FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2984100769> ?p ?o ?g. }

• W2984100769 abstract "Full text Figures and data Side by side Abstract Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract The second messenger c-di-GMP (or cyclic diguanylate) regulates biofilm formation, a physiological adaptation process in bacteria, via a widely conserved signaling node comprising a prototypical transmembrane receptor for c-di-GMP, LapD, and a cognate periplasmic protease, LapG. Previously, we reported a structure-function study of a soluble LapD•LapG complex, which established conformational changes in the receptor that lead to c-di-GMP-dependent protease recruitment (Chatterjee et al., 2014). This work also revealed a basal affinity of c-di-GMP-unbound receptor for LapG, the relevance of which remained enigmatic. Here, we elucidate the structural basis of coincidence detection that relies on both c-di-GMP and LapG binding to LapD for receptor activation. The data indicate that high-affinity for LapG relies on the formation of a receptor dimer-of-dimers, rather than a simple conformational change within dimeric LapD. The proposed mechanism provides a rationale of how external proteins can regulate receptor function and may also apply to c-di-GMP-metabolizing enzymes that are akin to LapD. https://doi.org/10.7554/eLife.21848.001 Introduction Bacteria exist either as free swimming, planktonic cells or as multi-cellular biofilms — aggregates adhered to biotic or abiotic surfaces, which are enveloped in a self-produced matrix composed of proteins, nucleic acids, and polysaccharides (Hall-Stoodley et al., 2004; Teschler et al., 2015). This matrix protects bacteria from hostile environments, rendering them more resistant to insult and, in the context of pathogenic microbes, more tolerant to antibiotics and the immune system. The transition between planktonic and sessile lifestyles is tightly regulated, typically involving the second messenger c-di-GMP (or cyclic diguanylate) (Hengge, 2009; Hengge et al., 2016; Römling et al., 2005; Wolfe and Visick, 2008). The cellular c-di-GMP level is governed by two enzyme classes with opposing activities: GGDEF domain-containing diguanylate cyclases and EAL or HD-GYP domain-containing phosphodiesterases, which synthesize and degrade the dinucleotide, respectively (Schirmer and Jenal, 2009). A wide variety of specific, c-di-GMP-binding receptors translate the second messenger signal into physiological responses (Chou and Galperin, 2016; Sondermann et al., 2012). Bacterial genomes can encode a large number of c-di-GMP-signaling proteins and this number scales roughly with the organism’s adaptivity (Galperin, 2005). A particularly interesting observation is that, despite identical catalytic activities, c-di-GMP-metabolizing enzymes often confer distinct signaling outcomes (Abel et al., 2011; Dahlstrom et al., 2015, 2016; Ha et al., 2014; Kulasakara et al., 2006; Lindenberg et al., 2013; Newell et al., 2011a). The mechanisms underlying this apparent signaling specificity are not well understood. Recent work by our lab and others have focused on a conserved signaling system comprising the Lap operon that controls cell adhesion and biofilm formation in gammaproteobacteria, including Pseudomonas fluorescens (Chatterjee et al., 2014; Navarro et al., 2011; Newell et al., 2009, 2011b), P. aeruginosa (Cooley et al., 2016 Rybtke et al., 2015), P. putida (Gjermansen et al., 2010), Bordetella bronchiseptica (Ambrosis et al., 2016), and Shewanella oneidensis (Zhou et al., 2015) (Figure 1A). At its center, the inner membrane protein LapD functions as a receptor with degenerate GGDEF and EAL domains, which together relay intracellular c-di-GMP concentrations to the periplasm. At high c-di-GMP levels, LapD sequesters the adhesin protein-specific, periplasmic protease LapG at the inner membrane via the receptor’s periplasmic domain. This step ensures that large adhesin proteins whose transcription is activated by the dinucleotide remain stably associated with the outer cell membrane. When c-di-GMP levels drop and adhesin expression ceases, LapD undergoes a conformational change, adopting an autoinhibited state with low affinity for LapG; freed