Activating an invertebrate bistable opsin with the all-trans 6.11 retinal analog.

Animal vision depends on opsins, a category of G protein-coupled receptor (GPCR) that achieves light sensitivity by covalent attachment to retinal. Typically binding as an inverse agonist, 11-cis retinal photoisomerizes to the all-trans isomer and activates the receptor, initiating downstream signaling cascades. Retinal bound to bistable opsins isomerizes back to the 11-cis state after absorption of a second photon, inactivating the receptor. Bistable opsins are essential for invertebrate vision and nonvisual light perception across the animal kingdom. While crystal structures are available for bistable opsins in the inactive state, it has proven difficult to form homogeneous populations of activated bistable opsins either via illumination or reconstitution with all-trans retinal. Here, we show that a nonnatural retinal analog, all-trans retinal 6.11 (ATR6.11), can be reconstituted with the invertebrate bistable opsin, Jumping Spider Rhodopsin-1 (JSR1). Biochemical activity assays demonstrate that ATR6.11 functions as a JSR1 agonist. ATR6.11 binding also enables complex formation between JSR1 and signaling partners. Our findings demonstrate the utility of retinal analogs for biophysical characterization of bistable opsins, which will deepen our understanding of light perception in animals.

A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron-binding protein FutA from Prochlorococcus.

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.

Structural basis of calmodulin modulation of the rod cyclic nucleotide-gated channel.

Calmodulin (CaM) regulates many ion channels to control calcium entry into cells, and mutations that alter this interaction are linked to fatal diseases. The structural basis of CaM regulation remains largely unexplored. In retinal photoreceptors, CaM binds to the CNGB subunit of cyclic nucleotide-gated (CNG) channels and, thereby, adjusts the channel's Cyclic guanosine monophosphate (cGMP) sensitivity in response to changes in ambient light conditions. Here, we provide the structural characterization for CaM regulation of a CNG channel by using a combination of single-particle cryo-electron microscopy and structural proteomics. CaM connects the CNGA and CNGB subunits, resulting in structural changes both in the cytosolic and transmembrane regions of the channel. Cross-linking and limited proteolysis-coupled mass spectrometry mapped the conformational changes induced by CaM in vitro and in the native membrane. We propose that CaM is a constitutive subunit of the rod channel to ensure high sensitivity in dim light. Our mass spectrometry-based approach is generally relevant for studying the effect of CaM on ion channels in tissues of medical interest, where only minute quantities are available.

Ultrafast structural changes direct the first molecular events of vision.

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.

Correction of rhodopsin serial crystallography diffraction intensities for a lattice-translocation defect.

Rhodopsin is a G-protein-coupled receptor that detects light and initiates the intracellular signalling cascades that underpin vertebrate vision. Light sensitivity is achieved by covalent linkage to 11-cis retinal, which isomerizes upon photo-absorption. Serial femtosecond crystallography data collected from rhodopsin microcrystals grown in the lipidic cubic phase were used to solve the room-temperature structure of the receptor. Although the diffraction data showed high completeness and good consistency to 1.8 Šresolution, prominent electron-density features remained unaccounted for throughout the unit cell after model building and refinement. A deeper analysis of the diffraction intensities uncovered the presence of a lattice-translocation defect (LTD) within the crystals. The procedure followed to correct the diffraction intensities for this pathology enabled the building of an improved resting-state model. The correction was essential to both confidently model the structure of the unilluminated state and interpret the light-activated data collected after photo-excitation of the crystals. It is expected that similar cases of LTD will be observed in other serial crystallography experiments and that correction will be required in a variety of systems.

Discovering cell-active BCL6 inhibitors: effectively combining biochemical HTS with multiple biophysical techniques, X-ray crystallography and cell-based assays.

