Summary Several recent studies describe the influence of the gut microbiota on host brain and behavior. However, the mechanisms responsible for microbiota-nervous system interactions are unknown. Using a combination of genetics, biochemistry, and crystallography, we identify and characterize two phylogenetically distinct enzymes found in the human microbiome that decarboxylate tryptophan to form the β-arylamine neurotransmitter tryptamine. Although this enzymatic activity is exceedingly rare among bacteria more broadly, analysis of the Human Microbiome Project data demonstrates that at least 10% of the human population harbors at least one bacterium encoding a tryptophan decarboxylase in their gut community. Our results uncover a previously unrecognized enzymatic activity that can give rise to host-modulatory compounds and suggests a potential direct mechanism by which gut microbiota can influence host physiology, including behavior.
Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.DOI: http://dx.doi.org/10.7554/eLife.07574.001
X-ray diffraction from protein crystals includes both sharply peaked Bragg reflections and diffuse intensity between the peaks. The information in Bragg scattering is limited to what is available in the mean electron density. The diffuse scattering arises from correlations in the electron density variations and therefore contains information about collective motions in proteins. Previous studies using molecular-dynamics (MD) simulations to model diffuse scattering have been hindered by insufficient sampling of the conformational ensemble. To overcome this issue, we have performed a 1.1-μs MD simulation of crystalline staphylococcal nuclease, providing 100-fold more sampling than previous studies. This simulation enables reproducible calculations of the diffuse intensity and predicts functionally important motions, including transitions among at least eight metastable states with different active-site geometries. The total diffuse intensity calculated using the MD model is highly correlated with the experimental data. In particular, there is excellent agreement for the isotropic component of the diffuse intensity, and substantial but weaker agreement for the anisotropic component. Decomposition of the MD model into protein and solvent components indicates that protein-solvent interactions contribute substantially to the overall diffuse intensity. We conclude that diffuse scattering can be used to validate predictions from MD simulations and can provide information to improve MD models of protein motions.diffuse scattering | protein crystallography | molecular-dynamics simulation | protein dynamics | staphylococcal nuclease P roteins explore many conformations while carrying out their functions in biological systems (1-3). X-ray crystallography is the dominant source of information about protein structure; however, crystal structure models usually consist of just a single major conformation and at most a small portion of the model as alternate conformations. Crystal structures therefore are missing many details about the underlying conformational ensemble (4).Proteins assembled in crystalline arrays, like proteins in solution, exhibit rich conformational diversity (4) and often can perform their native functions (5). Many methods have emerged for using Bragg data to model conformational diversity in protein crystals (6-17). The development of these methods has been important as conformational diversity can lead to inaccuracies in protein structure models (9,(18)(19)(20). A key limitation of using the Bragg data, however, is that different models of conformational diversity can yield the same mean electron density.Whereas the Bragg scattering only contains information about the mean electron density, diffuse scattering (diffraction resulting in intensity between the Bragg peaks) is sensitive to spatial correlations in electron density variations (21-28) and therefore contains information about the way that atomic positions vary together in protein crystals. Because models that yield the same mean electron density c...
X-ray diffraction has the potential to provide rich information about the structural dynamics of macromolecules. To realize this potential, both Bragg scattering, which is currently used to derive macromolecular structures, and diffuse scattering, which reports on correlations in charge density variations, must be measured. Until now, measurement of diffuse scattering from protein crystals has been scarce because of the extra effort of collecting diffuse data. Here, we present 3D measurements of diffuse intensity collected from crystals of the enzymes cyclophilin A and trypsin. The measurements were obtained from the same X-ray diffraction images as the Bragg data, using best practices for standard data collection. To model the underlying dynamics in a practical way that could be used during structure refinement, we tested translation-libration-screw (TLS), liquid-like motions (LLM), and coarse-grained normal-modes (NM) models of protein motions. The LLM model provides a global picture of motions and was refined against the diffuse data, whereas the TLS and NM models provide more detailed and distinct descriptions of atom displacements, and only used information from the Bragg data. Whereas different TLS groupings yielded similar Bragg intensities, they yielded different diffuse intensities, none of which agreed well with the data. In contrast, both the LLM and NM models agreed substantially with the diffuse data. These results demonstrate a realistic path to increase the number of diffuse datasets available to the wider biosciences community and indicate that dynamics-inspired NM structural models can simultaneously agree with both Bragg and diffuse scattering.protein dynamics | normal modes | structural biology | diffuse scattering | liquid-like motions X -ray crystallography can be a key tool for elucidating the structural basis of protein motions that play critical roles in enzymatic reactions, protein-protein interactions, and signaling cascades (1). X-ray diffraction yields an ensemble-averaged picture of the protein structure: each photon simultaneously probes multiple unit cells that can vary because of internal rearrangements or changes to the crystal lattice. Bragg analysis of X-ray diffraction only yields the mean charge density of the unit cell, however, which fundamentally limits the information that can be obtained about protein dynamics (2, 3).An inherent limitation in Bragg analysis is that models with different concerted motions can yield the same mean charge density (4). The traditional approach assumes a single structural model with individual atomic displacement parameters (B factors). Given enough data, anisotropic displacement parameters can be modeled. When the data are more limited, translation-libration-screw (TLS) refinement, which models rigid-body motions of subdomains (5), is often used [22% of Protein Data Bank (PDB) depositions (6, 7)]. Variations in the TLS domains can predict very different motions that agree equally well with the Bragg data (8, 9).Bragg analysis can be combined ...
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