The GPR52 receptor is a Class-A orphan G protein-coupled receptor (GPCR) whose endogenous ligand remains elusive. Highly expressed in the brain, it represents a promising therapeutic target for treating psychiatric disease and Huntington’s disease. However, tool ligand and drug discovery have been largely hampered by a lack of structural information due largely to the low homology (<20%) of GPR52 to any known GPCR structure. We reported three high resolution human GPR52 structures with and without a bound ligand. According to the structures, we observed a unique configuration of extracellular loop 2 (ECL2) that occupies the orthosteric pocket, a novel side pocket and a special winding mode for Transmembrane helix 5 (TM5). Mutagenesis and functional assay suggested the self-activation by ECL2. These findings provide unprecedented insights into the structural basis of GPR52 ligand recognition that will be valuable for GPR52 deorphanization and will guide the design of diverse ligands with distinct pharmacological properties that have not yet been possible.

Integrative modeling is an increasingly important tool in structural biology, providing structures by combining data from varied experimental methods and prior information. As a result, molecular architectures of large, heterogeneous, and dynamic systems, such as the ~52 MDa Nuclear Pore Complex, can be mapped with useful accuracy, precision, and completeness. Key challenges in improving integrative modeling include expanding model representations, increasing the variety of input data and prior information, quantifying a match between input information and a model in a Bayesian fashion, inventing more efficient structural sampling, as well as developing better model validation, analysis, and visualization. In addition, two community-level challenges in integrative modeling are being addressed under the auspices of the Worldwide Protein Data Bank (wwPDB). First, the impact of integrative structures is maximized by PDB-Dev, a prototype wwPDB repository for archiving, validating, visualizing, and disseminating integrative structures. Second, the scope of structural biology is expanded by linking the wwPDB resource for integrative structures with archives of data that have not been generally used for structure determination but are increasingly important for computing integrative structures, such as data from various types of mass spectrometry, spectroscopy, optical microscopy, proteomics, and genetics. To address the largest of modeling problems, a type of integrative modeling called metamodeling is being developed; metamodeling combines different types of input models as opposed to different types of data to compute an output model. Collectively, these developments will facilitate the structural biology mindset in cell biology and underpin spatiotemporal mapping of the entire cell.

Axonal degeneration is responsible for disease progression and accumulation of disability in many neurodegenerative conditions. Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1) is a nicotinamide adenine dinucleotide (NAD⁺)- cleaving enzyme whose activation triggers axon destruction [1-4]. Loss of the biosynthetic enzyme NMNAT2, which converts nicotinamide mononucleotide (NMN) to NAD⁺, activates SARM1 via an unknown mechanism. Using crystallography, cryo-EM, NMR and biochemical assays, we demonstrate that SARM1 is activated by an increase in the ratio of NMN to NAD⁺ and show that both metabolites compete for binding to the autoinhibitory N-terminal armadillo repeat (ARM) domain of SARM1 [5]. We show that NMN binding disrupts ARM-TIR interactions in the full-length SARM1 octamer, enabling its TIR domains to self-associate and form a catalytic site capable of cleaving NAD⁺ [5]. These structural insights identify SARM1 as a metabolic sensor of the NMN/NAD⁺ ratio, define the mechanism of SARM1 activation, and may enable a path to the development of allosteric inhibitors that block SARM1 activation.

[1] Essuman, K. et al.(2017). Neuron 93, 1334-1343.

[2] Essuman, K. et al. (2018). Curr. Biol. 28, 421-430.

[3] Horsefield, S. et al. (2019). Science 365, 793-799.

[4] Wan, L. et al. (2019). Science 365, 799-803.

[5] Figley, M. et al. (2021). Neuron 109, 1118–1136.

