ASC filament formation serves as a signal amplification mechanism for inflammasomes

A hallmark of inflammasome activation is the ASC speck, a micrometre-sized structure formed by the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), which consists of a pyrin domain (PYD) and a caspase recruitment domain (CARD). Here we show that assembly of the ASC speck involves oligomerization of ASC(PYD) into filaments and cross-linking of these filaments by ASC(CARD). ASC mutants with a non-functional CARD only assemble filaments but not specks, and moreover disrupt endogenous specks in primary macrophages. Systematic site-directed mutagenesis of ASC(PYD) is used to identify oligomerization-deficient ASC mutants and demonstrate that ASC speck formation is required for efficient processing of IL-1β, but dispensable for gasdermin-D cleavage and pyroptosis induction. Our results suggest that the oligomerization of ASC creates a multitude of potential caspase-1 activation sites, thus serving as a signal amplification mechanism for inflammasome-mediated cytokine production

Chaperone-Bound Clients: The Importance of Being Dynamic

Several recent atomic-resolution studies have resolved how chaperones interact with their client proteins. In some cases, molecular chaperones recognize and bind their clients in conformational ensembles that are locally highly dynamic and interconvert, while in other cases clients bind in unique conformations. The presence of a locally dynamic client ensemble state has important consequences, both for the interpretation of experimental data and for the functionality of chaperones, as local dynamics facilitate rapid client release, folding on and from the chaperone surface, and client recognition without shape complementarity. Facilitated by the local dynamics, at least some chaperones appear to specifically recognize energetically frustrated sites of partially folded client proteins, such that the release of frustration contributes to the interaction affinity

Molecular chaperones and their denaturing effect on client proteins

Advanced NMR methods combined with biophysical techniques have recently provided unprecedented insight into structure and dynamics of molecular chaperones and their interaction with client proteins. These studies showed that several molecular chaperones are able to dissolve aggregation-prone polypeptides in aqueous solution. Furthermore, chaperone-bound clients often feature fluid-like backbone dynamics and chaperones have a denaturing effect on clients. Interestingly, these effects that chaperones have on client proteins resemble the effects of known chaotropic substances. Following this analogy, chaotropicity could be a fruitful concept to describe, quantify and rationalize molecular chaperone function. In addition, the observations raise the possibility that at least some molecular chaperones might share functional similarities with chaotropes. We discuss these concepts and outline future research in this direction

Chaperones and chaperone-substrate complexes: dynamic playgrounds for NMR spectroscopists

The majority of proteins depend on a well-defined three-dimensional structure to obtain their functionality. In the cellular environment, the process of protein folding is guided by molecular chaperones to avoid misfolding, aggregation, and the generation of toxic species. To this end, living cells contain complex networks of molecular chaperones, which interact with substrate polypeptides by a multitude of different functionalities: transport them towards a target location, help them fold, unfold misfolded species, resolve aggregates, or deliver them towards a proteolysis machinery. Despite the availability of high-resolution crystal structures of many important chaperones in their substrate-free apo forms, structural information about how substrates are bound by chaperones and how they are protected from misfolding and aggregation is very sparse. This lack of information arises from the highly dynamic nature of chaperone-substrate complexes, which so far has largely hindered their crystallization. This highly dynamic nature makes chaperone-substrate complexes good targets for NMR spectroscopy. Here, we review the results achieved by NMR spectroscopy to understand chaperone function in general and details of chaperone-substrate interactions in particular. We assess the information content and applicability of different NMR techniques for the characterization of chaperones and chaperone-substrate complexes. Finally, we highlight three recent studies, which have provided structural descriptions of chaperone-substrate complexes at atomic resolution

Solution NMR studies of membrane-protein-chaperone complexes

The biosynthesis of the bacterial outer membrane depends on molecular chaperones that protect hydrophobic membrane proteins against aggregation while transporting them across the periplasm. In our ongoing research, we use high-resolution NMR spectroscopy in aqueous solution as the main technique to characterize the structures and biological functions of these membrane-protein-chaperone complexes. Here, we describe NMR studies addressing three functional aspects of periplasmic membrane-protein-chaperone complexes. Firstly, the Escherichia coli outer membrane protein OmpX binds to each of the two chaperones, Skp and SurA, in structurally at least partially similar states despite fundamental differences between the three-dimensional structures of the chaperones. Secondly, we show that the Skp-bound state of OmpX is equivalent to a chemically denatured state in terms of its refolding competence into detergent micelles in vitro. Thirdly, we use amino acid mutation analysis to show that the interaction of OmpX to Skp is not dominated by the two most hydrophobic segments of OmpX

