Conference Agenda

Cytochrome c oxidase or mitochondrial respiratory chain complex IV catalyses the transfer of electrons from cytochrome c in the intermembrane space, to molecular oxygen in the matrix and therefore contributes to the proton gradient that drives mitochondrial ATP synthesis. Complex IV dysfunction is a significant cause of human mitochondrial disease. Complex IV requires the incorporation of three copper ions, heme a and heme a₃ cofactors for the assembly and activity of the complex. Complex IV assembly factors are required for subunit maturation, co-factor incorporation and stabilization of intermediate assemblies of complex IV in humans. Loss-of-function mutations in several genes encoding complex IV assembly factors have been shown to result in diminished complex IV activity and severe pathologic conditions in affected infants [1].

Our study focuses on two mitochondrial complex IV assembly factors, Coa6 and Coa7, that are located in the intermembrane space of mitochondria and contain intramolecular disulfide bonds. Coa6 binds copper with femtomolar affinity and has been proposed to play a role in the biogenesis of the Cu_A site of complex IV [2,3]. The W59C pathogenic mutation in Coa6 does not affect copper binding or import of the protein into mitochondria but affects the maturation and stability of the protein [3]. The precise role of Coa7 in the biogenesis of complex IV is not completely understood. However, patients with Coa7 pathogenic mutations suffer from mitochondrial diseases owing to complex IV deficiency. This presentation will describe the crystal structures of the Coa7 and Coa6 (wild-type and the W59C mutant) proteins and implications for their roles in complex IV assembly and function.

References:

[1] Timon-Gomez, A., Nyvltova, E., Abriata, L. A., Vila, A. J., Hosler, J., and Barrientos, A. (2018) Mitochondrial cytochrome c oxidase biogenesis: Recent developments, Seminars in cell & developmental biology 76, 163-178.

[2] Stroud, D. A., Maher, M. J., Lindau, C., Vögtle, F. N., Frazier, A. E., Surgenor, E., … Ryan, M. T. (2015). COA6 is a mitochondrial complex IV assembly factor critical for biogenesis of mtDNA-encoded COX2. Human molecular genetics, 24(19), 5404–5415. doi:10.1093/hmg/ddv265

[3] Maghool, S., Cooray, N., Stroud, D. A., Aragão, D., Ryan, M. T., & Maher, M. J. (2019). Structural and functional characterization of the mitochondrial complex IV assembly factor Coa6. Life science alliance, 2(5), e201900458. doi:10.26508/lsa.2019004583.

Phosphoinositide-dependent protein kinase 1 (PDK1) is a master AGC kinase of the PI3K signalling pathway that phosphorylates at least other 23 AGC kinases, being PKB/Akt the most relevant substrate for growth and cell survival, and therefore a potential drug target for cancer treatment. Over the years, our laboratory used a chemical and structural biology approach to study and characterize in detail the allosteric regulation of the catalytic domain of PDK1. We developed small compounds that bind to a regulatory site we termed the PIF-pocket and activate PDK1, mimicking the mechanism of activation of AGC kinases by phosphorylation.

Using an integrative approach between biochemistry, crystallography and molecular dynamics, we showed how PS653, a small compound that binds to the active ATP-Binding site, displaces through a reverse allosteric mechanism the in vitro interaction between the PIF-pocket and PIFtide, which is a peptide derived from the hydrophobic motif of a PDK1 substrate. Thus, we not only demonstrated an allosteric regulation from a regulatory site to the active site, but also showed experimentally the existence of the reverse process [1]. This bidirectional allosteric mechanism of regulation between both pockets can therefore be modulated by small molecules that bind to their specific orthosteric site and either enhance or inhibit interactions at the allosteric site. Taking this into consideration, it is not surprising that while the pharmaceutical industry has been developing compounds that bind at the ATP-binding site of kinases, they unwillingly developed drugs that affect protein–protein interactions [2]. Moreover, we now provide further evidence of the bidirectional system using hydrogen/deuterium exchange (HDX) experiments and present a rather complete model for a kinase that can be modulated bidirectionally with small compounds. This concept of bidirectional allostery in kinases can be exploited to produce drugs that enhance or disrupt the formation of multi-protein complexes. Could this mechanism be already in use physiologically? We found out that adenosine binds at the ATP-binding site and allosterically enhances the interaction between PIFtide and PDK1, which demonstrates that bidirectional allostery is a phenomenum that can also be modulated by metabolites. But interestingly, adenine, AMP, ADP, or ATP do not produce this effect. The findings open the possibility that the physiological regulation of the kinase complexes may be modulated by metabolites and implies that the metabolic state of cells could be linked to the regulation of cell signalling.

