Active-Site pKa Determination for Photoactive Yellow Protein Rationalizes Slow Ground-State Recovery

The ability to avoid blue-light radiation is crucial for bacteria to survive. In Halorhodospira halophila, the putative receptor for this response is known as photoactive yellow protein (PYP). Its response to blue light is mediated by changes in the optical properties of the chromophore para-coumaric acid (pCA) in the protein active site. PYP displays photocycle kinetics with a strong pH dependence for ground-state recovery, which has remained enigmatic. To resolve this problem, a comprehensive pK(a) determination of the active-site residues of PYP is required. Herein, we show that Glu-46 stays protonated from pH 3.4 to pH 11.4 in the ground (pG) state. This conclusion is supported by the observed hydrogen-bonded protons between Glu-46 and pCA and Tyr-42 and pCA, which are persistent over the entire pH range. Our experimental results show that none of the active-site residues of PYP undergo pH-induced changes in the pG state. Ineluctably, the pH dependence of pG recovery is linked to conformational change that is dependent upon the population of the relevant protonation state of Glu-46 and the pCA chromophore in the excited state, collaterally explaining why pG recovery is slow

Comprehensive Determination of Protein Tyrosine pK(a) Values for Photoactive Yellow Protein Using Indirect C-13 NMR Spectroscopy

Upon blue-light irradiation, the bacterium Halorhodospira halophila is able to modulate the activity of its flagellar motor and thereby evade potentially harmful UV radiation. The 14 kDa soluble cytosolic photoactive yellow protein (PYP) is believed to be the primary mediator of this photophobic response, and yields a UV/Vis absorption spectrum that closely matches the bacterium's motility spectrum. In the electronic ground state, the para-coumaric acid (pCA) chromophore of PYP is negatively charged and forms two short hydrogen bonds to the side chains of Glu-46 and Tyr-42. The resulting acid triad is central to the marked pH dependence of the optical-absorption relaxation kinetics of PYP. Here, we describe an NMR approach to sequence-specifically follow all tyrosine side-chain protonation states in PYP from pH 3.41 to 11.24. The indirect observation of the nonprotonated (13)C(γ) resonances in sensitive and well-resolved two-dimensional (13)C-(1)H spectra proved to be pivotal in this effort, as observation of other ring-system resonances was hampered by spectral congestion and line-broadening due to ring flips. We observe three classes of tyrosine residues in PYP that exhibit very different pK(a) values depending on whether the phenolic side chain is solvent-exposed, buried, or hydrogen-bonded. In particular, our data show that Tyr-42 remains fully protonated in the pH range of 3.41–11.24, and that pH-induced changes observed in the photocycle kinetics of PYP cannot be caused by changes in the charge state of Tyr-42. It is therefore very unlikely that the pCA chromophore undergoes changes in its electrostatic interactions in the electronic ground state

H-1, C-13, and N-15 resonance assignment of photoactive yellow protein

<p>Photoactive yellow protein (PYP) is involved in the negative phototactic response towards blue light of the bacterium Halorhodospira halophila. Here, we report nearly complete backbone and side chain H-1, C-13 and N-15 resonance assignments at pH 5.8 and 20 A degrees C of PYP in its electronic ground state.</p>

Crystal Structures of Two Transcriptional Regulators from Bacillus cereus Define the Conserved Structural Features of a PadR Subfamily

PadR-like transcriptional regulators form a structurally-related family of proteins that control the expression of genes associated with detoxification, virulence and multi-drug resistance in bacteria. Only a few members of this family have been studied by genetic, biochemical and biophysical methods, and their structure/function relationships are still largely undefined. Here, we report the crystal structures of two PadR-like proteins from Bacillus cereus, which we named bcPadR1 and bcPadR2 (products of gene loci BC4206 and BCE3449 in strains ATCC 14579 and ATCC 10987, respectively). BC4206, together with its neighboring gene BC4207, was previously shown to become significantly upregulated in presence of the bacteriocin AS-48. DNA mobility shift assays reveal that bcPadR1 binds to a 250 bp intergenic region containing the putative BC4206–BC4207 promoter sequence, while in-situ expression of bcPadR1 decreases bacteriocin tolerance, together suggesting a role for bcPadR1 as repressor of BC4206–BC4207 transcription. The function of bcPadR2 (48% identical in sequence to bcPadR1) is unknown, but the location of its gene just upstream from genes encoding a putative antibiotic ABC efflux pump, suggests a role in regulating antibiotic resistance. The bcPadR proteins are structurally similar to LmrR, a PadR-like transcription regulator in Lactococcus lactis that controls expression of a multidrug ABC transporter via a mechanism of multidrug binding and induction. Together these proteins define a subfamily of conserved, relatively small PadR proteins characterized by a single C-terminal helix for dimerization. Unlike LmrR, bcPadR1 and bcPadR2 lack a central pore for ligand binding, making it unclear whether the transcriptional regulatory roles of bcPadR1 and bcPadR2 involve direct ligand recognition and induction.

Structural comparison between <i>bc</i>PadR1, <i>bc</i>PadR2 and LmrR.

