Intrinsic flexibility of <scp>NLRP</scp> pyrin domains is a key factor in their conformational dynamics, fold stability, and dimerization

Huber, Roland G.; Eibl, Clarissa; Fuchs, Julian E.

doi:10.1002/pro.2601

Cited by 25 publications

(35 citation statements)

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“…In 2014, we studied the binding stability of pathogenic ligands (imidozoquinoline and muramyl dipeptide) on NLRP3 LRR using molecular modeling, docking, and MD simulation approaches . Recently, Huber et al have described the intrinsic flexibility and behavior of NLRP14 PYD in different physiological condition . Wang and coauthors have studied the molecular switching of AIM2 PYD ‐helix‐α3 using a combined approach of NMR relaxation experiment and MD simulation …”

Section: Discussionmentioning

confidence: 78%

“…36 Recently, Huber et al have described the intrinsic flexibility and behavior of NLRP14 PYD in different physiological condition. 37 Wang and coauthors have studied the molecular switching of AIM2 PYD -helix-α3 using a combined approach of NMR relaxation experiment and MD simulation. 38 FIGURE 6 Illustration of conserved interactions between POP1 and NLRP3 PYD in A, type-II and B, type-III interfaces (acquired from Figures S4B and S5C, respectively) and their dynamic distances.…”

Section: Discussionmentioning

confidence: 99%

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POP1 might be recruiting its type‐Ia interface for NLRP3‐mediated PYD‐PYD interaction: Insights from MD simulation

Maharana

Vats

Gautam

et al. 2017

J of Molecular Recognition

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Inflammasomes are multiprotein caspase-activating complexes that enhance the maturation and release of proinflammatory cytokines (IL-1β and IL-18) in response to the invading pathogen and/or host-derived cellular stress. These are assembled by the sensory proteins (viz NLRC4, NLRP1, NLRP3, and AIM-2), adaptor protein (ASC), and effector molecule procaspase-1. In NLRP3-mediated inflammasome activation, ASC acts as a mediator between NLRP3 and procaspase-1 for the transmission of signals. A series of homotypic protein-protein interactions (NLRP3 :ASC and ASC :CASP1 ) propagates the downstream signaling for the production of proinflammatory cytokines. Pyrin-only protein 1 (POP1) is known to act as the regulator of inflammasome. It modulates the ASC-mediated inflammasome assembly by interacting with pyrin domain (PYD) of ASC. However, despite similar electrostatic surface potential, the interaction of POP1 with NLRP3 is obscured till date. Herein, to explore the possible PYD-PYD interactions between NLRP3 and POP1, a combined approach of protein-protein docking and molecular dynamics simulation was adapted. The current study revealed that POP1's type-Ia interface and type-Ib interface of NLRP3 might be crucial for 1:1 PYD-PYD interaction. In addition to type-I mode of interaction, we also observed type-II and type-III interaction modes in two different dynamically stable heterotrimeric complexes (POP1-NLRP3-NLRP3 and POP1-NLRP3-POP1). The inter-residual/atomic distance calculation exposed several critical residues that possibly govern the said interaction, which need further investigation. Overall, the findings of this study will shed new light on hitherto concealed molecular mechanisms underlying NLRP3-mediated inflammasome, which will have strong future therapeutic implications.

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Section: Discussionmentioning

confidence: 78%

Section: Discussionmentioning

confidence: 99%

POP1 might be recruiting its type‐Ia interface for NLRP3‐mediated PYD‐PYD interaction: Insights from MD simulation

Maharana

Vats

Gautam

et al. 2017

J of Molecular Recognition

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“…Furthermore, we characterized local dynamics of the backbone atoms of this residue by following a local alignment strategy (68). Additionally, we estimated dihedral entropies for all backbone torsions involving the conserved residue (69). Hydrogen bonds were extracted using cpptraj's default criteria.…”