LapG, in turn, processes the adhesin proteolytically, weakening cell adhesion and ultimately contributing to biofilm dispersal (Chatterjee et al., 2014; Navarro et al., 2011; Newell et al., 2011b; Cooley et al., 2016; Rybtke et al., 2015; Borlee et al., 2010; Martínez-Gil et al., 2014; Monds et al., 2007). Notably, our previous work identified a transient, yet detectable interaction of LapG with c-di-GMP-unbound LapD, suggesting that the protease may participate in an early event of LapD signaling (Chatterjee et al., 2014). Curiously, saturation binding of LapG to LapD was markedly lower in the absence of c-di-GMP compared to levels when both ligands, c-di-GMP and LapG, were present. The functional relevance and mechanistic role of this interaction, however, remained poorly defined. Figure 1 with 3 supplements see all Download asset Open asset SEC-MALS reveals a switch of LapD dimers to dimer-of-dimers upon ligand binding. (A) Working model for c-di-GMP-dependent regulation of the periplasmic protease LapG via the inner membrane protein LapD. Concerted conformational changes expose a periplasmic binding site for LapG on LapD, sequestering the protease away from its substrates, the adhesin proteins LapA in P. fluorescens or CdrA in P. aeruginosa. (DGC, diguanylate cyclase; red/orange asterisks indicate interaction helices in LapD/GcbC). (B). Size-exclusion chromatograms for detergent-solubilized LapD in different states. Samples were prepared as described in the Material and Methods. (Asterisks: c-di-GMP was included in the mobile phase). (C) Molecular weight of LapD in solution. Peak fractions were analyzed by in-line SEC-MALS. (Absorbance at 280 nm: Traces colored according to (B); molecular weight determination: Dark and light purple dots; theoretical molecular weights based on sequence: Horizontal dashed lines.) Data are representative of two biological replicates using independent protein preparations. https://doi.org/10.7554/eLife.21848.002 In addition, a subsystem of diguanylate cyclases feeds specifically into the P. fluoresens LapD, indicating apparent signaling specificity between enzymes and receptors involved in c-di-GMP signal transduction (Newell et al., 2011a). At least one of these enzymes, GcbC, fulfills a distinct role in contributing an activation signal that relies on protein–protein interactions with LapD (Dahlstrom et al., 2015, 2016) (Figure 1A). Yet, our previous structural analysis of LapD indicated that a helical motif mediating pairwise interactions with GcbC was occluded in the autoinhibited state (Figure 1—figure supplement 1). Furthermore, modeling LapD as a simple dimer also suggested global steric incompatibility between these two transmembrane proteins (Figure 1A). Here, we focus on the molecular basis of switching of the purified, full-length c-di-GMP receptor LapD. These follow-up studies reveal an unanticipated role for LapG, together with c-di-GMP, in establishing the signaling-competent conformation of LapD by inducing higher-order oligomerization of the receptor. On the basis of the results, significant modifications to our model include coincidence detection of dinucleotide and protease as well as bidirectional signaling across the membrane as integral steps in LapD activation, with implications for the origins of transmembrane c-di-GMP signaling and for the regulation of LapD via heterologous interactions with diguanylate cyclases (Dahlstrom et al., 2015, 2016). Results Activation of full-length LapD results in quaternary structure changes Previously, we showed that LapD has a reduced binding capacity for LapG in the absence of c-di-GMP (Chatterjee et al., 2014). This observation was based on an equilibrium-binding assay at a fixed LapD concentration and with LapG as the titrant. To further confirm a quantitative difference between c-di-GMP-bound and -unbound LapD with regard to LapG affinity, we developed a fluorescence anisotropy-based assay, which relies on LapG that is fluorescently labeled at the sole cysteine residue in the protein’s active site (Figure 1—figure supplement 2). Titration of purified, detergent-solubilized LapD to a fixed concentration of fluorescent LapG yielded saturation-binding data, revealing an