As a master kinase tightly regulated, PDK1 possesses a selective activation of substrates such as SGK or S6K, which in order to be phosphorylated require a docking interaction of their C-terminal hydrophobic motifs with the PIF-Pocket of PDK1. However, this is not the case of Akt/PKB, since it can be activated in a PIF-Pocket independent way. In this line, we and others showed that small compounds that bind to the PIFpocket of PDK1 block the phosphorylation of S6K, but do not affect the phosphorylation of PKB/Akt by PDK1[3]. However, up to date little is known about the mechanistic and structural details of PDK1 full length. We are currently using an interdisciplinary approach to understand how the full-length protein is regulated and if this regulation mechanism can be manipulated to specifically inhibit the activation of PKB/Akt. As a result of a medium-scale screening of small compounds, we validated a series of “hits” that modulate PDK1 structure by interaction at different sites on PDK1. We here present a series of results obtained using HDX and SAXS experiments on full length PDK1, as well as the crystal structure of the catalytic domain of PDK1 bound to a small compound that stabilizes a particular PDK1 conformation.

The new data is used to present an updated model on the molecular mechanism of regulation of full length PDK, in which we not only show the existence of bidirectional allostery but also the existence of 3 different conformations of full length PDK1.

[1] Schulze, J.O., Saladino, G., Busschots, K., Neimanis, S., Suess, E., Odadzic, D., Zeuzem, S., Hindie, V., Herbrand, A.K., Lisa, M.N., Alzari, P.M., Gervasio, F.L. and Biondi, R.M. Bidirectional Allosteric Communication between the ATP-Binding Site and the Regulatory PIF Pocket in PDK1 Protein Kinase. Cell Chem Biol, 2016. 23(10): p. 1193-1205.

[2] Leroux, A.E. and Biondi R.M, Renaissance of Allostery to Disrupt Protein Kinase Interactions. Trends Biochem Sci, 2020.

[3] Busschots, K., Lopez-Garcia, L.A., Lammi, C., Stroba, A., Zeuzem, S., Piiper, A., Alzari, P.M., Neimanis, S., Arencibia, J.M., Engel, M., Schulze, J.O. and Biondi, R.M., Substrate-Selective Inhibition of Protein Kinase PDK1 by Small Compounds that Bind to the PIF-Pocket Allosteric Docking Site. Chem Biol, 2012. 19(9): p. 1152-63

Bacterial oxidoreductase enzymes are found in the periplasm of bacteria and are involved in protein thiol oxidation, reduction and isomerisation. These proteins contribute to folding and correcting disulfide bonds in a wide range of substrates, including virulence factors. Among oxidoreductases, the disulfide bond forming protein A (DsbA) for example, has been thoroughly studied, characterised and shown to be involved in the virulence of multiple pathogenic bacteria. More recently, other oxidoreductases have received attention too. This is the case for the suppressor of copper sensitivity proteins (SCS). One member of this family, ScsC, has been found to contribute to copper resistance in Salmonella enterica serovar Typhimurium (Subedi et al., 2019), and, is involved in disulfide bond isomerisation in the periplasm of the bacterium Proteus mirabilis (Furlong et al., 2018). The C-terminal catalytic domain of ScsC has an architecture similar to that of DsbA, displaying a conserved thioredoxin fold, including a CXXC catalytic motif and an embedded α-helical domain, however the N-terminal domain, responsible for the quaternary structure of the protein, varies strongly in between the proteins from different bacteria species. In S. Typhimurium the protein is monomeric while in P. mirabilis, it is trimeric. More interestingly, the catalytic activity of the protein seems to depend on these C-terminal oligomerisation domains. Here we report the crystal structure of a new trimeric ScsC protein from the model bacterium Caulobacter crescentus, termed CcScsC. The trimerization domain of CcScsC is comprised of a long N-terminal α helix, which assemble via hydrophobic contact between the helices of the different protomers as well as a number of electrostatic interactions between their charged residues. In addition CcScsC is shown to bind copper (I) with picomolar affinity and to have isomerase activity comparable to the known bacterial isomerase E. coli DsbC. In conclusion, we report the structure of a trimeric bacterial oxidoreductase, with a role in protein thiol isomerisation and copper binding.

Furlong, E. J., Choudhury, H. G., Kurth, F., Duff, A. P., Whitten, A. E. & Martin, J. L. (2018). The Journal of biological chemistry 293, 5793-5805.