De novo cysteine biosynthesis is a pathway responsible for metabolizing inorganic sulfur to produce L-cysteine, which holds significance in the cellular activities of several organisms, majorly plants and microbes. Absence of this pathway in humans, accompanied by differential roles of accumulated L-cysteine in aiding adaptation of microbes in the harsh host environment, promoting toxin inactivation, biofilm formation, and development of antimicrobial resistance (AMR), make this pathway a lucrative target for novel therapeutics. The pathway is fuelled by a bienzyme complex, the Cysteine Synthase Complex (CSC), made up of CysE and CysK enzymes, where a hexameric CysE binds two CysK dimers, one on either end, to further cysteine production in a two-step fashion from L-serine. Recognition of industrial value in addition to the therapeutic potential of this pathway urges the need for further molecular investigations for its optimal exploitation. However, the large size of the molecular complex has put to question the feasibility of resolving its 3-D structure, partially reasoning the inability to obtain a 3-D structure even after nearly three decades of the complex’s discovery. Klebsiella pneumoniae, a pathogen with its multi-drug resistance (MDR) recognized in WHO’s list of six extensively MDR pathogens grouped as ES‘K’APE group of pathogens. Targeting the CSC of K. pneumoniae is a promising approach to up our armamentarium against the growing menace caused by the pathogen.

The success of rational structure-based drug discovery is driven by the availability of a crystal structure of the target protein complex. With a recent study reporting the SAXS-modelled CSC from E. coli, we aim to model the CSC complex using the K. pneumoniae CysE structure previously made available by our group, adopting a knowledge-based approach. The present poster reports the purification and characterization of recombinant CSC from K. pneumoniae, as an essential first step in progressing towards understanding its structure and biochemical function. Purified recombinant CSC was obtained and subjected to - (i) gel filtration chromatography to separate and estimate molar mass of the complex, (ii) dynamic light scattering for determining hydrodynamic radius and verifying homogenous population, and (iii) negative stain electron microscopy to visualize the purified complex. Simultaneous utilization of in silico tools integrated with techniques of structural biology is being carried to predict the 3-D structure of CSC, and identify residues involved in protein-protein interface stabilization. Following physical analysis and attempts to crystallize the CSC complex obtained from K. pneumoniae we observed heterogeneity in the CSC preparation, with a flexible/dynamic nature of the association between the two interacting proteins posing considerable challenges. Additionally, identification of interacting residues using in silico predictions would facilitate a better understanding of useful molecular recognition inferences within CSC, which presently are far from being well-understood.

Arsenic is a widely distributed toxic metalloid that poses a significant threat to human health by contaminating ground water systems [1]. Arsenic can exist in both organic and inorganic forms, with arsenite (AsO₃^3-) and arsenate (AsO₄^3-), being toxic species. Although arsenic is hazardous to human health, some prokaryotes have developed unique mechanisms that utilise arsenite (AsO₃^3-) and arsenate (AsO₄^3-) for respiration and therefore as energy sources.

The organism Rhizobium sp. NT-26, respires with arsenite and employs the arsenite oxidase enzyme (Aio) for its crucial respiratory activity, which catalyzes the oxidation of arsenite (AsO₃^3-) to arsenate (AsO₄^3-). The Aio enzyme consists of a large catalytic subunit (AioA), which contains a molybdenum centre and a 3Fe-4S cluster, and a small subunit (AioB) containing a Rieske 2Fe-2S cluster. Arsenite is oxidized to arsenate at the Mo site,concomitantly reducing Mo(VI) to Mo(IV) [2]. The electrons are then passed to the 3Fe-4S cluster, the Rieske cluster in AioB and finally to an electron acceptor, which is cytochrome c₅₅₁ (cyt c₅₅₁) [2, 3]. Structures of interprotein transfer complexes are interesting as they form via extensive electrostatic interactions, which are highly transient. Structural flexibility at the protein-protein interface promotes fast dissociation of the complex following electron transfer [4]. To date, the structure of the Aio and cyt c₅₅₁ complex has not been investigated, and the kinetics and thermodynamics of the Aio to cyt c₅₅₁ interaction are unknown. In this study, we describe the structure of the Aio/cyt c₅₅₁ complex, determined by X-ray crystallography. The structure provides insight into various types of interactions (hydrogen bonding, salt bridges and electrostatic interactions) of the complex that can be studied further to understand the mechanism and specificity between the partner proteins during electron transfer.