approximately 6-fold increase in LapG’s apparent affinity for LapD when c-di-GMP is present. Interestingly, the LapD titrations used here reached comparable maximum binding with and without c-di-GMP (Figure 1—figure supplement 2), in contrast to our previous assays in which LapG was the titrant which showed distinct maxima for LapD–LapG binding in the presence or absence of c-di-GMP (Chatterjee et al., 2014). The difference in binding observed when the fixed and titrated proteins are swapped suggests that LapG displays a constant, saturable binding site, whereas LapD’s binding capacity, and not only its affinity for LapG, is regulated by the dinucleotide. To begin investigating the structural underpinnings of LapD switching in the context of the intact receptor, we initially employed size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS). This approach enables assessment of the quaternary structure of detergent-solubilized, full-length LapD in response to c-di-GMP and LapG (Figure 1B and C, Figure 1—figure supplement 3). SEC-MALS is particularly advantageous for the study of membrane proteins because the respective scattering contributions of the protein and detergent components of the protein-detergent conjugate can be accurately deconvoluted to yield the molecular mass of the protein analyte (Gimpl et al., 2016) (Figure 1—figure supplement 3C). With this approach, purified apo-LapD was shown to be monodisperse and dimeric (Figure 1C, Figure 1—figure supplement 3C). Pre-incubation with LapG in the absence of c-di-GMP did not alter LapD’s elution time nor oligomerization state (Figure 1C), indicating that the interactions between the c-di-GMP-unbound receptor and the protease observed in saturation-binding assays ((Chatterjee et al., 2014); Figure 1—figure supplement 2) are likely transient. To probe how c-di-GMP influences LapD oligomerization, LapD was pre-incubated with dinucleotide and injected into the SEC-MALS system equilibrated with c-di-GMP in the mobile phase (Figure 1B). The LapD•c-di-GMP complex was predominantly dimeric, but a minor population of tetrameric LapD•c-di-GMP was also observed. Unexpectedly, when analyzed under identical conditions, the preformed LapG•LapD•c-di-GMP complex eluted predominantly as a higher-order species with a molecular weight corresponding to that of a LapD dimer-of-dimers (Figure 1C, Figure 1—figure supplement 3C). Omitting c-di-GMP in the mobile phase destabilized the complex, with LapD eluting as a dimeric species (Figure 1—figure supplement 3A and C). Together, these results provide compelling evidence that the LapG protease, together with c-di-GMP, plays a crucial role in the activation of LapD via induction of receptor oligomers. Cysteine crosslinking reveals multiple activation states of LapD The analysis described above suggests that native LapD can adopt at least three global states: (1) dimeric apo-LapD, (2) dimeric LapD•c-di-GMP, and (3) a LapD•c-di-GMP•LapG dimer-of-dimers complex. To characterize these states further, we turned to cysteine crosslinking, a time-honored approach to assess the conformation and dynamics of proteins with high spatial resolution (Bass et al., 2007; Butler and Falke, 1998). In this approach, a covalent crosslink forms upon mild oxidation, catalyzed by copper phenanthroline (Cu(Phen)2), only when two cysteine residues are in close spatial proximity (~2–3 Å). For visualization of crosslinked polypeptides in SDS-PAGE, we expressed LapD variants with a C-terminal monomeric superfolder-GFP (sfGFP) tag. Specific LapD–sfGFP detection in crude solubilized membrane fractions is based on in-gel fluorescence, with crosslinks introducing electrophoretic mobility shifts of the sfGFP-fusion protein. Furthermore, we mutated two native cysteine residues in the soluble domains of LapD (C304 and C397) to alanine and serine, respectively, to eliminate non-specific crosslinks. This LapD variant (‘TMcys’) retained the two native cysteine residues in the transmembrane helices (C12 and C158). Using a specific LapG photo-crosslinking assay (Chatterjee et al., 2014), we showed that LapD-TMcys was able to bind LapG in a c-di-GMP-dependent manner similar to wild-type LapD, as was a variant lacking all four native