Subedi, P., Paxman, J. J., Wang, G., Ukuwela, A. A., Xiao, Z. & Heras, B. (2019). The Journal of biological chemistry 294 15876-15888

L-Asparaginases from bacterial periplasm (e.g. EcAII) have high L-asparagine affinity and are used as potent antileukemic drugs. Plants possess a different, Ntn class of asparaginases, which are also found in bacteria (e.g. EcAIII). It was predicted ~20 years ago that Rhizobium etli, a bacterial symbiont of legume plants that is capable of nitrogen fixation, will possess yet another, R.etli-type L-asparaginase. The crystal structure of this enzyme, ReAII, reveals a dimeric protein that is indeed completely different from the EcAII and EcAIII prototypes, with structural resemblance to some serine β-lactamases and glutaminases. The presumed active site is organized around S48, which is surrounded by three tightly H-bonded water molecules and is further H-bonded to N134. Near-by there is a tandem of Cys residues coordinating a zinc cation. The coordination sphere is completed by a water molecule and a Lys side chain. Another Lys residue penetrates the active site to provide an H-bond link to S48. C225 of this Cys-rich protein also bears an unknown posttranslational modification.

Version:1.0 StartHTML:0000000183 EndHTML:0000038935 StartFragment:0000038461 EndFragment:0000038895 SourceURL:file:///Z:/home/mariusz/Dysk-E/mariusz/ABSTRACTS/IUCr-2020-mj.docx which is surrounded by three tightly H-bonded water molecules and is further

Pentatricopeptide (PPR) proteins with a C-terminal DYW domain have been characterized as site-specific factors for C to U RNA editing in plant mitochondria and plastids. While substrate recognition is conferred by the repetitive pentatricopeptide (PPR) tract, the exact role of the DYW domain, which can be also recruited to an editing site in trans, has not been clarified. The DYW domain, which was named by the highly conserved last three amino acids, aspartate, tyrosine, and tryptophan, has been proposed as the best candidate to elicit deamination employing a HxE(x)nCxxC zinc ion binding signature. Since DYW domains share a low sequence conservation with known deaminase structures (from 5 to 19% residue identities), modelling attempts have been conducted albeit with a limited reliability. Lastly, missing structural information has left the exact function and catalytic properties of DYW domains within the RNA editosome open.

We present structures and functional data of a DYW domain in an inactive ground state and a catalytically activated conformation. DYW domains harbour a cytidine deaminase fold and a C-terminal DYW motif, with catalytic and structural Zn atoms, respectively. The deaminase fold is interrupted by a conserved domain, which regulates the active site sterically via a large-scale conformational change and mechanistically via the Zn coordination geometry. Thus, we coined this novel domain 'gating domain' and the accompanying unusual metalloprotein regulation principle of DYW proteins 'gated Zn-shutter'. An autoinhibited ground state and its activation by the presence of either ATP, GTP or the inhibitor tetrahydro uridine is consolidated by differential scanning fluorimetry as well as in vivo and in vitro RNA editing assays. Our observations explain three decades of prior failed attempts to establish an in vitro RNA editing assay and impaired nucleotide binding of DYW domains. In vivo, the framework of an active plant RNA editosome triggers the release of DYW autoinhibition to ensure a controlled and coordinated deamination likely playing a key role in mitochondrial and chloroplast homeostasis.

Human myeloperoxidase (MPO) was first isolated in 1941 from purulent pleuritis fluid from tuberculosis patients. When neutrophilic polymorphonuclear leukocytes (neutrophils) entrap microbial or other invasive particulates, they release MPO during degranulation. In a respiratory burst of highly reactive oxygen species, MPO catalyzes the production of hypohalous acids, primarily hypochlorous acid in physiologic situations, from hydrogen peroxide. Mammal MPO crystal structures were progressively acquired and encoded in PDB with partial glycosylation identification. Actually, the N-glycan composition of native MPO had been thoroughly investigated with mass spectrometry and shows 5 N-glycans at positions 323, 355, 391, 483 and 729 [1]. MPO’s enzymatic activity was shown to be modulated by hyper-truncation of 2 out of 5 N-glycosylation sites [2].

In our obtained crystal structure at 2.6 Å resolution containing 4 disulfide-linked homodimers of MPO (Fig. 1), an interesting collection of glycans have been characterized using the iterative process of crystallographic refinement and model building. We compared those with the glycans from proteomics studies and from 18 human MPO structures in the PDB. We made use of the Symbol Nomenclature for Glycans (SNFG) to illustrate congruence in the experimental data. In conclusion, we found each of the 5 glycosylation sites either non-glycosylated or glycosylated with hyper-truncated paucimannosidic, high-mannose and complex N-glycans, with the N-acetyl-β-D-glucosamine (GlcNAc) core-type asparagine-linked glycans on Asn355 or Asn391 sites [2] gate-keeping the funnel towards the ROS-activated heme group. Our results perfectly illustrate the power of protein crystallography to resolve protein glycosylation.