Section: Methodsmentioning

confidence: 99%

Dynamics of an Active-Site Flap Contributes to Catalysis in a JAMM Family Metallo Deubiquitinase

et al. 2015

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The endosome-associated deubiquitinase (DUB) AMSH is a member of the JAMM family of zinc-dependent metallo isopeptidases with high selectivity for Lys63-linked polyubiquitin chains, which play a key role in endosomal-lysosomal sorting of activated cell surface receptors. The catalytic domain of the enzyme features a flexible flap near the active site that opens and closes during its catalytic cycle. Structural analysis of its homologs, AMSH-LP (AMSH-like protein) and the fission yeast counterpart, Sst2, suggest that a conserved Phe residue in the flap may be critical for substrate binding and/or catalysis. To gain insight into the contribution of this flap in substrate recognition and catalysis, we generated mutants of Sst2 and characterized them using a combination of enzyme kinetics, x-ray crystallography, molecular dynamics simulations, and isothermal titration calorimetry (ITC). Our analysis shows that the Phe residue in the flap contributes key interactions during the rate-limiting step but not to substrate binding, since mutants of Phe403 exhibit a defect only in kcat but not in KM. Moreover, ITC studies show Phe403 mutants have similar KD for ubiquitin compared to the wild-type enzyme. The x-ray structures of both Phe403Ala and the Trp mutant, in both the free and ubiquitin bound form, reveal no appreciable structural change that might impair substrate or alter product binding. We observed that the side chain of the Trp residue is oriented identically with respect to the isopeptide moiety of the substrate as the Phe residue in the wild-type enzyme, so the loss of activity seen in this mutant cannot be explained by the absence of a group with the ability to provide van der Waals interactions that facilitate the catalysis of the Lys63-linked diubiquitin. Molecular dynamics simulations indicate that the flap in the Trp mutant is quite flexible, allowing almost free rotation of the indole side chain. Therefore, it is possible that these different dynamic properties of the flap in the Trp mutant, compared to the wild-type enzyme, manifest as a defect in interactions that facilitate the rate-limiting step. Consistent with this notion, the Trp mutant was able to cleave Lys48-linked and Lys11-linked diubiquitin better than the wild-type enzyme, indicating altered mobility and hence reduced selectivity.

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“…We employed an extensive equilibration protocol 34 before performing unrestrained sampling simulations for 100 ns in NpT ensemble at 300 K and 1 bar. Additionally, we calculated thermodynamic entropies from the distributions of protein backbone torsion angles and summarized the three individual degrees of freedom to residue-wise and total entropies 36 Shorter sampling periods (i.e.…”

Section: Molecular Dynamics Simulationsmentioning

confidence: 99%

Experimental and computational evidence on conformational fluctuations as a source of catalytic defects in genetic diseases

et al. 2016

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Theoretical and experimental evidences have shown that protein function, regulation and degradation are intrinsicallylinked to the dynamic and fluctuating nature of protein ensembles. However, the effect of missense mutations on catalytic performance are often interpreted from conformational analyses derived from X-ray crystallography, molecular dynamics and modeling, while effects on conformational fluctuations at the active site as the source of catalytic defects are rarely investigated. Here, we explore the role of conformational fluctuations in the catalytic efficiency of WT and three missense mutations in the UDP-Galactose 4´-epimerase (GALE) protein causing type III galactosemia. Using comprehensive molecular dynamics simulations and small angle X-ray scattering we correlate low NAD+ binding affinity in some mutants with an increased population of non-competent conformations for NAD+ binding. Proteolysis studies combined with thermodynamic calculations reveal that mutations affecting GALE catalytic performance favor larger conformational fluctuations at the N-terminal domain and NAD+ binding site, shifting the equilibrium towards non-binding competent states in the native ensemble. Therefore, we provide a novel ensemble-based thermodynamic mechanism to explain catalytic defects caused by missense mutations that links large and transient conformational fluctuations and loss of catalytic efficiency and substrate/coenzyme affinity. In the context of this mechanism, we propose that allosteric ligands aimed at modulating these transient conformational fluctuations might correct catalytic defects in inherited metabolic diseases, representing a different approach to current small ligand therapies which target the low stability, but notcatalytic defects, of mutations

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Intrinsic flexibility of NLRP pyrin domains is a key factor in their conformational dynamics, fold stability, and dimerization

Cited by 25 publications

References 50 publications

POP1 might be recruiting its type‐Ia interface for NLRP3‐mediated PYD‐PYD interaction: Insights from MD simulation

POP1 might be recruiting its type‐Ia interface for NLRP3‐mediated PYD‐PYD interaction: Insights from MD simulation

Dynamics of an Active-Site Flap Contributes to Catalysis in a JAMM Family Metallo Deubiquitinase

Experimental and computational evidence on conformational fluctuations as a source of catalytic defects in genetic diseases

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