cysteine residues (‘cysless’) (Figure 2—figure supplement 1A). Site-directed cysteine mutations were introduced into the TMcys–sfGFP fusion background in order to analyze LapD conformational changes in different states of activation. One central strategy in our studies is based on the mutation A602C, which allows us to probe EAL domain dimerization and, in conjunction with S229C, signaling (S) helix–EAL domain contacts, reporting on active and autoinhibited LapD, respectively (Figure 2A). We confirmed by SEC-MALS analysis that LapD–sfGFP, LapDTMcys, and a representative variant with an engineered cysteine, LapDTMcys-A602C, formed constitutive dimers in their respective apo-states, similar to wild-type LapD, and that neither the sfGFP fusion nor cysteine mutagenesis perturbed the receptor notably (Figure 2—figure supplement 1B). Figure 2 with 1 supplement see all Download asset Open asset Cysteine crosslinking reveals distinct conformations of apo-, c-di-GMP, and c-di-GMP–LapG-bound LapD. (A) Overview of native and engineered cysteine residues used in crosslinking studies. The composite model of LapD shown is based on the available crystal structures of autoinhibited (top) and c-di-GMP-bound (bottom) domains. (B) Detection of intramolecular and intradimer disulfide bonds. Upper panel: LapDTMcys–sfGFP variants with the indicated cysteine mutation. Colored arrows and asterisks mark specific crosslinking bands. Only the band with grey marking could not be assigned unambiguously to a specific crosslinking sites. Note: The W125E variant of LapD migrates slightly slower than its native counterparts (also seen in Figure 2—figure supplement 1A). Lower panel: LapDTMcys–sfGFP variants with native transmembrane cysteine residues mutated. Experiments with individual protein variants (wild-type and mutants) were repeated at least three times using independent protein preparations. Primary data reproduced here are representative of each replicate. https://doi.org/10.7554/eLife.21848.006 Apo-LapD: only a fraction of apo-LapD protomers adopt the autoinhibited conformation at a given time We first probed the apo-state of LapD in which the cytoplasmic EAL domain is thought to engage with the S helix (Navarro et al., 2011) (Figure 2A). In the corresponding crystal structure, residues S229 of the S helix and A602 of the EAL domain of the same polypeptide chain are in close proximity. Hence, cysteine mutations that are introduced at these positions allow us to assess their propensity to support disulfide formation as a reporter for the autoinhibited conformation of LapD. Upon expressing LapDTMcys-S229C-A602C (fused to sfGFP), there is a low, but detectable level of spontaneous disulfide-bond formation in the absence of oxidant, which was sensitive to reducing agent (Figure 2B; red arrow/asterisk). Short copper-catalyzed oxidation (~10 min) yields crosslinks of only about half of apo-LapD protomers. This result suggests that conformational heterogeneity exists within apo-LapD and only a certain population of protomers adopt the autoinhibited state at a given time. c-di-GMP-bound LapD: dinucleotide binding leads to EAL domain dimerization and is accompanied by reorientation of transmembrane helices Addition of c-di-GMP to the S229C-A602C variant prior to copper oxidation abolishes the oxidant-dependent, intra-molecular crosslinking. Instead, new bands with slower electrophoretic mobility are observed (Figure 2B; blue and grey markings), which account for the entire LapD population (minus the spontaneously crosslinked fraction that is locked in the autoinhibited state prior to oxidation). One of these bands (blue marking) is rationalized as a homotypic crosslink between corresponding cysteine residues at position 602, as residue A602 is also central to the canonical EAL domain dimerization interface observed in the LapDEAL•c-di-GMP complex structure (Figure 2A, bottom) (Navarro et al., 2011). This interpretation is supported by the presence of a crosslinking adduct with comparable electrophoretic mobility using LapDTMcys-A602C, which lacks the S229C mutation, and by the absence of such a band in LapDTMcys, which lacks both S229C and A602C, despite otherwise identical experimental conditions (Figure 2B). We hypothesized that the origin of two other predominant bands with slower mobility in the LapDTMcys-S229C-A602C•c-di-GMP sample stems from movements in the transmembrane helices upon activation, which may orient the native cysteine residues C12 and/or C158 into crosslinking-competent positions. Indeed, the exposure of c-di-GMP-activated LapDTMcys to oxidant gave rise to two crosslinking adducts with slower electrophoretic mobility (green and magenta markings, Figure 2B). This observation indicates that the transmembrane domain cysteine residues contribute to the disulfide-mediated banding pattern in the presence of c-di-GMP. To identify explicitly the relevant crosslinks involving the transmembrane helices, we mutated these two native cysteine residues individually to alanine and subjected them to oxidative crosslinking. No crosslinked bands were observed in the case of LapDTMcys-C158A, whereas LapDTMcys-C12A supported a single crosslinking adduct (Figure 2B, bottom panel). Hence, the dominant adduct (green marking) pertains to covalent bonds between corresponding C158 residues in a LapD dimer, whereas the lower, weaker band (magenta marking) stems from a crosslink between C12 and C158 (Figure 2, bottom panel). Taken together, the shift to a LapD species with slower electrophoretic mobility upon incubation with c-di-GMP can, for the most part, be explained by cysteine crosslinking caused by movements in the transmembrane helices (green/magenta markings) and EAL domain dimerization (blue marking), as well as a subpopulation presumably containing multiple crosslinks (grey marking) (Figure 2B). LapD•c-di-GMP•LapG: LapG contributes to LapD activation by inducing distinct conformational changes Using A602C crosslinking as an analytical tool, we next probed the effect of LapG on the activation of LapD complexes. Notably, crosslinking via corresponding A602C residues in the LapD•c-di-GMP•LapG complex increases relative to the level observed in the absence of LapG (Figure 2B, top panel; blue markings). This apparent close proximity of A602C residues is indicative of the formation of canonical, c-di-GMP-dependent EAL domain dimers (Navarro et al., 2011; Barends et al., 2009; Minasov et al., 2009; Sundriyal et al., 2014). Concomitantly, LapG addition prevented C158–C158 (green marking) and C12–C158 (magenta marking) crosslinks between transmembrane helices, as well as other, higher molecular weight (gray marking) crosslinks (Figure 2B). Addition of LapG in the absence of c-di-GMP also causes a conformational change that is not compatible with C158–C158 crosslinking (Figure 2B, bottom panel), suggesting that LapG contributes to LapD structural transitions independently of c-di-GMP. Mutating the main anchor residue for LapG on LapD’s periplasmic domain binding, W125 (Chatterjee et al., 2014; Navarro et al., 2011; Chatterjee et al., 2012), abolished these LapG-induced effects on cysteine crosslinking but not those dependent on c-di-GMP (compare LapDTMcys-A602C and LapDTMcys-W125E-A602C; Figure 2B, top panel). This control establishes specificity and implicates the crystallographic LapD–LapG interface in LapG’s effect on LapD conformation. Together with the SEC-MALS data, these observations unequivocally demonstrate that the transition from dimeric LapD•c-di-GMP to tetrameric LapD•c-di-GMP•LapG is coincident with EAL domain dimerization. Interestingly, only ~50% of the EAL domains appear to be engaged in dimers, suggesting that like LapD in the autoinhibited state, activated LapD is based on asymmetric LapD dimers as the minimal unit. EAL domain dimerization mediates the formation of LapD dimer-of-dimers Our previous model assumed that EAL domain dimerization occurs within a LapD dimer (Figures 1A and 3A, left panel), a notion that needed to be revisited in light of the LapD dimer-of-dimers observed in the presence of LapG and c-di-GMP (Figure 1C). An alternative model consistent with the above and previous findings (Navarro et al., 2011) is one in which EAL domains bridge two LapD dimers rather than interacting within the same LapD dimer (Figure 3A, right panel). To distinguish between these two possibilities, we mixed LapD variants with distinct molecular