Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase.

The Escherichia coli inducible lysine decarboxylase, LdcI/CadA, together with the inner-membrane lysine-cadaverine antiporter, CadB, provide cells with protection against mild acidic conditions (pH∼5). To gain a better understanding of the molecular processes underlying the acid stress response, the X-ray crystal structure of LdcI was determined. The structure revealed that the protein is an oligomer of five dimers that associate to form a decamer. Surprisingly, LdcI was found to co-crystallize with the stringent response effector molecule ppGpp, also known as the alarmone, with 10 ppGpp molecules in the decamer. ppGpp is known to mediate the stringent response, which occurs in response to nutrient deprivation. The alarmone strongly inhibited LdcI enzymatic activity. This inhibition is important for modulating the consumption of lysine in cells during acid stress under nutrient limiting conditions. Hence, our data provide direct evidence for a link between the bacterial acid stress and stringent responses.

Crystallization and preliminary X-ray analysis of the inducible lysine decarboxylase from Escherichia coli.

The decameric inducible lysine decarboxylase (LdcI) from Escherichia coli has been crystallized in space groups C2 and C222(1); the Ta6Br12(2+) cluster was used to derivatize the C2 crystals. The method of single isomorphous replacement with anomalous scattering (SIRAS) as implemented in SHELXD was used to solve the Ta6Br12(2+)-derivatized structure to 5 A resolution. Many of the Ta6Br12(2+)-binding sites had twofold and fivefold noncrystallographic symmetry. Taking advantage of this feature, phase modification was performed in DM. The electron-density map of LdcI displays many features in agreement with the low-resolution negative-stain electron-density map [Snider et al. (2006), J. Biol. Chem. 281, 1532-1546].

Crystal and solution structures of 7-amino-actinomycin D complexes with d(TTAGBrUT), d(TTAGTT) and d(TTTAGTTT).

The formation of the complex of 7-amino-actinomycin D with potentially single-stranded DNA has been studied by X-ray crystallography in the solid state, by NMR in solution and by molecular modelling. The crystal structures of the complex with 5'-TTAG[Br(5)U]T-3' provide interesting examples of MAD phasing in which the dispersive component of the MAD signal was almost certainly enhanced by radiation damage. The trigonal and orthorhombic crystal modifications both contain antibiotic molecules and DNA strands in the form of a 2:4 complex: in the orthorhombic form there is one such complex in the asymmetric unit, while in the trigonal structure there are four. In both structures the phenoxazone ring of the first drug intercalates between a BrU-G (analogous to T-G) wobble pair and a G-T pair where the T is part of a symmetry-related molecule. The chromophore of the second actinomycin intercalates between the BrU-G and G-BrU wobble pairs of the partially paired third and fourth strands. The base stacking also involves (A*T)*T triplets and Watson-Crick A-T pairs and leads to similar complex three-dimensional networks in both structures, with looping-out of unpaired bases. Although the available NOE constraints of a solution containing the antibiotic and d(TTTAGTTT) strands in the ratio 1:1 are insufficient to determine the structure of the complex from the NMR data alone, they are consistent with the intercalation geometry observed in the crystal structure. Molecular-dynamics (MD) trajectories starting from the 1:2 complexes observed in the crystal showed that although the thymines flanking the d(AGT) core are rather flexible and the G-T pairing is not permanently preserved, both strands remain bound to the actinomycin by strong interactions between it and the guanines between which it is sandwiched. Similar strong binding (hemi-intercalation) of the actinomycin to a single guanine was observed in the MD trajectories of a 1:1 complex. The dominant interaction is between the antibiotic and guanine, but the complexes are stabilized further by promiscuous base-pairing.

Solution and structure of an alternating D,L-peptide.

The crystal structure of H-(L-Tyr-D-Tyr)(4)-L-Lys-OH has been determined to 1.3 A resolution. The D,L-alternating peptide crystallizes in the tetragonal system, space group P4(3)2(1)2, with unit-cell parameters a = b = 27.99 (3), c = 78.93 (8) A. The crystals contain two molecules in the asymmetric unit that form a double-stranded right-handed antiparallel beta-helix. The structure has been solved by SIRAS using a crystal soaked in an iodide-containing solution for 1 min. The programs SHELXD and SHELXE were used to determine the iodide substructure and also the experimental electron-density map. Using the coordinates of known D,L-peptides deposited in the PDB, several attempts were made to solve the structure by molecular-replacement techniques. Although the backbone of the MR model selected shows great similarity and was used to trace the actual peptide structure in the map, it was not possible to obtain the correct solution before the experimental phases became available. The correct fragment orientations are easily determined, but the same does not apply to the translation search. Nevertheless, insights into fragment search and expansion were gained from the tests described in this paper. The correlation coefficient calculated with the resolution shell of data around 2.4 A, a distance corresponding to most 1-3 interatomic vectors, is a particularly good discriminator of correct orientations in the rotation search of small fragments.