Figure 1. (a) Glycosylation as part of protein crystal structures (2 MPO dimers), (b) a sweet handshake holds the dimer together

[1] Van Antwerpen, P., Slomianny, M. C., et al., (2010) Glycosylation pattern of mature dimeric leukocyte and recombinant monomeric myeloperoxidase: glycosylation is required for optimal enzymatic activity. J Biol Chem 285, 16351-16359. 10.1074/jbc.M109.089748

[2] Tjondro, H. C., Ugonotti, J., et al., (2020) Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase. J Biol Chem 10.1074/jbc.M109.089748

Keywords: myeloperoxidase; glycosylation; crystal structure, N-glycans

Neural precursor cell expressed developmentally down-regulated 4 ligase (Nedd4-2) is an E3 ubiquitin ligase that targets proteins for ubiquitination and endocytosis, thereby regulating numerous ion channels, membrane receptors and tumor suppressors. In turn, Nedd4-2 activity is regulated by autoinhibition, calcium binding, oxidative stress, substrate binding (through its WW domains), phosphorylation and 14-3-3 protein binding [1-3]. However, the structural basis of 14-3-3-mediated Nedd4-2 regulation remains poorly understood.

Here, we combined several techniques of integrative structural biology to characterize Nedd4-2 and its complex with 14-3-3. The results from our binding affinity and crystallographic analyses demonstrate that phosphorylated Ser³⁴² and Ser⁴⁴⁸ are the key residues that facilitate 14-3-3 protein binding to Nedd4-2 and that Ser⁴⁴⁸ is the dominant site. Moreover, 14-3-3 protein binding induces a structural rearrangement of Nedd4-2 by inhibiting interactions between its structured domains, including the N- and C-lobes of the catalytic HECT domain. Overall, our findings provide the first structural glimpse into the 14-3-3-mediated Nedd4-2 regulation and highlight the potential of the Nedd4-2:14-3-3 complex as a pharmacological target for Nedd4-2-associated diseases such as hypertension, epilepsy, kidney disease and cancer.

[1] P. Goel, J. A. Manning, and S. Kumar, Gene, 557, no. 1, pp. 1–10, Feb. 2015.

[2] H. He, C. Huang, Z. Chen, H. Huang, X. Wang and J. Chen, Biomed Pharmacother, 125, no. 1, pp. 109983, Feb. 2020.

[3] J. A. Manning and S. Kumar, Trends Biochem. Sci., 43, no. 8, pp. 635–647, Aug. 2018.

This study was supported by the Czech Science Foundation (Project 20-00058S), the Czech Academy of Sciences (Research Projects RVO: 67985823 of the Institute of Physiology) and by Grant Agency of Charles University (Project 740119).

Low G+C Gram-positive Firmicutes, such as the clinically important pathogens Staphylococcus aureus and Bacillus cereus, use the low-molecular weight (LMW) thiol bacillithiol (BSH) as a defense mechanism to buffer the intracellular redox environment and counteract oxidative stress encountered by human neutrophils during infections. The protein bacillithiol disulfide reductase Bdr has recently been shown to function as an essential NADPH-dependent reductase of oxidized bacillithiol disulfide (BSSB) resulting from stress responses and is crucial in maintaining the reduced pool of BSH and cellular redox balance. We have solved the first structures of Bdrs, namely from S. aureus and B. cereus [1]. Our analyses reveal a uniquely organized biological tetramer; however, the monomeric subunit has high structural similarity to other flavoprotein disulfide reductases. The absence of a redox active cysteine in the vicinity of the FAD isoalloxazine ring implies a new direct disulfide reduction mechanism, which is backed by the presence of a potentially gated channel, serving as a putative binding site for BSSB in proximity to the FAD cofactor. We also report enzymatic activity for both Bdrs, which along with the structures presented in this work provide important structural and functional insight into a new class of FAD-containing NADPH-dependent oxidoreductases, related to the emerging fight against pathogenic bacteria.

[1] Hammerstad, M., Gudim, I. & Hersleth, H.-P. (2020). Biochemistry. 59, 4793-4798.

Polysaccharide lyases are the biologically and industrially important enzymes, which catalyze non-hydrolytic degradation of polysaccharides via a beta-elimination reaction mechanism. There are 40 families of PLs in the CAZY database; classified based on their secondary structure elements and the folds. The PL-5 family enzyme adopts (alpha/alpha)_5,5 fold with an N-terminal lid-loop interaction giving rise to a pseudo-toroid architecture. Our research group work is focused on delineating the structure-function-dynamics for PL-5 family enzymes. To this end, the biochemical characterization has been carried on the selected among PL-5 enzymes to identify substrate specificity and enzyme efficiency. We have determined the X-ray crystal structures of the enzymes in apo and substrate-bound forms to understand structural aspects of substrate acquisition and specificity as a function of pH and the enzyme-substrate interactions. Further, the molecular dynamic simulation performed on the X-ray structures suggest the potential dynamics in loop configuration of the molecule to a closed and open state; providing mechanistic insights into functioning, and the mechanism of substrate acquisition and product expulsion in the PL-5 family enzymes.