APSY-NMR with proteins: practical aspects and backbone assignment

Automated projection spectroscopy (APSY) is an NMR technique for the recording of discrete sets of projection spectra from higher-dimensional NMR experiments, with automatic identification of the multidimensional chemical shift correlations by the dedicated algorithm GAPRO. This paper presents technical details for optimizing the set-up and the analysis of APSY-NMR experiments with proteins. Since experience so far indicates that the sensitivity for signal detection may become the principal limiting factor for applications with larger proteins or more dilute samples, we performed an APSY-NMR experiment at the limit of sensitivity, and then investigated the effects of varying selected experimental parameters. To obtain the desired reference data, a 4D APSY-HNCOCA experiment with a 12-kDa protein was recorded in 13min. Based on the analysis of this data set and on general considerations, expressions for the sensitivity of APSY-NMR experiments have been generated to guide the selection of the projection angles, the calculation of the sweep widths, and the choice of other acquisition and processing parameters. In addition, a new peak picking routine and a new validation tool for the final result of the GAPRO spectral analysis are introduced. In continuation of previous reports on the use of APSY-NMR for sequence-specific resonance assignment of proteins, we present the results of a systematic search for suitable combinations of a minimal number of four- and five-dimensional APSY-NMR experiments that can provide the input for algorithms that generate automated protein backbone assignment

Residual structure in a peptide fragment of the outer membrane protein X under denaturing conditions: a molecular dynamics study

The Escherichia coli outer membrane protein X (OmpX) contains two polypeptide segments that present nonrandom residual structure in 8M aqueous urea, whereas the remainder of the protein is in a flexibly disordered conformation (Tafer et al. in Biochemistry 43:860-869, 2004). In the present study, the results of two long-timescale (0.4μs) unrestrained explicit-solvent molecular dynamics (MD) simulations of a tetradecapeptide representative of one of these two segments in 8M aqueous urea are reported and analyzed. The two simulations were initiated either from the conformation of the corresponding segment in an NMR model structure of the unfolded protein or from an entirely extended configuration. The sampled conformational ensembles agree qualitatively with the experimentally observed NOEs, but not quantitatively, suggesting that a number of relevant configurations were not visited on the 2×0.4μs timescale. Major conformational transitions occur on the 0.1μs timescale, and the ensembles corresponding to the two independent simulations overlap only to a limited extent. However, both simulations show in multiple events the reversible formation and disruption of α-helical secondary structure (characteristic of the urea-denatured state) and β-turn secondary structure (characteristic of the native state). Events of helix formation are correlated with the appearance of hydrogen bonds between two side chains (Asp75-Ser78) and of a persistent hydrophobic contact (Trp76-Tyr80). They also evidence a peculiar helix stabilization and N-terminal capping role for a negatively charged residue (Asp75). These features are in good qualitative agreement with the NMR model for the structured state of the corresponding segment in the urea-denatured protein. The analysis of the simulations provides a detailed picture of the structural and dynamic features of the considered peptide at atomic resolution that is of high relevance in the understanding of the OmpX folding proces

4D APSY-HBCB(CG)CDHD experiment for automated assignment of aromatic amino acid side chains in proteins

A four-dimensional (4D) APSY (automated projection spectroscopy)-HBCB(CG)CDHD experiment is presented. This 4D experiment correlates aromatic with aliphatic carbon and proton resonances from the same amino acid side chain of proteins in aqueous solution. It thus allows unambiguous sequence-specific assignment of aromatic amino acid ring signals based on backbone assignments. Compared to conventional 2D approaches, the inclusion of evolution periods on 1Hβ and 13Cδ efficiently removes overlaps, and provides two additional frequencies for consequent automated or manual matching. The experiment was successfully applied to three proteins with molecular weights from 6 to 13kDa. For the complementation of the assignment of the aromatic resonances, TOCSY- or COSY-based versions of a 4D APSY-HCCHaro sequence are propose

Redefining Molecular Chaperones as Chaotropes

Molecular chaperones are the key instruments of bacterial protein homeostasis. Chaperones not only facilitate folding of client proteins, but also transport them, prevent their aggregation, dissolve aggregates and resolve misfolded states. Despite this seemingly large variety, single chaperones can perform several of these functions even on multiple different clients, thus suggesting a single biophysical mechanism underlying. Numerous recently elucidated structures of bacterial chaperone-client complexes show that dynamic interactions between chaperones and their client proteins stabilize conformationally flexible non-native client states, which results in client protein denaturation. Based on these findings, we propose chaotropicity as a suitable biophysical concept to rationalize the generic activity of chaperones. We discuss the consequences of applying this concept in the context of ATP-dependent and -independent chaperones and their functional regulation

Automated Resonance Assignment of Proteins: 6 DAPSY-NMR

The 6-dimensional(6D) APSY-seq-HNCOCANH NMR experiment correlates two sequentially neighbor in gamidemoieties in proteins via the C′ and Cα nuclei, with efficient suppression of the back transfer from Cα to the originating amidemoiety. The automatic analysis of two-dimensional(2D) projections of this 6D experiment with the use of GAPRO (Hilleretal., 2005) provides a high-precision 6D peak list, which permits automated sequential assignments of proteins with the assignment software GARANT (Bartels et al., 1997). The procedure was applied to two proteins, the 63-residue 434-repressor(1-3) and the 115-residue TM1290. For both proteins, complete sequential assignments for all NMR-observable backbone resonances were obtained, and the polypeptide segments thus identified could be unambiguously located in the amino acid sequence. These results demonstrate that APSY-NMR spectroscopy in combination with a suitable assignment algorithm can provide fully automated sequence-specific backbone assignments of small protein