A Photoswitchable Neurotransmitter Analogue Bound to Its Receptor

Incorporation of the azobenzene derivative <i>gluazo</i>, a synthetic photochromic ligand, into a kainate receptor allows for the optical control of neuronal activity. The crystal structure of <i>gluazo</i> bound to a dimeric GluK2 ligand-binding domain reveals one monomer in a closed conformation, occupied by <i>gluazo</i>, and the other in an open conformation, with a bound buffer molecule. The glutamate group of <i>gluazo</i> interacts like the natural glutamate ligand, while its <i>trans</i>-azobenzene moiety protrudes into a tunnel. This elongated cavity presumably cannot accommodate a <i>cis</i>-azobenzene, which explains the reversible activation of the receptor upon photoisomerization

Structural Basis for the Specific Cotranslational Incorporation of <i>p</i>‑Boronophenylalanine into Biosynthetic Proteins

The site-specific incorporation of the non-natural amino acid <i>p</i>-boronophenylalanine (Bpa) into recombinant proteins enables the development of novel carbohydrate-binding functions as well as bioorthogonal chemical modification. To this end, Bpa is genetically encoded by an amber stop codon and cotranslationally inserted into the recombinant polypeptide chain at the ribosome by means of an artificial aminoacyl-tRNA synthetase (aaRS) in combination with a compatible suppressor tRNA. We describe the crystal structure of an aaRS specific for Bpa, which had been engineered on the basis of the TyrRS from <i>Methanocaldococcus jannaschii</i>, in complex with both Bpa and AMP. The substrates are bound in an orientation resembling the aminoacyl-AMP mixed anhydride intermediate and engaged in a network of four hydrogen bonds that allows specific recognition of the boronate moiety by the aaRS. The key determinant of this interaction is the coplanar alignment of its Glu162 carboxylate group with Bpa, which results in a double hydrogen bond with the boronic acid substituent. Our structural study elucidates how a small set of five side chain exchanges within the TyrRS active site can switch its substrate specificity to the hydrophilic amino acid Bpa, thus stimulating the reprogramming of other aaRS to recruit useful non-natural amino acids for next-generation protein engineering

Molecular Design of ⁶⁸Ga- and ⁸⁹Zr-Labeled Anticalin Radioligands for PET-Imaging of PSMA-Positive Tumors

Anticalin proteins directed against the prostate-specific membrane antigen (PSMA), optionally having tailored plasma half-life using PASylation technology, show promise as radioligands for PET-imaging of xenograft tumors in mice. To investigate their suitability, the short-circulating unmodified Anticalin was labeled with 68Ga (τ1/2 = 68 min), using the NODAGA chelator, whereas the half-life extended PASylated Anticalin was labeled with 89Zr (τ1/2 = 78 h), using either the linear chelator deferoxamine (Dfo) or a cyclic derivative, fusarinine C (FsC). Different PSMA targeting Anticalin versions (optionally carrying the PASylation sequence) were produced carrying a single exposed N- or C-terminal Cys residue and site-specifically conjugated with the different radiochelators via maleimide chemistry. These protein conjugates were labeled with radioisotopes having distinct physical half-lives and, subsequently, applied for PET-imaging of subcutaneous LNCaP xenograft tumors in CB17 SCID mice. Uptake of the protein tracers into tumor versus healthy tissues was assessed by segmentation of PET data as well as biodistribution analyses. PET-imaging with both the 68Ga-labeled plain Anticalin and the 89Zr-labeled PASylated Anticalin allowed clear delineation of the xenograft tumor. The radioligand A3A5.1-PAS(200)-FsC·89Zr, having an extended plasma half-life, led to a higher tumor uptake 24 h p.i. compared to the 68Ga·NODAGA-Anticalin imaged 60 min p.i. (2.5% ID/g vs 1.2% ID/g). Pronounced demetallation was observed for the 89Zr·Dfo-labeled PASylated Anticalin, which was ∼50% lower in the case of the cyclic radiochelator FsC (p < 0.0001). Adjusting the plasma half-life of Anticalin radioligands using PASylation technology is a viable approach to increase radioisotope accumulation within the tumor. Furthermore, 89Zr-ImmunoPET-imaging using the FsC radiochelator is superior to that using Dfo. Our strategy for the half-life adjustment of a tumor-targeting Anticalin to match the physical half-life of the applied radioisotope illustrates the potential of small binding proteins as an alternative to antibodies for PET-imaging

Comparison of <i>B. subtilis</i> YxeF NMR structure and <i>B. amyloliquefaciens</i> A7ZAF5 homology model.