The spread of resistances against antibiotics in bacteria is a serious global problem. In order to prevent the transfer of resistances it is crucial to understand the involved processes. Conjugative DNA transfer is the most important means to transfer antibiotic resistance genes among bacteria. It is present in Gram- positive (G⁺) and in Gram- negative (G^-) bacteria.

I am working on a Type IV Secretion System (T4SS) encoded on the broad-host-range plasmid pIP501 from Enterococcus faecalis. It can spread among different types of bacterial hosts and hence plays an important role in the propagation of multi drug resistant germs. Enterococci are abundant among humans and animals, which intensifies the problem. To date most of the structural information stems from G^- T4SS. Deciphering the mechanisms involved and solving the structure of the pore forming complex (PFC) would be of great help in the war against multidrug resistant bacteria.
Alongside the structural elucidation of the PFC, I am working on a DNA binding protein, namely TraM, which is a putative member of the PFC. The investigation of TraM includes biophysical, biochemical and structural characterization.
We are on the way to determine the residues of TraM, which are involved in DNA binding. We designed two different N-terminal constructs varying in length, TraM94 and TraM167. The structure of TraM94 was recently solved in our group. In contrast to the monomeric TraM94 TraM167 is a trimer in solution like the C-terminal domain of TraM, whose structure was solved in our group already some years ago.

Glutathione transferases (GSTs) are widespread enzymes involved in a number of catalytic and non-catalytic processes (1 and reference herein). They are mainly known as enzymes of the cellular phase II detoxification system where they catalyse the nucleophilic addition of glutathione (GSH) to a variety of small non-polar compounds. GSTs have been extensively investigated in animals and plants because of their great relevance to human health and agriculture. In contrast, studies in bacteria remain scarce, especially in the cyanobacteria phylum, which encompasses oxygenic photosynthetic prokaryotes with a wide range of morphologies and ecologies. They have key roles in global carbon and nitrogen cycles, contribute strongly to the fixation of atmospheric CO₂ and to its storage in ocean sediments (carbon sinks).

The best-studied cyanobacterium Synechocystis PCC6803, which has 6 GSTs, is an attractive organism for deciphering both GST selectivity and redundancy. Preliminary studies show that these GSTs play the expected roles in stress protection. Furthermore, a knockout mutant can be restored by human GST counterparts, demonstrating the conservation of functions throughout evolution (2). SynGSTC1 is involved in the detoxication of methylglyoxal, a toxic by-product of the cellular metabolism of most organisms (3). We solved the first crystal structure of a GST from cyanobacteria, namely that of SynGSTC1. It shows the putative active site signature SRAS and belongs to the Chi class of GSTs (Figure 1) and its sequence length is shorter by about 30 residues when compared to the usual GST length (~ 220 aa). SynGSTC1 adopts the canonical GST fold that consists of two domains (Figure 1) and exhibits structural similarities with the Ure2p class of GSTs (4). The structure-function relationships of SynGSTC1 will be presented using innovative tools based on molecular dynamic simulations and charge-density of ultra-high resolution structures.

Iron is an essential trace element required for a multitude of cellular processes [1]. When in excess iron becomes toxic, therefore its intracellular concentration must be strictly regulated by a number of interacting mechanisms [2]. Ferritin is a ubiquitous iron-storage protein that forms a highly conserved 24-subunit spherical cage-like structure. Ferritin catalyses the oxidation of iron (II) to iron (III) by dioxygen at a di-iron site called the ferroxidase centre, and the newly oxidised iron (III) is then sequestered as a mineral core to prevent cellular damage [3]. As part of a greater study to understand iron transport we utilise the model organism, Caenorhabditis elegans, to investigate and elucidate these processes.

C. elegans contains two ferritin proteins, FTN-1 and FTN-2, that are orthologous to the human ferritins [4]. FTN-1 and FTN-2 both exhibit ferroxidase activity, although FTN-2 catalyses the oxidation of iron (II) at a rate significantly faster than FTN-1. All residues involved in catalysis are conserved between FTN-1 and FTN-2, suggesting that these mechanistic anomalies are due to structural differences at a location distinct to the ferroxidase centre. To address this, we solved the structures of both FTN-1 and FTN-2 by X-ray crystallography to 1.84 Å and 1.47 Å resolution respectively, and the structure of FTN-2 by cryo electron microscopy to 1.88 Å. FTN-1 and FTN-2 both adopt the conserved 24-subunit cage-like structure and bind one metal in the higher affinity “A site” of the di-iron ferroxidase centre of each chain [3]. Further comparative analyses using both X-ray crystallography and electron microscopy techniques, reveal the structural features that influence iron influx, catalysis and transfer to the mineral core.