weight, LapD–sfGFP and non-fluorescent LapD, which separately form constitutive dimers in their respective apo-state (Figure 2—figure supplement 1B). As seen before (e.g. Figure 2B), oxidation of LapDTMcys-A602C–sfGFP in the presence of c-di-GMP and LapG produced a crosslinking band that migrates just below the 250 kDa marker (Figure 3B; dark blue arrow). Addition of LapDTMcys lacking sfGFP prior to oxidation decreases crosslinking efficiency, indicating a competitive effect between LapDTMcys-A602C–sfGFP and LapDTMcys dimers. To unambiguously demonstrate that this competition is due to the formation of a LapD dimer-of-dimers, we replaced non-fluorescent LapDTMcys with non-fluorescent LapDTMcys-A602C. Upon oxidation, we observe two crosslinking bands that correspond to LapDTMcys-A602C–sfGFP and LapDTMcys-A602C–sfGFP•LapDTMcys-A602C dimers-of-dimers (dark and light blue arrow, respectively), for which the difference in molecular weight after SDS denaturation is roughly a single sfGFP moiety. Importantly, these specific crosslinking events occur readily with detergent-solubilized LapD at low protein concentration (~1 μM). Qualitatively similar results were obtained when proteins are embedded in a lipid bilayer of fused membrane fractions from cells separately expressing LapDTMcys-A602C–sfGFP or non-fluorescent LapDTMcys variants (see Material and Methods; Figure 3B). In summary, these data provide a structural and mechanistic basis for the dimer-of-dimers observed in the SEC-MALS data. Figure 3 Download asset Open asset LapD•c-di-GMP•LapG dimer-of-dimers also form in a membrane environment. (A) Two LapD constructs, one genetically fused to sfGFP and one non-fluorescent, both harboring the A602C mutation, were engineered and expressed separately. In previously proposed models, EAL domain dimerization was thought to occur within a single LapD dimer (left). Under this model, one would only expect a single band shift corresponding to c-di-GMP- and LapG-activated LapD–sfGFP dimers upon oxidation, even if the sfGFP-fused and non-fluorescent variants were present in the membrane. Alternatively, the EAL domains could dimerize across two LapD dimers (right) to form a dimer-of-dimers. Under the latter model, when the two constructs are mixed, activated with c-di-GMP and LapG, oxidized with a disulfide-promoting copper catalyst, denatured in SDS-PAGE, and imaged by in-gel fluorescence, a faster migrating covalent heterodimeric adduct consisting of LapD–sfGFP and LapD (dark) should be observed in addition to the slower migrating complex containing just LapD–sfGFP homodimeric adduct. (B) Complex formation is mediated by dimerization of EAL domains across two LapD dimers rather than within the same LapD dimer. The two LapD variants shown in (A) were expressed separately. Crosslinking via corresponding A602C residues was induced after incubation with c-di-GMP and LapG, either in detergent-solubilized samples (left panel) or upon fusing membrane fractions from the two cultures (right panel). In both detergent and membranes, SDS-PAGE analysis of this crosslinking experiment shows that a heterodimeric adduct containing both LapD–sfGFP and non-fluorescent LapD (lighter blue triangle; asterisk denotes residue from a non-fluorescent LapD) is observed in addition to a species containing only LapD–sfGFP (darker blue triangle). In detergent, non-fluorescent LapD lacking the A602C mutation serves as a competitor, reducing LapD-A602C–sfGFP crosslinking efficiency. Representative data from two independent, biological replicates are shown. https://doi.org/10.7554/eLife.21848.008 Modeling of small angle X-ray scattering (SAXS) data elucidates the conformational transitions upon LapD activation In order to obtain further structural evidence for LapD’s global conformational changes, we modeled SAXS data collected on detergent-solubilized LapD in four distinct states (Figure 4A; Figure 4—figure supplement 1): (1) apo-LapD trapped in the c-di-GMP-insensitive, fully autoinhibited conformation via an intramolecular crosslink between S229C and A602C (see Materials and Methods, Figure 2, Figure 2—figure supplement 1A, and Figure 4); (2) Apo-LapD; (3) LapD•c-di-GMP; (4) LapD•c-di-GMP•LapG. The first two