Structural Biology Planet

A pentad mutation of GAPR-1 causes structural changes, and shifts the GAPR-1 monomer-dimer equilibrium toward dimerization, which together may prevent its binding to Beclin 1.

Two forms of the HicAB toxin–antitoxin complex from E. coli were purified and crystals were obtained of one of the forms.

The interaction of DAPI with d(CGTGAATTCACG) results in displacement of the the ordered spine waters and confers hydrophobic behaviour on the DNA.

Three-dimensional nanometre-sized crystals of macromolecules currently resist structure elucidation by single-crystal X-ray crystallography. Here, a single nanocrystal with a diffracting volume of only 0.14 µm3, i.e. no more than 6 × 105 unit cells, provided sufficient information to determine the structure of a rare dimeric polymorph of hen egg-white lysozyme by electron crystallography. This is at least an order of magnitude smaller than was previously possible. The molecular-replacement solution, based on a monomeric polyalanine model, provided sufficient phasing power to show side-chain density, and automated model building was used to reconstruct the side chains. Diffraction data were acquired using the rotation method with parallel beam diffraction on a Titan Krios transmission electron microscope equipped with a novel in-house-designed 1024 × 1024 pixel Timepix hybrid pixel detector for low-dose diffraction data collection. Favourable detector characteristics include the ability to accurately discriminate single high-energy electrons from X-rays and count them, fast readout to finely sample reciprocal space and a high dynamic range. This work, together with other recent milestones, suggests that electron crystallography can provide an attractive alternative in determining biological structures.

The Pfp1 protease, a cysteine protease of unknown specificity from the hyperthermophilic archaeon Thermococcus thioreducens, was crystallized in two distinctive crystal forms: from concentrated citrate in one case and PEG in the other. X-ray data were collected from both crystal forms at room temperature to about 1.9 Å resolution using a laboratory source and detector, and the structures were solved by molecular replacement using the Pfp1 protease from Pyrococcus horikoshii as the search model. In the T. thioreducens protease structures, Cys18 residues on adjacent molecules in the asymmetric units form intermolecular disulfide bonds, thereby yielding hexamers composed of three cross-linked, quasi-dyad-related dimers with crystallographically exact threefold axes and exhibiting almost exact 32 symmetry. The corresponding residue in P. horikoshii Pfp1 is Tyr18. An individual active site containing Cys100 and His101 also includes a Glu74 residue contributed by a quasi-twofold-related, non-cross-linked subunit. Two catalytic triads are therefore closely juxtaposed about the quasi-twofold axis at the interface of these subunits, and are relatively sequestered within the hexamer cavity. The cysteine in the active site is observed to be oxidized in both of the crystal forms that were studied.

An alternative rational approach to improve protein crystals by using single-site mutation of surface residues is proposed based on the results of a statistical analysis using a compiled data set of 918 independent crystal structures, thereby reflecting not only the entropic effect but also other effects upon protein crystallization. This analysis reveals a clear difference in the crystal-packing propensity of amino acids depending on the secondary-structural class. To verify this result, a systematic crystallization experiment was performed with the biotin carboxyl carrier protein from Pyrococcus horikoshii OT3 (PhBCCP). Six single-site mutations were examined: Ala138 on the surface of a β-sheet was mutated to Ile, Tyr, Arg, Gln, Val and Lys. In agreement with prediction, it was observed that the two mutants (A138I and A138Y) harbouring the residues with the highest crystal-packing propensities for β-sheet at position 138 provided better crystallization scores relative to those of other constructs, including the wild type, and that the crystal-packing propensity for β-sheet provided the best correlation with the ratio of obtaining crystals. Two new crystal forms of these mutants were obtained that diffracted to high resolution, generating novel packing interfaces with the mutated residues (Ile/Tyr). The mutations introduced did not affect the overall structures, indicating that a β-sheet can accommodate a successful mutation if it is carefully selected so as to avoid intramolecular steric hindrance. A significant negative correlation between the ratio of obtaining amorphous precipitate and the crystal-packing propensity was also found.

Two nonstructural proteins encoded by Zika virus strain MR766 RNA, a methyltransferase and a helicase, were crystallized and their structures were solved and refined at 2.10 and 2.01 Å resolution, respectively. The NS5 methyltransferase contains a bound S-adenosyl-l-methionine (SAM) co-substrate. The NS3 helicase is in the apo form. Comparison with published crystal structures of the helicase in the apo, nucleotide-bound and single-stranded RNA (ssRNA)-bound states suggests that binding of ssRNA to the helicase may occur through conformational selection rather than induced fit.

The structure of 4′,6-diamidine-2-phenylindole (DAPI) bound to the synthetic B-DNA oligonucleotide d(CGTGAATTCACG) has been solved in space group P212121 by single-crystal X-ray diffraction at a resolution of 2.2 Å. The structure is nearly isomorphous to that of the previously reported crystal structure of the oligonucleotide d(CGTGAATTCACG) alone. The adjustments in crystal packing between the native DNA molecule and the DNA–DAPI complex are described. DAPI lies in the narrow minor groove near the centre of the B-DNA fragment, positioned over the A–T base pairs. It is bound to the DNA by hydrogen-bonding and van der Waals interactions. Comparison of the two structures (with and without ligand) shows that DAPI inserts into the minor groove, displacing the ordered spine waters. Indeed, as DAPI is hydrophobic it confers this behaviour on the DNA and thus restricts the presence of water molecules.

Toxin–antitoxin (TA) systems are widespread in both bacteria and archaea, where they enable cells to adapt to environmental cues. TA systems play crucial roles in various cellular processes, such as programmed cell death, cell growth, persistence and virulence. Here, two distinct forms of the type II toxin–antitoxin complex HicAB were identified and characterized in Escherichia coli K-12, and both were successfully overexpressed and purified. The two proposed forms, HicABL and HicABS, differed in the presence or absence of a seven-amino-acid segment at the N-terminus in the antitoxin HicB. The short form HicABS readily crystallized under the conditions 0.1 M Tris–HCl pH 8.0, 20%(w/v) PEG 6000, 0.2 M ammonium sulfate. The HicABS crystal diffracted and data were collected to 2.5 Å resolution. The crystal belonged to space group I222 or I212121, with unit-cell parameters a = 67.04, b = 66.31, c = 120.78 Å. Matthews coefficient calculation suggested the presence of two molecules each of HicA and HicBS in the asymmetric unit, with a solvent content of 55.28% and a Matthews coefficient (VM) of 2.75 Å3 Da−1.