By suppressing gene transcription through the recruitment of corepressor proteins, B-cell lymphoma 6 (BCL6) protein controls a transcriptional network required for the formation and maintenance of B-cell germinal centres. As BCL6 deregulation is implicated in the development of Diffuse Large B-Cell Lymphoma, we sought to discover novel small molecule inhibitors that disrupt the BCL6-corepressor protein-protein interaction (PPI). Here we report our hit finding and compound optimisation strategies, which provide insight into the multi-faceted orthogonal approaches that are needed to tackle this challenging PPI with small molecule inhibitors. Using a 1536-well plate fluorescence polarisation high throughput screen we identified multiple hit series, which were followed up by hit confirmation using a thermal shift assay, surface plasmon resonance and ligand-observed NMR. We determined X-ray structures of BCL6 bound to compounds from nine different series, enabling a structure-based drug design approach to improve their weak biochemical potency. We developed a time-resolved fluorescence energy transfer biochemical assay and a nano bioluminescence resonance energy transfer cellular assay to monitor cellular activity during compound optimisation. This workflow led to the discovery of novel inhibitors with respective biochemical and cellular potencies (IC50s) in the sub-micromolar and low micromolar range.

Optimizing Shape Complementarity Enables the Discovery of Potent Tricyclic BCL6 Inhibitors.

To identify new chemical series with enhanced binding affinity to the BTB domain of B-cell lymphoma 6 protein, we targeted a subpocket adjacent to Val18. With no opportunities for strong polar interactions, we focused on attaining close shape complementarity by ring fusion onto our quinolinone lead series. Following exploration of different sized rings, we identified a conformationally restricted core which optimally filled the available space, leading to potent BCL6 inhibitors. Through X-ray structure-guided design, combined with efficient synthetic chemistry to make the resulting novel core structures, a >300-fold improvement in activity was obtained by the addition of seven heavy atoms.

Trapping and structural characterisation of a covalent intermediate in vitamin B6 biosynthesis catalysed by the Pdx1 PLP synthase.

The Pdx1 enzyme catalyses condensation of two carbohydrates and ammonia to form pyridoxal 5-phosphate (PLP) via an imine relay mechanism of carbonyl intermediates. The I333 intermediate characterised here using structural, UV-vis absorption spectroscopy and mass spectrometry analyses rationalises stereoselective deprotonation and subsequent substrate assisted phosphate elimination, central to PLP biosynthesis.

Into Deep Water: Optimizing BCL6 Inhibitors by Growing into a Solvated Pocket.

We describe the optimization of modestly active starting points to potent inhibitors of BCL6 by growing into a subpocket, which was occupied by a network of five stably bound water molecules. Identifying potent inhibitors required not only forming new interactions in the subpocket but also perturbing the water network in a productive, potency-increasing fashion while controlling the physicochemical properties. We achieved this goal in a sequential manner by systematically probing the pocket and the water network, ultimately achieving a 100-fold improvement of activity. The most potent compounds displaced three of the five initial water molecules and formed hydrogen bonds with the remaining two. Compound 25 showed a promising profile for a lead compound with submicromolar inhibition of BCL6 in cells and satisfactory pharmacokinetic (PK) properties. Our work highlights the importance of finding productive ways to perturb existing water networks when growing into solvent-filled protein pockets.

Achieving In Vivo Target Depletion through the Discovery and Optimization of Benzimidazolone BCL6 Degraders.

Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein-protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.

Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis.

Substrate channeling has emerged as a common mechanism for enzymatic intermediate transfer. A conspicuous gap in knowledge concerns the use of covalent lysine imines in the transfer of carbonyl-group-containing intermediates, despite their wideuse in enzymatic catalysis. Here we show how imine chemistry operates in the transfer of covalent intermediates in pyridoxal 5'-phosphate biosynthesis by the Arabidopsis thaliana enzyme Pdx1. An initial ribose 5-phosphate lysine imine is converted to the chromophoric I320 intermediate, simultaneously bound to two lysine residues and partially vacating the active site, which creates space for glyceraldehyde 3-phosphate to bind. Crystal structures show how substrate binding, catalysis and shuttling are coupled to conformational changes around strand ß6 of the Pdx1 (ßα)8-barrel. The dual-specificity active site and imine relay mechanism for migration of carbonyl intermediates provide elegant solutions to the challenge of coordinating a complex sequence of reactions that follow a path of over 20 Å between substrate- and product-binding sites.