states were stably monodisperse after purification, and could be analyzed directly. The two c-di-GMP-containing states, however, contained minor peaks based on our SEC-MALS data (Figure 1C). To avoid complications associated with such polydisperse samples and to capitalize on the observation that the predominant species could be separated by gel filtration, these samples were injected into an in-line size-exclusion chromatography system mirroring our SEC-MALS experiments. Guinier analysis of these samples collected on the SEC-SAXS setup (Malaby et al., 2015) revealed that they were monodisperse, and were therefore suitable for further analysis. Interpretation of the SAXS data was facilitated by having experimentally determined crystallographic structures of isolated domains of LapD and by the recently developed program MEMPROT, which models a detergent corona around transmembrane domains (Pérez and Koutsioubas, 2015). Figure 4 with 4 supplements see all Download asset Open asset Modeling of SAXS data for distinct LapD states illustrates the conformational changes upon receptor activation. (A) Primary SAXS data. Solid lines, experimental scattering curves of LapD in the states indicated; dashed lines, theoretical scattering curves of the three-dimensional envelopes shown in panel (C) with χ2 values listed to the right. (B) Real-space pair-wise distance distribution functions for each state of LapD. (C) Modeling of SAXS data. Top: Ab initio three-dimensional envelopes calculated on the basis of the experimental scattering data. Dotted circles and boxes highlight areas of density that change between different states. Middle: Crystal structures of individual domains of LapD docked manually into the envelopes depict interpretations of the ab initio envelope models. Gray spheres represent the detergent corona that surrounds the transmembrane domain. Bottom: Cartoon models of LapD domain movements in each state based on the SAXS data (bottom left inset: SDS-PAGE of purified trapped-inactive LapD used for SAXS analysis). Source files of SAXS data and envelope data are available in Figure 4—source data 2. https://doi.org/10.7554/eLife.21848.009 Figure 4—source data 1 Statistics associated with the analysis of LapD small-angle X-ray scattering data. All analyses were performed using the indicated programs included in the ATSAS 2.7.1 software package (Petoukhov et al., 2012). Rg, radius of gyration; Dmax, maximal particle dimension; Porod volume, volume of scattering particle; NSD, normalized spatial discrepancy; Rflex and Rsigma, as previously defined (Tria et al., 2015). https://doi.org/10.7554/eLife.21848.010 Download elife-21848-fig4-data1-v2.xlsx Figure 4—source data 2 SAXS data and envelope model files related to Figure 4. The zip archive contains buffer-subtracted scattering raw data (folder ‘raw_data’, extension ‘.dat’) and GNOM files (folder ‘gnom_output’, extension ‘.out’) that contain original data, real space distance distribution functions, and associated statistics. The folder ‘damfilt_pdb’ contains the final files (extension ‘.pdb’) of the envelope modeling. https://doi.org/10.7554/eLife.21848.011 Download elife-21848-fig4-data2-v2.zip Analysis of distance distribution functions suggests a slightly more compact structure of the trapped-inactive LapD with a measurably shorter Dmax and smaller Porod volume compared to native apo-LapD (Figure 4B, left panel; Figure 4—source data 1). These observations are reflected in ab initio three-dimensional envelopes in which the trapped-inactive LapD lacks the extended density toward the bottom of the molecule seen in native apo-LapD (Figure 4C, dotted circles). Manually docking individual LapD domains suggests that the trapped-inactive LapD envelope has dimensions consistent with both protomers' adopting the autoinhibited conformation that was observed in the crystal structure of this module. On the other hand, the native apo-LapD envelope is consistent with a heterogeneous state in which one protomer adopts the autoinhibited conformation w" @default.
• W2984100769 created "2019-11-22" @default.
• W2984100769 creator A5026956570 @default.
• W2984100769 creator A5028476200 @default.
• W2984100769 creator A5050599439 @default.
