Project description:In response to cell death signals, an active apoptosome is assembled from Apaf-1 and procaspase-9 (pc-9). Here we report a near atomic structure of the active human apoptosome determined by cryo-electron microscopy. The resulting model gives insights into cytochrome c binding, nucleotide exchange and conformational changes that drive assembly. During activation an acentric disk is formed on the central hub of the apoptosome. This disk contains four Apaf-1/pc-9 CARD pairs arranged in a shallow spiral with the fourth pc-9 CARD at lower occupancy. On average, Apaf-1 CARDs recruit 3 to 5 pc-9 molecules to the apoptosome and one catalytic domain may be parked on the hub, when an odd number of zymogens are bound. This suggests a stoichiometry of one or at most, two pc-9 dimers per active apoptosome. Thus, our structure provides a molecular framework to understand the role of the apoptosome in programmed cell death and disease.
Project description:ROMK (Kir1.1) potassium channels are closed by internal acidification with a pKa of 6.7 +/- 0.01 in 100 mM external K and a pKa of 7.0 +/- 0.01 in 1 mM external K. Internal acidification in 1 mM K (but not 100 mM K) not only closed the pH gate but also inactivated Kir1.1, such that realkalization did not restore channel activity until high K was returned to the bath. We identified a new putative intersubunit salt bridge (R128-E132-Kir1.1b) in the P-loop of the channel near the selectivity filter that affected the K sensitivity of the inactivation process. Mutation of either R128-Kir1.1b or E132-Kir1.1b caused inactivation in both 1 mM and 100 mM external K during oocyte acidification. However, 300 mM external K (but not 200 mM Na + 100 mM K) protected both E132Q and R128Y from inactivation. External application of a modified honey-bee toxin, tertiapin Q (TPNQ), also protected Kir1.1 from inactivation in 1 mM K and protected E132Q and R128Y from inactivation in 100 mM K, which suggests that TPNQ binding to the outer mouth of the channel stabilizes the active state. Pretreatment of Kir1.1 with external Ba prevented Kir1.1 inactivation, similar to pretreatment with TPNQ. In addition, mutations that disrupted transmembrane helix H-bonding (K61M-Kir1.1b) or stabilized a selectivity filter to helix-pore linkage (V121T-Kir1.1b) also protected both E132Q and R128Y from inactivation in 1 mM K and 100 mM K. Our results are consistent with Kir inactivation arising from conformational changes near the selectivity filter, analogous to C-type inactivation.
Project description:G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human ?(2) adrenergic receptor (?(2)AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive ?(2)AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11?Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.
Project description:Using grazing incidence X-rays and X-ray photoelectron spectroscopy during the mass transfer limited catalytic oxidation of CO, the long-range surface structure of Pd(100) was investigated. Under the reaction conditions of 50:4 O2 to CO, 300 mbar pressure, and temperatures between 200 and 450 °C, the surface structure resulting from oxidation and the subsequent oxide reduction was elucidated. The reduction cycle was halted, and while under reaction conditions, angle-dependent X-ray photoelectron spectroscopy close to the critical angle of Pd and modeling of the data was performed. Two proposed models for the system were compared. The suggestion with the metallic islands formed on top of the oxide island was shown to be consistent with the data.
Project description:The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody. Comparisons between inactive- and active-state opioid receptor structures reveal substantial conformational changes in the binding pocket and intracellular and extracellular regions. Extensive structural analysis and experimental validation illuminate key residues that propagate larger-scale structural rearrangements and transducer binding that, collectively, elucidate the structural determinants of KOP pharmacology, function, and biased signaling. These molecular insights promise to accelerate the structure-guided design of safer and more effective κ-opioid receptor therapeutics.
Project description:The disordered and basic C-terminal 14 residues of human troponin T (TnT) are essential for full inhibition of actomyosin ATPase activity at low Ca2+ levels and for limiting activation at saturating Ca2+. In previous studies, stepwise truncation of the C-terminal region of TnT increased activity in proportion to the number of positive charges eliminated. To define key basic residues more closely, we generated phosphomimetic-like mutants of TnT. Phosphomimetic mutants were chosen because of reports that phosphorylation of TnT, including sites within the C terminal region, depressed activity, contrary to our expectations. Four constructs were made where one or more Ser and Thr residues were replaced with Asp residues. The S275D and T277D mutants, near the IT helix and adjacent to basic residues, produced the greatest activation of ATPase rates in solution; the effects of the S275D mutant were recapitulated in muscle fiber preparations with enhanced myofilament Ca2+ sensitivity. Actin filaments containing S275D TnT were also shown to be incapable of populating the inactive state at low Ca2+ levels. Actin filaments containing both S275D/T284D were not statistically different from those containing only S275D in both solution and cardiac muscle preparation studies. Finally, actin filaments containing T284D TnT, closer to the C-terminus and not adjacent to a basic residue, had the smallest effect on activity. Thus, the effects of negative charge placement in the C-terminal region of TnT were greatest near the IT helix and adjacent to a basic residue.
