We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with ...the lipid substrate, diC4-PIP₂, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP₂ molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.
This paper describes the use of barium chloride to produce a heavy‐atom derivative of xylanase II crystals from Trichoderma longibrachiatum, which was obtained either by cocrystallization or soaking. ...SAD phasing led to interpretable electron‐density maps that allowed unambiguous chain tracing. In the best case, with a data set collected at 9.5 keV, 88% of the residues were built, with 83% of the side chains assigned. The barium ions are found to mainly interact with main‐chain carbonyl groups and water molecules. It is suggested that barium ions could also be used as a potential anomalous scatterer in the quick cryosoaking procedure for phasing.
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Continuous irradiation of a single protein crystal alters its physical structure. This typically manifests as photoelectric chemical transformations and a loss of diffraction, which can ...be counterproductive to structure determination. Serial crystallography methods overcome these effects by recording partial datasets from multiple crystals, effectively spreading the dose over many crystals. As the number of crystals per dataset increases, however, new methods are needed to rapidly replenish samples in the X-ray beam. Acoustic droplet ejection (ADE) technology offers a robust and reliable method for transferring crystals. Acoustic injectors based on ADE technology use focused sound waves to deliver crystals contained within picoliter and nanoliter droplets, and can generate hundreds of droplets each second. Acoustic injectors have been tested at the National Synchrotron Light Source, where crystal-containing droplets were deposited on a goniometer-mounted conveyor belt. The conveyor belt enabled translation and rotation in the X-ray beam, permitting small wedges of data to be collected on each crystal. Crystals were also directly injected into oncoming X-ray pulses at the Linac Coherent Light Source, producing diffraction patterns from several samples under conditions of ambient temperature and pressure. Development is underway to incorporate acoustic injectors into the microfocus and automated macromolecular crystallography beamlines at the National Synchrotron Light Source II.
We demonstrate a general strategy for determining structures from showers of microcrystals. It uses acoustic droplet ejection to transfer 2.5 nL droplets from the surface of microcrystal slurries, ...through the air, onto mounting micromesh pins. Individual microcrystals are located by raster-scanning a several-micrometer X-ray beam across the cryocooled micromeshes. X-ray diffraction data sets merged from several micrometer-sized crystals are used to determine 1.8 Ǻ resolution crystal structures.
Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this ...technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal‐improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s−1) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl‐shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high‐throughput screening.
The tumor suppressor protein Merlin inhibits cell proliferation upon establishing cell-cell contacts. Because Merlin has high level of sequence similarity to the Ezrin-Radixin-Moesin family of ...proteins, the structural model of Ezrin-Radixin-Moesin protein autoinhibition and cycling between closed/resting and open/active conformational states is often employed to explain Merlin function. However, recent biochemical studies suggest alternative molecular models of Merlin function. Here, we have determined the low-resolution molecular structure and binding activity of Merlin and a Merlin(S518D) mutant that mimics the inactivating phosphorylation at S518 using small-angle neutron scattering and binding experiments. Small-angle neutron scattering shows that, in solution, both Merlin and Merlin(S518D) adopt a closed conformation, but binding experiments indicate that a significant fraction of either Merlin or Merlin(S518D) is capable of binding to the target protein NHERF1. Upon binding to the phosphatidylinositol 4,5-bisphosphate lipid, the wild-type Merlin adopts a more open conformation than in solution, but Merlin(S518D) remains in a closed conformation. This study supports a rheostat model of Merlin in NHERF1 binding and contributes to resolving a controversy about the molecular conformation and binding activity of Merlin.
Nutrient import across Gram-negative bacteria’s outer membrane is powered by the proton-motive force, delivered by the cytoplasmic membrane protein complex ExbB–ExbD–TonB. Having purified the ExbB
4
...–ExbD
2
complex in the detergent dodecyl maltoside, we substituted amphipol A8-35 for detergent, forming a water-soluble membrane protein/amphipol complex. Properties of the ExbB
4
–ExbD
2
complex in detergent or in amphipols were compared by gel electrophoresis, size exclusion chromatography, asymmetric flow field-flow fractionation, thermal stability assays, and electron microscopy. Bound detergent and fluorescently labeled amphipol were assayed quantitatively by 1D NMR and analytical ultracentrifugation, respectively. The structural arrangement of ExbB
4
–ExbD
2
was examined by EM, small-angle X-ray scattering, and small-angle neutron scattering using a deuterated amphipol. The amphipol-trapped ExbB
4
–ExbD
2
complex is slightly larger than its detergent-solubilized counterpart. We also investigated a different oligomeric form of the two proteins, ExbB
6
–ExbD
4
, and propose a structural arrangement of its transmembrane
α
-helical domains.
Protein phosphatase 1 (PP1) interacts with ∼200 regulatory proteins to form holoenzymes, which target PP1 to specific locations and regulate its specificity. While it is known that many PP1 ...regulatory proteins are dynamic in the unbound state, much less is known about the residual flexibility after PP1 holoenzyme formation. Here, we have used small angle X-ray scattering to investigate the flexibility of the PP1:spinophilin holoenzyme in solution. Collectively, our data shows that the PP1:spinophilin holoenzyme is dynamic in solution, which allows for an increased capture radius of spinophilin and is likely important for its biological role.
MINT-8057915: PP1-alpha (uniprotkb:P62136) and Spinophilin (uniprotkb:O35274) bind (MI:0407) by x ray scattering (MI:0826)
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