• W2984100769 date "2016-12-14" @default.
• W2984100769 modified "2023-09-27" @default.
• W2984100769 title "Author response: Coincidence detection and bi-directional transmembrane signaling control a bacterial second messenger receptor" @default.
• W2984100769 doi "https://doi.org/10.7554/elife.21848.018" @default.
• W2984100769 hasPublicationYear "2016" @default.
• W2984100769 type Work @default.
• W2984100769 sameAs 2984100769 @default.
• W2984100769 citedByCount "0" @default.
• W2984100769 crossrefType "peer-review" @default.
• W2984100769 hasAuthorship W2984100769A5026956570 @default.
• W2984100769 hasAuthorship W2984100769A5028476200 @default.
• W2984100769 hasAuthorship W2984100769A5050599439 @default.
• W2984100769 hasBestOaLocation W29841007691 @default.
• W2984100769 hasConcept C104317684 @default.
• W2984100769 hasConcept C105580179 @default.
• W2984100769 hasConcept C118892022 @default.
• W2984100769 hasConcept C142724271 @default.
• W2984100769 hasConcept C163235415 @default.
• W2984100769 hasConcept C170493617 @default.
• W2984100769 hasConcept C185592680 @default.
• W2984100769 hasConcept C204787440 @default.
• W2984100769 hasConcept C207579572 @default.
• W2984100769 hasConcept C24530287 @default.
• W2984100769 hasConcept C2779832538 @default.
• W2984100769 hasConcept C54355233 @default.
• W2984100769 hasConcept C62478195 @default.
• W2984100769 hasConcept C70721500 @default.
• W2984100769 hasConcept C71924100 @default.
• W2984100769 hasConcept C86803240 @default.
• W2984100769 hasConcept C95444343 @default.
• W2984100769 hasConceptScore W2984100769C104317684 @default.
• W2984100769 hasConceptScore W2984100769C105580179 @default.
• W2984100769 hasConceptScore W2984100769C118892022 @default.
• W2984100769 hasConceptScore W2984100769C142724271 @default.
• W2984100769 hasConceptScore W2984100769C163235415 @default.
• W2984100769 hasConceptScore W2984100769C170493617 @default.
• W2984100769 hasConceptScore W2984100769C185592680 @default.
• W2984100769 hasConceptScore W2984100769C204787440 @default.
• W2984100769 hasConceptScore W2984100769C207579572 @default.
• W2984100769 hasConceptScore W2984100769C24530287 @default.
• W2984100769 hasConceptScore W2984100769C2779832538 @default.
• W2984100769 hasConceptScore W2984100769C54355233 @default.
• W2984100769 hasConceptScore W2984100769C62478195 @default.
• W2984100769 hasConceptScore W2984100769C70721500 @default.
• W2984100769 hasConceptScore W2984100769C71924100 @default.
• W2984100769 hasConceptScore W2984100769C86803240 @default.
• W2984100769 hasConceptScore W2984100769C95444343 @default.
• W2984100769 hasLocation W29841007691 @default.
• W2984100769 hasOpenAccess W2984100769 @default.
• W2984100769 hasPrimaryLocation W29841007691 @default.
• W2984100769 hasRelatedWork W148421061 @default.
• W2984100769 hasRelatedWork W1603274157 @default.
• W2984100769 hasRelatedWork W2030655819 @default.
• W2984100769 hasRelatedWork W2124232297 @default.
• W2984100769 hasRelatedWork W2141891129 @default.
• W2984100769 hasRelatedWork W2270573517 @default.
• W2984100769 hasRelatedWork W2412992299 @default.
• W2984100769 hasRelatedWork W3092561524 @default.
• W2984100769 hasRelatedWork W3147082385 @default.
• W2984100769 hasRelatedWork W2125157906 @default.
• W2984100769 isParatext "false" @default.
• W2984100769 isRetracted "false" @default.
• W2984100769 magId "2984100769" @default.
• W2984100769 workType "peer-review" @default.