FtsY_NG bound with fragments.

<p>(A) FtsY_NG bound to Fragment 1 (shown in green) in the Trp343 and Phe365 binding sites. (B and C) FtsY_NG:Fragment 1 interaction profile in (B) Trp343 binding site and (C) Phe365 binding site. (D) FtsY_NG bound to Fragment 2 (shown in orange) in the Trp343 and Phe365 binding sites. (E and F) FtsY_NG:Fragment 2 interaction profile in (E) Trp343 binding site and (F) Phe365 binding site. (G) FtsY_NG bound to Fragment 3 (shown in pink) in the Trp343 and Phe365 binding sites. (H and I) FtsY_NG:Fragment 3 interaction profile in (H) Trp343 binding site and (I) Phe365 binding site. Fragments are displayed as sticks with Fragment 1 shown in green, Fragment 2 in orange, Fragment 3 in pink and the amino acids interacting with them shown as blue sticks. Interactions are water bridge (grey line), hydrophobic (red dashed line), hydrogen bond (blue line) and π-stacking (green dashed line). Distances for interactions are indicated in the figure. mF_o-DF_c fragment electron density map is shown in grey and contoured at 3σ level in (A), (D) and (G).</p

Chemical environment surrounding the fragment-binding sites.

<p>In Trp343 binding site: (A) Fragment 1 (green), (B) Fragment 2 (orange) and (C) Fragment 3 (magenta). (D−F) In Phe365 binding site: (D) Fragment 1, (E) Fragment 2 and (F) Fragment 3. FtsY_NG is shown as surface with amino acids that form the surrounding fragment binding site labelled and shown as blue sticks. Fragment 1 is shown in green, Fragment 2 in orange and Fragment 3 in magenta. R represents positions to be modified according to the Phe365 binding site.</p

STD, WATERlogsy and CPMG ligand-detected experiments carried out on Fragment 2 in the absence (green traces) and presence (orange traces) of FtsY_NG.

<p>The top (black) trace is the ¹H 1D NMR spectrum of Fragment 2 for reference. (B−D) ¹⁵N-TROSY-HSQC spectrum of ¹⁵N-FtsY_NG alone (blue) and following addition of (B) Fragment 2 (red), (C) GTP analogue (red) and (D) 4.5S RNA (red). Arrows indicate the peaks that have shifted during the titration with green arrows highlighting the same peaks that have shifted in the fragment titration and the 4.5S RNA titration. (E) Chemical structures of Fragments 1, 2 and 3.</p

Discovery of fragments that target key interactions in the signal recognition particle (SRP) as potential leads for a new class of antibiotics

<div><p>Given the increasing incidence of antibiotic resistance, antibiotics that employ new strategies are urgently needed. Bacterial survival is dependent on proper function of the signal recognition particle (SRP) and its receptor (FtsY). A unique set of interactions in FtsY:SRP-RNA represents a promising candidate for new antibiotic development as no antibiotic targets this complex and these interactions are functionally replaced by protein:protein interactions in eukaryotes. We used a Fragment Based Drug Design (FBDD) approach to search for new compounds that can bind FtsY, and have identified three lead fragments. <i>In vitro</i> and <i>in vivo</i> analyses have shown that despite a high micromolar binding affinity, one fragment has some antimicrobial properties. X-ray structures of <i>E</i>. <i>coli</i> FtsY:fragments reveal the fragments bind in the targeted RNA interaction site. Our results show that FBDD is a suitable approach for targeting FtsY:SRP-RNA for antibiotic development and opens the possibility of targeting protein:RNA interactions in general.</p></div

The Structure of an LIM-Only Protein 4 (LMO4) and Deformed Epidermal Autoregulatory Factor-1 (DEAF1) Complex Reveals a Common Mode of Binding to LMO4

<div><p>LIM-domain only protein 4 (LMO4) is a widely expressed protein with important roles in embryonic development and breast cancer. It has been reported to bind many partners, including the transcription factor Deformed epidermal autoregulatory factor-1 (DEAF1), with which LMO4 shares many biological parallels. We used yeast two-hybrid assays to show that DEAF1 binds both LIM domains of LMO4 and that DEAF1 binds the same face on LMO4 as two other LMO4-binding partners, namely LIM domain binding protein 1 (LDB1) and C-terminal binding protein interacting protein (CtIP/RBBP8). Mutagenic screening analysed by the same method, indicates that the key residues in the interaction lie in LMO4_LIM2 and the N-terminal half of the LMO4-binding domain in DEAF1. We generated a stable LMO4_LIM2-DEAF1 complex and determined the solution structure of that complex. Although the LMO4-binding domain from DEAF1 is intrinsically disordered, it becomes structured on binding. The structure confirms that LDB1, CtIP and DEAF1 all bind to the same face on LMO4. LMO4 appears to form a hub in protein-protein interaction networks, linking numerous pathways within cells. Competitive binding for LMO4 therefore most likely provides a level of regulation between those different pathways.</p></div

Engineering tethered LMO4_LIM2•DEAF1_404–418 and DEAF1_404–418•LMO4_LIM2 complexes.

