Abstract P-4: Robust Method for Background Subtraction in Serial X-ray Diffraction Data

Background: Membrane receptors play an important role in signal transduction across the cell membrane in all living organisms. Their structural studies have been enabled by multiple technological breakthroughs in their heterologous expression, stabilization, crystallization, and crystallographic data collection as well as in cryogenic electron microscopy (cryoEM). During the last decade, serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has enabled structure determination of previously inaccessible proteins, including several G-protein-coupled receptors (GPCR), that produce only micrometer-sized crystals, thus paving the way towards understanding their activation mechanism and rational drug discovery. In addition to experimental difficulties, membrane protein structure determination is also often accompanied by data processing challenges. In particular, the lipidic cubic phase that serves as a carrier for membrane protein microcrystals, as well as various XFEL beam-shaping devices may generate substantial background scattering that could complicate the structure factor extraction from the diffraction images. Methods: In this work, we tested an adaptation of the denoising algorithm via matrix decomposition to XFEL-SFX data. We benchmarked its performance using high-background data from PAL-XFEL and established its applicability to serial crystallography image denoising, as well as compared it to the CrystFEL-based image denoising algorithm. Results: We find that, although the decomposition-based image denoising does not outperform CrystFEL median subtraction, it performs better than the integration without any additional subtraction. We find the non-negative matrix factorization performing better than more traditional singular-value decomposition methods, both in terms of visual interpretability and final data quality. Conclusion: We hope that this work will draw attention to background subtraction methods in structural biology, and will pave the way towards processing of most challenging datasets in structural biology, in particularly, those collected from membrane proteins

Small-wedge synchrotron and serial XFEL datasets for Cysteinyl leukotriene GPCRs

Structural studies of challenging targets such as G protein-coupled receptors (GPCRs) have accelerated during the last several years due to the development of new approaches, including small-wedge and serial crystallography. Here, we describe the deposition of seven datasets consisting of X-ray diffraction images acquired from lipidic cubic phase (LCP) grown microcrystals of two human GPCRs, Cysteinyl leukotriene receptors 1 and 2 (CysLT1R and CysLT2R), in complex with various antagonists. Five datasets were collected using small-wedge synchrotron crystallography (SWSX) at the European Synchrotron Radiation Facility with multiple crystals under cryo-conditions. Two datasets were collected using X-ray free electron laser (XFEL) serial femtosecond crystallography (SFX) at the Linac Coherent Light Source, with microcrystals delivered at room temperature into the beam within LCP matrix by a viscous media microextrusion injector. All seven datasets have been deposited in the open-access databases Zenodo and CXIDB. Here, we describe sample preparation and annotate crystallization conditions for each partial and full datasets. We also document full processing pipelines and provide wrapper scripts for SWSX and SFX data processing

Structure-Based Virtual Screening of Ultra-Large Library Yields Potent Antagonists for a Lipid GPCR

Cysteinyl leukotriene G protein-coupled receptors, CysLT1R and CysLT2R, regulate bronchoconstrictive and pro-inflammatory effects and play a key role in allergic disorders, cardiovascular diseases, and cancer. CysLT1R antagonists have been widely used to treat asthma disorders, while CysLT2R is a potential target against uveal melanoma. However, very few selective antagonist chemotypes for CysLT receptors are available, and the design of such ligands has proved to be challenging. To overcome this obstacle, we took advantage of recently solved crystal structures of CysLT receptors and an ultra-large Enamine REAL library, representing a chemical space of 680 M readily available compounds. Virtual ligand screening employed 4D docking models comprising crystal structures of CysLT1R and CysLT2R and their corresponding ligand-optimized models. Functional assessment of the candidate hits yielded discovery of five novel antagonist chemotypes with sub-micromolar potencies and the best Ki = 220 nM at CysLT1R. One of the hits showed inverse agonism at the L129Q constitutively active mutant of CysLT2R, with potential utility against uveal melanoma

Modulation of Toll-like receptor 1 intracellular domain structure and activity by Zn2+ ions

Toll-like receptors (TLRs) play an important role in the innate immune response. While a lot is known about the structures of their extracellular parts, many questions are still left unanswered, when the structural basis of TLR activation is analyzed for the TLR intracellular domains. Here we report the structure and dynamics of TLR1 toll-interleukin like (TIR) cytoplasmic domain in crystal and in solution. We found that the TLR1-TIR domain is capable of specific binding of Zn with nanomolar affinity. Interactions with Zn are mediated by cysteine residues 667 and 686 and C667 is essential for the Zn binding. Potential structures of the TLR1-TIR/Zn complex were predicted in silico. Using the functional assays for the heterodimeric TLR1/2 receptor, we found that both Zn addition and Zn depletion affect the activity of TLR1, and C667A mutation disrupts the receptor activity. Analysis of C667 position in the TLR1 structure and possible effects of C667A mutation, suggests that zinc-binding ability of TLR1-TIR domain is critical for the receptor activation

Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET

Single-molecule FRET experiments with human A2A adenosine receptor in its apo and agonist-bound states in lipid nanodiscs provide insights into its conformational dynamics and activation

Structural basis for receptor selectivity and inverse agonism in S1P5 receptors

The bioactive lysophospholipid sphingosine-1-phosphate (S1P) acts via five different subtypes of S1P receptors (S1PRs) - S1P1-5. S1P5 is predominantly expressed in nervous and immune systems, regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders, as well as carcinogenesis. Several S1PR therapeutic drugs have been developed to treat these diseases; however, they lack receptor subtype selectivity, which leads to side effects. In this article, we describe a 2.2 Å resolution room temperature crystal structure of the human S1P5 receptor in complex with a selective inverse agonist determined by serial femtosecond crystallography (SFX) at the Pohang Accelerator Laboratory X-Ray Free Electron Laser (PAL-XFEL) and analyze its structure-activity relationship data. The structure demonstrates a unique ligand-binding mode, involving an allosteric sub-pocket, which clarifies the receptor subtype selectivity and provides a template for structure-based drug design. Together with previously published S1PR structures in complex with antagonists and agonists, our structure with S1P5-inverse agonist sheds light on the activation mechanism and reveals structural determinants of the inverse agonism in the S1PR family

Metabolic Fate of Human Immunoactive Sterols in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) infection is among top ten causes of death worldwide, and the number of drug-resistant strains is increasing. The direct interception of human immune signaling molecules by Mtb remains elusive, limiting drug discovery. Oxysterols and secosteroids regulate both innate and adaptive immune responses. Here we report a functional, structural, and bioinformatics study of Mtb enzymes initiating cholesterol catabolism and demonstrated their interrelation with human immunity. We show that these enzymes metabolize human immune oxysterol messengers. Rv2266 – the most potent among them – can also metabolize vitamin D3 (VD3) derivatives. High-resolution structures show common patterns of sterols binding and reveal a site for oxidative attack during catalysis. Finally, we designed a compound that binds and inhibits three studied proteins. The compound shows activity against Mtb H37Rv residing in macrophages. Our findings contribute to molecular understanding of suppression of immunity and suggest that Mtb has its own transformation system resembling the human phase I drug-metabolizing system