SummaryPhage-inducible chromosomal islands (PICIs) represent a novel and universal class of mobile genetic elements, which have broad impact on bacterial virulence. In spite of their relevance, how the Gram-negative PICIs hijack the phage machinery for their own specific packaging and how they block phage reproduction remains to be determined. Using genetic and structural analyses, we solve the mystery here by showing that the Gram-negative PICIs encode a protein that simultaneously performs these processes. This protein, which we have named Rpp (for redirecting phage packaging), interacts with the phage terminase small subunit, forming a heterocomplex. This complex is unable to recognize the phage DNA, blocking phage packaging, but specifically binds to the PICI genome, promoting PICI packaging. Our studies reveal the mechanism of action that allows PICI dissemination in nature, introducing a new paradigm in the understanding of the biology of pathogenicity islands and therefore of bacterial pathogen evolution.
The SH3 domain of the c-Src tyrosine kinase (c-Src-SH3) aggregates to form intertwined dimers and amyloid fibrils at mild acid pHs. In this work, we show that a single mutation of residue Gln128 of this SH3 domain has a significant effect on: (i) its thermal stability; and (ii) its propensity to form amyloid fibrils. The Gln128Glu mutant forms amyloid fibrils at neutral pH but not at mild acid pH, while Gln128Lys and Gln128Arg mutants do not form these aggregates under any of the conditions assayed. We have also solved the crystallographic structures of the wild-type (WT) and Gln128Glu, Gln128Lys and Gln128Arg mutants from crystals obtained at different pHs. At pH 5.0, crystals belong to the hexagonal space group P6522 and the asymmetric unit is formed by one chain of the protomer of the c-Src-SH3 domain in an open conformation. At pH 7.0, crystals belong to the orthorhombic space group P212121, with two molecules at the asymmetric unit showing the characteristic fold of the SH3 domain. Analysis of these crystallographic structures shows that the residue at position 128 is connected to Glu106 at the diverging β-turn through a cluster of water molecules. Changes in this hydrogen-bond network lead to the displacement of the c-Src-SH3 distal loop, resulting also in conformational changes of Leu100 that might be related to the binding of proline rich motifs. Our findings show that electrostatic interactions and solvation of residues close to the folding nucleation site of the c-Src-SH3 domain might play an important role during the folding reaction and the amyloid fibril formation.
B-phycoerythrin from the red alga Porphyridium cruentum was crystallized using the technique of capillary counter-diffusion. Crystals belonging to the space group R3 with almost identical unit cell constants and diffracting to 1.85 and 1.70 Å were obtained at pH values of 5 and 8, respectively. The most important difference between structures is the presence of the residue His88a in two different conformations at pH 8. This residue is placed next to the chromophore phycoerythrobilin PEB82a and the new conformation results in the relocation of the hydrogen-bond network and hydration around PEB82a, which probably contributes to the observed pH dependence of the optical spectrum associated with this chromophore.Comparison with the structures of B-phycoerythrin from other red algae shows differences in the conformation of the A-ring of the chromophore PEB139a. This conformational difference in B-phycoerythrin from P. cruentum enables the formation of several hydrogen bonds that connect PEB139a with the chromophore PEB158b at the (ab) 3 hexamer association interface. The possible influence of these structural differences on the optical spectrum and the ability of the protein to perform energy transfer are discussed, with the two pH-dependent conformations of His88a and PEB82a being proposed as representing critical structural features that are correlated with the pH dependence of the optical spectrum and transient optical states during energy transfer. DatabaseStructural data have been deposited in the Protein Data Bank under accession numbers 3V58 and 3V57. Structured digital abstract• B-phycoerythrin beta and B-phycoerythrin alpha bind by x-ray crystallography (View interaction)Abbreviations PDB, Protein Data Bank; PEB, phycoerythrobilin; PUB, phycourobilin.
Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine, the first step in the synthesis of catecholamine neurotransmitters. The protein contains a 159-residue regulatory domain (RD) at its N-terminus that forms dimers in solution; the N-terminal region of RDTyrH (residues 1-71) is absent in the solution structure of the domain. We have characterized the conformational stability of two species of RDTyrH (one containing the N-terminal region and another lacking the first 64 residues) to clarify how that N-terminal region modulates the conformational stability of RD. Under the conditions used in this study, the RD species lacking the first 64 residues is a monomer at pH 7.0, with a small conformational stability at 25 °C (4.7 ± 0.8 kcal mol(-1)). On the other hand, the entire RDTyrH is dimeric at physiological pH, with an estimated dissociation constant of 1.6 μM, as determined by zonal gel filtration chromatography; dimer dissociation was spectroscopically silent to circular dichroism but not to fluoresecence. Both RD species were disordered below physiological pH, but the acquisition of secondary native-like structure occurs at pHs lower than those measured for the attainment of tertiary native- and compactness-like arrangements.
The atomic resolution crystal structures of complexes between the SH3 domain of the c-Src tyrosine kinase and two high-affinity peptides belonging to class I and class II have been solved. The crystals of the Thr98Asp and Thr98Glu mutants in complex with the APP12 peptide (APPLPPRNRPRL) belonged to the trigonal space group P3121 and in both cases the asymmetric unit was composed of one molecule of the SH3-APP12 complex. The crystals of the Thr98Glu mutant in complex with the VSL12 peptide (VSLARRPLPLP) belonged to the trigonal space group P3221 and the asymmetric unit was also composed of a single molecule of the SH3-VSL12 complex. All crystals were obtained in the presence of PEG 300 under the same conditions as reported for the intertwined dimeric structure of the c-Src SH3 domain, but the presence of the peptide stabilizes the monomeric form of the domain. These structures allow a detailed analysis of the role of salt bridges, cation-π interactions and hydrogen bonds in the binding of proline-rich motifs to the c-Src SH3 domain. Moreover, these crystallographic structures allow the role of water molecules in the binding of these motifs to the c-Src SH3 domain to be studied for the first time.
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