These structural insights will further our understanding of the mechanisms that ferritin utilizes to regulate iron storage and its role in the iron homeostasis. These findings will have further implications for diagnosis and treatment of haemochromatosis, anaemia and other iron related diseases.

[1] Anderson, G.J. & Frazer, D.M. (2017). Am. J. Clin. Nutr. 106, 1559S-1566S.

[2] Aisen, P., Enns, C. & Wessling-Resnick, M. (2001). Int. J. Biochem. Cell Biol. 33 (10), 940-959.

[3] Ebrahimi, K.H., Hagedoorn, P. & Hagen, W.R. (2015). Chem Rev. 115 (1), 295-326.[4] Anderson, C.P. & Leibold, E.A. (2014). Front Pharmacol. 5 (113).

Ferredoxin (Fd) is a redox protein containing the iron-sulfur cluster as the active site, and distributed in various organisms; archaea, bacteria, plants and animals. Specific Fd located in the stroma of chloroplast or cyanobacteria is called “plant-type” and possesses a [2Fe-2S] cluster ligated by four conserved cysteine residues. It carries one electron from Photosystem I reaction centre on the thylakoid membrane to several Fd-dependent enzymes. Unique feature of this plant-type Fd is its low redox-potential around -400 mV, which can reduce NADP+ to NADPH (E_m = -350 mV) in vivo¹. However in vitro, it means that the chemically reduced samples are easily oxidized by air and, on the other hand, the modern strong X-ray beam could reduce the oxidized form of crystallized sample². Previous structural analyses of Ser46, Phe64 and Glu93 mutants showed the structural basis for the redox potential increase of mutants by 50~90 mV³, while these mutated residues were completely conserved among the plant-type Fds³. Furthermore, X-ray crystallography on oxidized and reduced Fd from Anabaena showed that the peptide bond next to S46 flipped upon partial reduction⁴. Although several X-ray structures of plant-type Fds including above were available in the PDB, their redox states were not precisely controlled and probably in the mixed states. Consequently, it is not clear how dissociation/association between the plant-type Fd and partner proteins is controlled by one electron redox on the [2Fe-2S] cluster. Here, we solved the X-ray structures of oxidized Fd with minimum X-ray dose and fully reduced Fd from cyanobacterium Thermosynechococcus elongatus (TeFd) at 0.78 and 0.77 Å resolution, respectively. Both oxidized/reduced crystals had a space group of C2 and all used crystals were isomorphous. Our high-resolution structures newly reveal the redox-linked repositioning of Ser46, Phe64 and Glu93 (Fig 1). To investigate the reason how these structural changes are originated from the small but significant structural change in the [2Fe-2S] cluster, we solved the crystal structures of the oxidized/reduced S46A or F64A mutants of TeFd at 0.95~1.05 Å resolution, independently (Fig 2). All our obtained high-resolution structures imply how the small structural changes of the cluster are spatially amplified and propagated through the peptide chain. The detail of our discussion will be presented in our presentation.

[1] Cammack, R., Rao, K. K. & Bargeron, C. P. (1977) Biochem. J. 186 (2), 205–209.

[2] Ohnishi, Y., Muraki, N., Kiyota, D., Okumura, H., Baba, S., Kawano, Y., Kumasaka, T., Tanaka, H. & Kurisu, G. (2020) J. Biochem., 167, 549–555.

[3] Holden, H. M., Jacobson, B. L., Hurley, J. K., Tollin, G., Oh, B.-H., Skjeldal, L., Chae, Y. K., Cheng, H., Xia, B. & Markley, J. L. (1994)Journal of Bioenergetics and Biomembrane, 26 (1).

[4] Morales, R., Chron, M. H., Hudry-Clergeon, G., Pétillot, Y., Norager, S., Medina, M. & Frey, M. (1999) Biochemistry 38 (48), 15764–15773.