<p>(A) Superpositions of the wHTH domains of <i>bc</i>PadR1 (light-green), <i>bc</i>PadR2 (red), LmrR (blue), and the following homologs: MexR (green), SmtB (magenta), BlaI (cyan), RTP (orange), and Pex (gray) with the secondary structure elements indicated and labeled. (B) Superposition of the single <i>bc</i>PadR1, <i>bc</i>PadR2 and LmR subunits. (D) Superposition of the <i>bc</i>PadR1, <i>bc</i>PadR2 and LmR dimers.</p

Genomic neighborhood of the <i>bc</i>PadR1 and <i>bc</i>Padr2 encoding genes and analysis of promoter binding.

<p>(A) Organization of the BC4206(<i>bc</i>PadR1)-BC4207 operon of <i>B. cereus</i> ATCC 14579, the putative BCE3449(<i>bc</i>PadR2)-BCE3448-BCE3447-BCE3446 regulon of <i>B. cereus</i> ATCC 10987 and the <i>lmrR</i>-<i>lmrCD</i> regulon of <i>L. lactis</i>. The relative scale of the genes and intergenic regions is proportional to nucleotide length. Boxes indicate the operon/regulon boundaries. The PadR-encoding genes are colored in yellow, while their (putative) target genes, encoding resistance-associated membrane proteins, are in green. (B) The sequence of the intergenic region between BC4205 and BC4206 that is used for the EMSA experiments. Putative -35 and -10 promoter sequences of BC4206 are underlined, the start codons of BC4205 and BC4206 are highlighted in bold, and the amino acids of the coded proteins are indicated above the DNA sequence. The putative binding site of <i>bc</i>PadR1 (homologous to the canonical ATGT/ACAT inverted sequence motif) is highlighted in blue. (C) EMSA experiments with <i>bc</i>PadR1. DNA fragments encompassing the promoter regions of BC4206 (lanes 1–10) and BC4029 (lanes 11–13, negative control) were prepared by PCR and end labeled with ³³P. DNA binding was assayed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048015#s2" target="_blank">Materials and Methods</a>. Lanes 1, 10 and 11 contain the free DNA probe. Samples run on lanes 2 and 12 contain 0.5 µM; lane 3 1 µM; lanes 4–9 and 13 2 µM of purified <i>bc</i>PadR1 protein. Lanes 5 to 9 contains samples including increasing concentrations of AS-48 from 0.5 pM to 0.69 µM.</p

Data collection and refinement.

*<p>Values in parentheses refer to the highest resolution shell.</p>§<p>The R-factor is calculated for all measured reflections in the specified resolution range, while the R_free is calculated for reflections belonging to a random test set not used in refinement (10% of the data).</p

Modeling of the DNA-bound <i>bc</i>PadR complexes.

<p>(A) Superposition of the <i>bc</i>PadR1 (light-green) and <i>bc</i>PadR2 (red) dimers onto DNA-bound RTP (orange, PDB code 1F4K). (B) Superposition of the <i>bc</i>PadR1 (light-green) and <i>bc</i>PadR2 (red) dimers onto DNA-bound BlaI (cyan, PDB code 1XSD). Below the superpositions are structure-based sequence alignments including secondary structures. Residues of RTP and BlaI which participate in DNA binding are indicated with small spheres using the following color scheme: green, interacting with the phosphate backbone; blue, interacting with the base moiety and yellow, interacting with the ribose backbone).</p

Ribbon representations of the <i>bc</i>PadR dimers and selected structural homolog.

<p>(A) <i>bc</i>PadR1, (B) <i>bc</i>PadR2, (C) LmrR (PDB code 3F8B), and (D) RTP, a replication terminator protein from <i>Bacillus subtilis</i> (PDB code 1F4K). For each dimer, one of the subunits is shown in a rainbow color gradient from the N-terminus (blue) to the C-terminus (red), whereas the other subunit is colored grey. Helices involved in dimerization are indicated. Residues Trp91 of <i>bc</i>PadR1, Trp93 of <i>bc</i>PadR2, and Trp96 of LmrR are drawn in stick representation.</p

Multiple sequence alignment of <i>bc</i>PadR1, <i>bc</i>PadR2, and other members of the PadR-s2 subfamily.

<p>Only sequences for which structures are available in the PDB are shown in the alignment. The PadR-s2 proteins with unpublished structures are addressed by their PDB entry names: 1XMA, a putative transcriptional regulator from <i>Clostridium thermocellum</i>; 3HHH, a putative transcriptional regulator from <i>Enterococcus faecalis</i> V583; 3L7W, uncharacterized protein SMU.1704 from <i>Streptococcus mutans</i> UA159; 3RI2, a putative transcriptional regulator from <i>Eggerthella lenta</i> DSM 2243. Residues that participate in dimerization (for <i>bc</i>PadR1, <i>bc</i>PadR2, and LmrR) and/or have a role in drug binding (only for LmrR) are indicated by small spheres below the sequences. The consensus sequence is derived from a multiple sequence alignment of 2156 PadR-s2 proteins using as criteria that the conserved residue(s) should be present in at least 50% of the sequences.</p