<p>Surface electrostatic potential calculated for (A) the YxeF NMR structure (first conformer of ensemble deposited in the PDB) and (B) the homology model of A7ZAF5 by using the program GRASP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Petrey2" target="_blank">[56]</a> accessed through the protein function annotation server MarkUs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Petrey1" target="_blank">[55]</a>. The homology model was calculated using the SWISS-MODEL server in alignment mode <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Altschul1" target="_blank">[60]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Arnold1" target="_blank">[61]</a> and Verify3D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Luthy1" target="_blank">[63]</a>, Procheck <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Laskowski1" target="_blank">[64]</a> and ProsaII <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Sippl1" target="_blank">[65]</a> all atom z-scores (-1.12, −3.43 and −1.61, respectively) were obtained using the PSVS server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Bhattacharya2" target="_blank">[66]</a> and are indicative of a good quality model. In (C) and (D), ribbon drawings are shown for the structures of YxeF and A7ZAF5 in the same orientation, that is, viewed on the open end of the β-barrels. The acidic residues giving rise to the negative potential inside the cavities are depicted in licorice representation and are labeled (black for YxeF, red for A7ZAF5). (E) Pfam multiple alignment of the sequences of all members of PF11631. Except for YxeF (P54945), the sequences are labeled with their UniProt <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-UniProt1" target="_blank">[25]</a> IDs (D4G3V0, E8VFY0, E0TYE6, D5MWC1, E3E109, A7ZAF5, E1UTS8). Amino acid background colors reflect average similarity inferred from the Blosum62 matrix, ranging from ‘most conserved’ (black) to ‘least conserved’ (white). YxeF and A7ZAF5 are highlighted in bold on the left and the region of the alignment used for building the comparative model of A7ZAF5 from the YxeF structure is enclosed by red boxes. The acidic residues labeled in (C) and (D) are marked with black (YxeF) and red (A7ZAF5) asterisks, respectively, above or below the alignment.</p

2D [¹⁵N,¹H] HSQC spectra of lipoprotein YxeF.

<p>(A) Spectrum recorded for the sample used for NMR structure determination at 750 MHz ¹H resonance frequency. Resonance assignments are indicated using the one-letter amino acid code. Signals arising from side chains (Asn H^δ2/N^δ2, Gln H^ε2/N^ε2, Arg H^ε/N^ε and Trp H^ε1/N^ε1) are labeled with (*) and folded signals are designated with (†) next to the residue number. Signals arising from the His purification tag were not sequence specifically assigned. The spectral region indicated by dotted lines comprises most of the signals arising from the β-barrel (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone-0037404-g002" target="_blank">Figure 2</a>) and is displayed for the spectra shown in (B). Those were recorded at different temperatures at 500 MHz ¹H resonance frequency (see text).</p

Comparison of β-barrels.

<p>Ribbon drawings of β-barrels of avidin (PDB ID 1AVD, green) in (A) and, after rotation by 180°, in (D); bacterial lipocalin Blc from <i>E. coli</i> (PDB ID 3MBT, orange) in (B) and (E); YxeF in (C) and (F) (PDB ID 2JOZ, blue). For clarity, the disordered terminal polypeptide segments of YxeF, as well as the corresponding segments in avidin and Blc, are not shown. In (A)–(C), β-strands A and H are labeled, while in (D)–(F) β-strand D is indicated.</p

Comparison of YxeF NMR structure (PDB ID 2JOZ, coded in blue) and Blc X-ray crystal structure (PDB ID 3MBT, orange).