Green chemistry paradigm has been raised to reduce environmental damages during process of products. American Chemical Society suggested 12 principals such as atom economy, safter chemicals, energy efficiency, degradable products and less hazardous chemical syntheses [1]. One of alternatives for green chemistry is using biocatalysts, whose endogenous characters are attractive in the following aspects: decrease synthesis procedures, less side products, and mild reaction condition. Microbial enzymes are one of promising sources for biocatalysts in industrial processes to produce biofuel from biomass and building blocks [2]. We identified the structure of the carbohydrate esterase, SmAcE1 from Sinorhizobium meliloti [3] . The crystal structure of SmAcE1 was determined at 2.05 Å resolution, and revealed that it belonged to an α/β hydrolase fold in GDSL superfamily. It formed a hexameric structure by dimer of trimers with supporting of size exclusion chromatography analysis. Catalytic triad (Ser15, His195 and Asp192) and an oxyanion hole-forming SGNH (Ser15, Gly57, Asn97 and His195) were also conserved in its three dimensional structure. The docking analysis to acetylate substrates showed the hydrophilic residues on its surface are important in substrate binding. The models from crystal structure and docking analysis suggest the industrially applicable potency of SmAcE1 after enhancement of its selectivity and activity by further structure-based engineering.

Deoxyhypusine synthase (DHS) is a transferase catalysing the formation of deoxyhypusine, which is the first, rate-limiting step of unique post-translational modification: hypusination. DHS catalyzes the transfer of 4-aminobutyl moiety of spermidine to a specific lysine of eIF5A precursor in an NAD-dependent manner. This modification occurs exclusively on only one protein: eukaryotic translation initiation factor 5A (eIF5A) and it is essential for cell proliferation [1]. Malfunctions of the hypusination pathway, including those caused by mutations within the DHS encoding gene, are associated with such conditions as cancer or neurodegeneration [2].

The presented study aimed to investigate substrate specificity of the first step of hypusination using macromolecular crystallography as the main tool and additionally to assess the impact of newly recognized pathological mutations in DHS coding gene on protein stability, activity and structure.

Human DHS wild type and its two mutants were expressed, purified and crystallized. Our attempts lead to six high-resolution crystal structures of DHS wt in apo form and complexes with natural substrates. Based on crystal structures and activity tests it was shown that despite almost identical binding of spermidine and spermine, probably only spermidine can serve as a proper substrate of deoxyhypusine formation. Furthermore, it was shown that against the previous studies, no conformational changes occur in the DHS structure upon spermidine-binding [3].

Availability of high-quality structural data will aid the design of novel DHS inhibitors for potential applications in cancer therapy and can significantly advance our understanding of newly recognized genetic DHS disorder.

1. Park MH, Wolff EC. Hypusine, a polyamine-derived amino acid critical for eukaryotic translation. J Biol Chem. 2018;293(48):18710-18718. 2. Ganapathi M, Padgett LR, Yamada K, et al. Recessive Rare Variants in Deoxyhypusine Synthase, an Enzyme Involved in the Synthesis of Hypusine, Are Associated with a Neurodevelopmental Disorder. Am J Hum Genet. 2019;104(2):287-298. 3. Wątor E, Wilk P, Grudnik P. Half Way to Hypusine-Structural Basis for Substrate Recognition by Human Deoxyhypusine Synthase. Biomolecules. 2020;10(4):522.

The research has been supported by National Science Centre (NCN, Poland) research grant no. 2019/33/B/NZ1/01839 to P.G and 2019/35/N/NZ1/02805 to E.W.

Hyoscyamine 6β-hydroxylase (H6H) is a bifunctional enzyme that catalyzes two final steps in the scopolamine biosynthesis pathway in the Solanaceae family [1]. It performs hydroxylation of (2’S)-hyoscyamine at the C6 position of the tropane moiety, which yields (6S,2'S)-6β-hydroxyhyoscyamine, and subsequent dehydrogenation of (6S,2'S)-6β-hydroxyhyoscyamine into (2’S)-scopolamine with formation of an epoxide (Figure 1). However, it was recently shown that H6H can also catalyze production of (6R, 2'S)-6β-hydroxyhyoscyamine from (2’S)-hyoscyamine at small scale [2].

H6H belongs to the family of non-heme 2-oxoglutarate/Fe(II)-dependent dioxygenases that share conserved double-stranded β-helix motif, so-called jelly-roll fold, composed of eight antiparallel β-strands. Here, we present crystal structures of H6H from Datura metel and its truncated version in complexes with 2-oxoglutarate, hyoscyamine and 6β-hydroxyhyoscyamine [3]. Through analysis of the substrate binding pocket, we point out crucial residues in hyoscyamine binding and explain results of previous studies on the substrate preference of H6H.

Figure 1. Two final steps in the biosynthesis of scopolamine - both catalyzed by H6H. MarvinSketch was used to draw structures and reactions [4].

[1] Hashimoto T, Yamada Y. Plant Physiol. 1986;81(2):619–625.

[2] Pan J, Wenger ES, Matthews ML, et al. J Am Chem Soc. 2019;141(38):15153–15165.