[1] H. V. Aposhian, and M. M. Aposhian, “Arsenic toxicology: five questions,” Chem Res Toxicol, vol. 19, no. 1, pp.1-15, Jan, 2006.

[2] T. P. Warelow, M. Oke, B. Schoepp-Cothenet, J. U. Dahl, N. Bruselat, G. N. Sivalingam, S. Leimkühler, K.

Thalassinos, U. Kappler, and J. H. Naismith, “The respiratory arsenite oxidase: structure and the role of residues surrounding the rieske cluster,” PLoS One, vol. 8, no. 8, pp. e72535, 2013.

[3] P. J. Ellis, T. Conrads, R. Hille, and P. Kuhn, “Crystal structure of the 100 kDa arsenite oxidase from Alcaligenes faecalis in two crystal forms at 1.64 Å and 2.03 Å,” Structure, vol. 9, no. 2, pp. 125-132, 2001.

[4] D. Leys, and N. S. Scrutton, “Electrical circuitry in biology: emerging principles from protein structure,” Curr Opin Struct Biol, vol. 14, no. 6, pp. 642-7, Dec, 2004.

α-L-Fucosidases (EC 3.2.1.51) catalyse hydrolysis of the α-L-fucosyl moiety from the non-reducing terminus of oligosaccharides and glycoconjugates. New representatives are sought for their unique functional properties or particular specificity, especially in connection with the transglycosylation ability to enable targeted modification of compounds for biomedical applications. They belong to several glycosyl hydrolase families, GH29, GH95, GH139, GH141, and GH151, and utilize either the retaining or inverting mechanism. While members of some families, e.g. GH29, have been studied thoroughly, the structural information and mechanistic details for other families, including GH151, are missing.

In our previous studies [1,2] and our more recent results we bring structural and functional insights into the mechanism, active site complementation and specificity of two isoenzymes from bacterium Paenibacillus thiaminolyticus. The proteins were characterised using a range of biophysical techniques, small angle X-ray scattering, X-ray crystallography, and in silico analysis (substrate docking), together with assays of α-L-fucose hydrolysis and transglycosylation ability. The crystal structure of α-L-fucosidase isoenzyme 1 (GH29) showed a new and unusual organization of the enzyme in a hexamer, with the active sites exposed to the surrounding environment and suggested active site complementation. Mutagenesis and catalytic assays confirmed the first case of active site complementation in α-L-fucosidases [2]. Our recent crystal structure of isoenzyme 2 from the same bacterium brings the first structural insight into the GH151 family, with unexpected oligomerization, enclosure of the active site inside the oligomer, and, again, proven active site complementation. Mutations modifying the complemented amino acid lead to changes in the catalytic properties of both enzymes. The comparison on the level of structure, functional, and biophysical data for the two isoenzymes brings answers to some principal questions regarding α-L-fucosidase substrate specificity and raises new questions about the functionality and stability of complemented active sites within these families of α-L-fucosidases.

This work was supported by the Czech Academy of Sciences (86652036), by the European Regional Development Fund (CZ.02.1.01/0.0/0.0/15_003/0000447, CZ.02.1.01/0.0/0.0/16_013/0001776, CZ.1.05/1.1.00/02.0109), by the Ministry of Education, Youth and Sports of the Czech Republic (LM2015043 and LM2018127, support of Biocev-CMS – Crystallization, Biophysics and Diffraction facilities of CIISB, part of Instruct-ERIC).

1. Benešová E, Lipovová P, Krejzová J, Kovaľová T, Buchtová P, Spiwok V & Králová B (2015) α-L-Fucosidase isoenzyme iso2 from Paenibacillus thiaminolyticus. BMC Biotechnol. 15, 36.

2. Kovaľová T, Kovaľ T, Benešová E, Vodičková P, Spiwok V, Lipovová P, Dohnálek J (2019) Active site complementation and hexameric arrangement in the GH family 29; a structure–function study of α-L-fucosidase isoenzyme 1 from Paenibacillus thiaminolyticus. Glycobiology, 29(1), 59–73.