<p>(A) Schematics of full-length LMO4 (blue) and DEAF1 (orange) and engineered ‘intramolecular complexes’ of LMO4_LIM2 and DEAF1_404–418. The complexes are tethered via a glycine-serine linker (red) either from the C-terminus of LMO4 to the N-terminus of DEAF1 or vice versa. SAND, coiled-coil (CC) and MYND domains, and nuclear localisation (NLS) and nuclear export (NES) signals in DEAF1 and the LIM1 and LIM2 domains in LMO4 are indicated. (B) MALLS analysis of tethered constructs as indicated; protein concentrations at the detectors are 30 µM. Lines represent the refractive index and calculated molecular weights are shown as symbols. Monomeric BSA (blue) was used as a standard. (C) ¹⁵N-HSQC spectra of LMO4_LIM2•DEAF1_404–418 (black) and DEAF1_404–418•LMO4_LIM2 (red) were recorded in 20 mM sodium acetate at pH 5.0, 35 mM NaCl and 0.5 mM TCEP-HCl at 298 K on a 600 MHz spectrometer.</p

NMR restraints and refinement statistics for LMO4_LIM2DEAF1_404–418.

a<p>There were no dihedral angle violations >5°.</p>b<p>Full parameter and topology files are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109108#pone.0109108.s001" target="_blank">File S1</a>.</p>c<p>Regions of LMO4 between residues 86–139 and of DEAF1 between residues 404–414 including S208 of the glycine-serine linker were considered to be structured because the residues contained within had sum of angle order parameters (φ + ψ)>1.8 except for residues 103–105 of LMO4 and residues 404, 406 and 407 of DEAF1.</p>d<p>Distance violations were restricted to disordered regions of the protein.</p><p>NMR restraints and refinement statistics for LMO4_LIM2DEAF1_404–418.</p

Yeast two-hybrid and mutagenic analysis of LMO4-DEAF1 binding.

<p>(A) Data showing the interaction of DEAF1_45–566 (DEAF1) or control (empty) with the tandem LIM domains of LMO4 (LMO4), the isolated LIM domains of LMO4 (LIM1 and LIM2) or a pre-formed LMO4•LDB1_LID complex. These were spotted onto low-stringency interaction plates or growth control plates. “Empty” refers to pGAD10 vector lacking an insert. (B) Summary of yeast two-hybrid work. Surface residues of LMO4 that when mutated strongly affected (red), attenuated (orange) or had no effect (blue) on interaction with DEAF1 are mapped onto the structure of LMO4•LDB1_LID (1RUT). Non-mutated residues are in white, and LDB1_LID is shown as dark sticks. (C) Mutagenic scanning of the minimal LMO4-binding domain of DEAF1 (in the DEAF1_{404–438_457–479} construct). Residues in DEAF1_404–438 were systematically mutated to alanine or glycine in sets of three (or two) as indicated and analysed for binding to LMO4 using yeast two-hybrid assays. Co-transformants were spotted onto selective media (low, medium and high stringency plates) as well as growth control plates. The sequence of DEAF1 is coloured according to whether the mutation strongly affected binding (red), attenuated binding (orange) or had no effect (blue) compared to wild-type positive control on each plate (“wt”). Thick white lines indicate separate plates.</p

Relaxation analysis of LMO4_LIM2•DEAF1_404–418.

<p>(A) Longitudinal (<i>T</i>₁), (B) transverse (<i>T</i>₂) relaxation time constants, (C) heteronuclear NOEs, calculated as the ratio of peak intensities with and without proton saturation, all at 600 MHz. (D) Lipari-Szabo (S²) parameters for each assigned backbone amide group in LMO4_LIM2•DEAF1_404–418 calculated from data recorded at 600 MHz and 800 MHz, using the program relax. Error bars represent one standard deviation from the curve fit for each residue. Background colours indicate regions belonging to LMO4 (blue), DEAF1 (yellow) or the glycine-serine linker (G/S; grey).</p

LMO4 is a protein-protein interaction network hub linking multiple cellular processes.

<p>Protein-protein interaction network assembled from data reported for mouse and human LMO4 proteins from the STRING protein-protein interaction database, plus additional papers cited in the introduction. Bold lines indicate protein-protein interactions that have been characterised structurally. Other lines indicate reported interactions that have different levels of evidence and some of these lines may represent indirect interactions. Proteins are loosely grouped into cellular processes.</p