Project description:A reaction's transition state (TS) structure plays a critical role in determining reactivity and has important implications for the design of catalysts, drugs, and other applications. Here, we explore TS structure in the enzyme alkaline phosphatase using hybrid Quantum Mechanics/Molecular Mechanics simulations. We find that minor perturbations to the substrate have major effects on TS structure and the way the enzyme stabilizes the TS. Substrates with good leaving groups (LGs) have little cleavage of the phosphorus-LG bond at the TS, while substrates with poor LGs have substantial cleavage of that bond. The results predict nonlinear free energy relationships for a single rate-determining step, and substantial differences in kinetic isotope effects for different substrates; both trends were observed in previous experimental studies, although the original interpretations differed from the present model. Moreover, due to different degrees of phosphorus-LG bond cleavage at the TS for different substrates, the LG is stabilized by different interactions at the TS: while a poor LG is directly stabilized by an active site zinc ion, a good LG is mainly stabilized by active site water molecules. Our results demonstrate the considerable plasticity of TS structure and stabilization in enzymes. Furthermore, perturbations to reactivity that probe TS structure experimentally (i.e., substituent effects) may substantially perturb the TS they aim to probe, and thus classical experimental approaches such as free energy relations should be interpreted with care.
Project description:X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbing atom and therefore allows bond distances and angles of the surrounding atomic cluster to be measured with atomic resolution. By contrast, the accuracy and resolution of metalloprotein active sites obtainable from x-ray crystallography are often insufficient to analyze the electronic properties of the metals that are essential for their biological functions. Here, we demonstrate that the combination of both methods on the same metalloprotein single crystal yields a structural model of the protein with exceptional active-site resolution. To this end, we have collected an x-ray diffraction data set to 1.4-A resolution and Fe K-edge polarized x-ray absorption near edge structure (XANES) spectra on the same cyanomet sperm whale myoglobin crystal. The XANES spectra were quantitatively analyzed by using a method based on the multiple scattering approach, which yielded Fe-heme structural parameters with +/-(0.02-0.07)-A accuracy on the atomic distances and +/-7 degrees on the Fe-CN angle. These XANES-derived parameters were subsequently used as restraints in the crystal structure refinement. By combining XANES and x-ray diffraction, we have obtained an cyanomet sperm whale myoglobin structural model with a higher precision of the bond lengths and angles at the active site than would have been possible with crystallographic analysis alone.
Project description:Traffic disturbances (i.e. pollution, light, noise, and vibrations) often extend into the area surrounding a road creating a 'road-effect zone'. Habitat within the road-effect zone is degraded or, in severe cases, completely unsuitable for wildlife, resulting in indirect habitat loss. This can have a disproportionate impact on wildlife in highly modified landscapes, where remaining habitat is scarce or occurs predominantly along roadside reserves. In this study, we investigated the road-effect zone for insectivorous bats in highly cleared agricultural landscapes by quantifying the change in call activity with proximity to three major freeways. The activity of seven out of 10 species of bat significantly decreased with proximity to the freeway. We defined the road-effect zone to be the proximity at which call activity declined by at least 20% relative to the maximum detected activity. The overall road-effect zone for bats in this region was 307 m, varying between 123 and 890 m for individual species. Given that this road-effect zone exceeds the typical width of the roadside verges (<50 m), it is possible that much of the vegetation adjacent to freeways in this and similar landscapes provides low-quality habitat for bats. Without accounting for the road-effect zone, the amount of habitat lost or degraded due to roads is underestimated, potentially resulting in the loss of wildlife, ecosystem services and key ecosystem processes (e.g. predator-prey or plant-pollinator interactions) from the landscape. We suggest all future environmental impact assessments include quantifying the road-effect zone for sensitive wildlife, in order to best plan and mitigate the impact of roads on the environment. Mitigating the effects of new and existing roads on wildlife is essential to ensure enough high-quality habitat persists to maintain wildlife populations.
Project description:Plants dissipate excess excitation energy as heat by non-photochemical quenching (NPQ). NPQ has been thought to resemble in vitro aggregation quenching of the major antenna complex, light harvesting complex of photosystem II (LHC-II). Both processes are widely believed to involve a conformational change that creates a quenching centre of two neighbouring pigments within the complex. Using recombinant LHC-II lacking the pigments implicated in quenching, we show that they have no particular role. Single crystals of LHC-II emit strong, orientation-dependent fluorescence with an emission maximum at 680 nm. The average lifetime of the main 680 nm crystal emission at 100 K is 1.31 ns, but only 0.39 ns for LHC-II aggregates under identical conditions. The strong emission and comparatively long fluorescence lifetimes of single LHC-II crystals indicate that the complex is unquenched, and that therefore the crystal structure shows the active, energy-transmitting state of LHC-II. We conclude that quenching of excitation energy in the light-harvesting antenna is due to the molecular interaction with external pigments in vitro or other pigment-protein complexes such as PsbS in vivo, and does not require a conformational change within the complex.