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

A new twist of rubredoxin function in M. tuberculosis

Electron transfer mediated by metalloproteins drives many biological processes. Rubredoxins are a ubiquitous [1Fe-0S] class of electron carriers that play an important role in bacterial adaptation to changing environmental conditions. In Mycobacterium tuberculosis, oxidative and acidic stresses as well as iron starvation induce rubredoxins expression. However, their functions during M. tuberculosis infection are unknown. In the present work, we show that rubredoxin B (RubB) is able to efficiently shuttle electrons from cognate reductases, FprA and FdR to support catalytic activity of cytochrome P450s, CYP124, CYP125, and CYP142, which are important for bacterial viability and pathogenicity. We solved the crystal structure of RubB and characterized the interaction between RubB and CYPs using site-directed mutagenesis. Mutations that not only neutralize single charge but also change the specific residues on the surface of RubB did not dramatically decrease activity of studied CYPs. Together with isothermal calorimetry (ITC) experiments, the obtained results suggest that interactions are transient and not highly specific. The redox potential of RubB is −264 mV vs. Ag/AgCl and the measured extinction coefficients are 9931 M−1cm−1 and 8371 M−1cm−1 at 380 nm and 490 nm, respectively. Characteristic parameters of RubB along with the discovered function might be useful for biotechnological applications. Our findings suggest that a switch from ferredoxins to rubredoxins might be crucial for M. tuberculosis to support CYPs activity during the infection

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

XANES Measurements for Studies of Adsorbed Protein Layers at Liquid Interfaces

X-ray absorption near edge structure (XANES) spectra for protein layers adsorbed at liquid interfaces in a Langmuir trough have been recorded for the first time. We studied the parkin protein (so-called E3 ubiquitin ligase), which plays an important role in pathogenesis of Parkinson disease. Parkin contains eight Zn binding sites, consisting of cysteine and histidine residues in a tetracoordinated geometry. Zn K-edge XANES spectra were collected in the following two series: under mild radiation condition of measurements (short exposition time) and with high X-ray radiation load. XANES fingerprint analysis was applied to obtain information on ligand environments around zinc ions. Two types of zinc coordination geometry were identified depending on X-ray radiation load. We found that, under mild conditions, local zinc environment in our parkin preparations was very similar to that identified in hemoglobin, treated with a solution of ZnCl2 salt. Under high X-ray radiation load, considerable changes in the zinc site structure were observed; local zinc environment appeared to be almost identical to that defined in Zn-containing enzyme alkaline phosphatase. The formation of a similar metal site in unrelated protein molecules, observed in our experiments, highlights the significance of metal binding templates as essential structural modules in protein macromolecules

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