrane of the compartment. We found that the protonation of histidine side chains triggers the conformational changes required for membrane interaction. All histidines are involved in a concerted manner, but none is indispensable. However, the preponderance of each histidine varies according to the transition observed. The pair His 223 -His 257 and His 251 are the most sensitive triggers for the formation of the molten globule state in solution, whereas His 322 -His 323 and His 251 are the most sensitive triggers for membrane binding. Interestingly, the histidines are located at key positions throughout the structure of the protein, in hinges and at the interface between each of the three layers of helices forming the domain. Their protonation induces local destabilizations, disrupting the tertiary structure and favoring membrane interaction. We propose that the selection of histidine residues as triggers of membrane interaction enables the T domain to initiate translocation at the rather mild pH found in the endosome, contributing to toxin efficacy.

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Molecular Mechanism Underlying the Thermal Stability and pH-induced Unfolding of CHABII

Cited by 40 publications

References 78 publications

NMR and MD Studies Reveal That the Isolated Dengue NS3 Protease Is an Intrinsically Disordered Chymotrypsin Fold Which Absolutely Requests NS2B for Correct Folding and Functional Dynamics

NMR and MD Studies Reveal That the Isolated Dengue NS3 Protease Is an Intrinsically Disordered Chymotrypsin Fold Which Absolutely Requests NS2B for Correct Folding and Functional Dynamics

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

Concerted Protonation of Key Histidines Triggers Membrane Interaction of the Diphtheria Toxin T Domain

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