<p>(A) Structure-based sequence alignment between YxeF and Blc obtained with the program DALI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037404#pone.0037404-Holm1" target="_blank">[16]</a>. The three structurally conserved regions (SCR1-3) typically found in lipocalins (see text) are boxed (continuous line for SCR1, which appears to be conserved in YxeF; dashed line for SCR2 and SCR3). Conserved residues being part of the calycin signature motif resulting in an interaction between Gly 36-X-Trp 38 in SCR1 and Arg 128 in SCR3 (see text) are highlighted using red boxes. Residues being part of the second hydrophobic core of Blc [see also (D] are highlighted using cyan boxes. (B) Superposition of the Trp and Arg residues being part of the calycin Gly-X-Trp and Arg motif in Blc (licorice representation, orange) and YxeF (line representation, all NMR conformers, blue). The superposition is obtained after superposition of the X-ray structure of Blc with each conformer of the NMR solution structure of YxeF (residues 32–132). (C) Structural superposition generated by the program DALI viewed from the open end of the β-barrels (for YxeF residues 32–132 were considered). In Blc, box 1 identifies the C-terminally located α-helix and box 2 the C-terminal β-strand, which are packed against the outside of the β-barrel and thereby form a second hydrophobic core (see D). (D) Ribbon drawing of the Blc structure with licorice representation of hydrophobic residues (in cyan) located in the C-terminal α-helix and on the outside of the β-barrel forming a second hydrophobic core [see also (C)].</p

Statistics of YxeF(19–144) NMR Structure.

a<p>Relative to pairs with non-degenerate chemical shifts.</p>b<p>Residues 37–40, 53–58, 62–69, 73–76, 80–86, 92–98, 105–110, 116–120, and 123–128.</p>c<p>Residues 33–40, 45–48, 51–87, 93–96, 99–101, 104–111, 115–131.</p>d<p>Backbone and side-chain heavy atoms of residues 37–39, 54–59, 63–64, 66, 68, 75, 77, 81–82, 84–86, 93–96, 98, 105–108, 115–118. Best-defined side chains are those exhibiting a displacement of less than 1 Å for their side chain heavy atoms after superposition of the β-strands for minimal r.m.s.d.</p

Schematic representation of secondary structure element topologies.

<p>(A) YxeF, (B) lipocalins and (C) fatty acid-binding proteins. β-strands are represented by arrows, α-helices by rectangles, and 3₁₀-helices by ellipses. N- and C-termini are indicated as N and C respectively, and the ‘Ω-type’ loop L1 shared by YxeF and lipocalins is labeled.</p

Serial magnetic cell enrichment of naturally occurring regulatory T cells.

<p>(a) Serial positive magnetic enrichment of CD4⁺CD25⁺CD45RA⁺ regulatory T cells (nTregs) from PBMCs. For pre-selection of CD4⁺ cells, PBMCs were first incubated with anti-CD4 Fab-multimers conjugated with <i>Strep</i>-Tactin-functionalized magnetic beads. The resulting positive fraction was then liberated from surface-bound label by D-biotin treatment and washed to remove anti-CD4 reagents. The second purification step comprised the selection for CD25 positive cells from the pre-selected CD4⁺ cell pool via specific anti-CD25 Fab bound to <i>Strep</i>-Tactin coated magnetic beads. Cell bound reagents were again removed from the resulting positive fraction by addition of D-biotin. In a third purification step, CD45RA⁺ cells were isolated from the enriched CD4⁺CD25⁺ cell population by using CD45RA-specific Fab-multimers conjugated to <i>Strep</i>-Tactin-coated magnetic beads. Living lymphocytes in the respective fractions of each selection step are shown. One representative experiment from five independent blood donors is shown. (b) Intracellular FoxP3 staining of triple positive enriched CD4⁺CD25⁺CD45RA⁺ regulatory T cells. (c) Overlay of the enriched CD4⁺CD25⁺CD45RA⁺ cell population (black dots) derived from serial magnetic selection as shown in (a) and the corresponding starting population (underlying grey dots). (d) Summary of cell purities obtained within each purification step of multiparameter magnetic bead-based nTregs purifications as performed in (a) with PBMCs derived from 5 different blood donors (left graph, mean values are indicated). In the right graph, yields (in %) of the target nTregs are shown; mean value is indicated. For all samples analyzed by flow cytometry, at least 50.000 events have been acquired.</p