[3] Kluza A, Wojdyla Z, Mrugala B, et al. Dalton Trans. 2020 Apr 7;49(14):4454-4469.

[4] MarvinSketch version 18.20, ChemAxon, 2018.

Keywords: hyoscyamine 6β-hydroxylase; scopolamine biosynthesis; metalloenzymes

This research project was supported by SONATA-BIS grant no.UMO-2014/14/E/NZ1/00053 from the National Science Centre, Poland. AK would like to acknowledge the support of PROM Programme – International Scholarship Exchange of PhD Candidates and Academic Staff, co-financed granted from the European Union, including the European Social Fund within the framework of the Knowledge Education Development Operational Programme, non-competitive project entitled: International Scholarship Exchange of PhD Candidates and Academic Staff, contract number PPI/PRO/2019/1/00021/U/00001.

Hyoscyamine 6β-hydroxylase (H6H) is a bifunctional 2-oxoglutarate/Fe(II)-dependent dioxygenase that catalyzes the two final steps in the biosynthesis of scopolamine [1], that is a regioselective hydroxylation of hyoscyamine at the C6 position, followed by a formation of the epoxide ring utilising the installed hydroxy group [2]. The combination of crystallographic and computational studies on H6H:hyoscyamine complex provided insight into the substrate binding and the selectivity of the enzymatic reaction [3].

The QM/MM studies reveal that the regioselectivity of the hydroxylation reaction is dictated by only a few residues (i.e. Lys-129, Tyr-326, Lys-330), which promote the reaction occurring at the C6 site and at the same time hinder the alternative channel proceeding at the neighbouring (C7) position. Notably, the electronic properties of the reactants, that is hyoscyamine and the active site, do not favour any of the reaction channels, which suggests that switching regioselectivity of the oxygen rebound and thus obtaining other potentially useful alkaloids, may be achieved by targeting the residues in vicinity of the reactants.

[1] Hashimoto T., Matsuda J. & and Y. Yamada Y. (1993), FEBS Lett. 329, 35–39

[2] Li L., Van Belkum M.J. & Vederas J.C. (2012) Bioorg. Med. Chem. 20, 4356–4363

[3] Kluza A., Wojdyla Z., Mrugala B., et al. (2020) Dalton T. 49, 4454-4469

Keywords: metalloenzymes, reaction mechanisms, reaction regioselectivity, computation

This research project was supported by the National Science Centre, Poland and PL-Grid Infrastructure and in part by Project PROM - International scholarship exchange of PhD candidates and academic staff, financed by the European Social Fund implemented operational programme Knowledge Education Development, project: International scholarship exchange of PhD candidates and academic staff, contract number PPI/PRO/2019/1/00021/U/001.

Heme oxygenase-1 (HO-1) catalyzes the heme degradation using seven electrons supplied by NADPH–cytochrome P450 oxidoreductase (CPR) where FAD and FMN are bound as co-enzymes. Electrons flow from NADPH to heme in the redox partner via FAD and FMN. Previous biophysical analyzes such as SAXS and FRET suggest the existence of a dynamic equilibrium between the open and the closed forms of CPR in which orientations of FMN and FAD-binding domains are different [1]. We previously determined the crystal structure of the open-form stabilized CPR (ΔTGEE) in complex with heme–HO-1 at 4.3 Å resolution and demonstrated that ΔTGEE is tightly bound to heme–HO-1 while the reduction in heme–HO-1 using ΔTGEE is markedly slow because FAD is too far from FMN for electron transfer between them [2].

Here we characterized the enzymatic activity and the reduction kinetics of HO-1 using the closed-form stabilized CPR (147CC514) where the disulfide bond between FAD and FMN binding domains was introduced. We also analyzed the interaction between 147CC514 and heme–HO-1 by analytical ultracentrifugation [3]. The results indicate that HO-1 activity coupled with 147CC514 is markedly weaker than that coupled with CPR and the interaction between 147CC514 and heme–HO-1 is considerably weak. In addition, we examined the coupling of the redox and the structural states by full-scale molecular dynamics (MD) simulation of CPR (total 86.4 μs) [4]. Our MD result demonstrated that CPR has a tendency to open in the fully-reduced state while the major form of CPR is the closed form both in the fully-oxidized and fully-reduced states. We also found a correlation between the FAD-FMN distance and the predicted FMN-HO-1 distance, which is embedded in the equilibrium thermal fluctuation of CPR. Thus, the redox coupled transition between the open and the closed forms of CPR is indispensable for the smooth electron transfer from CPR to heme–HO-1 complex.

Further, we prepared the fusion protein of ΔTGEE and HO-1 referring to the previously reported structure of ΔTGEE in complex with heme–HO-1 and determined its fusion protein structure in complex with heme at 3.25 Å resolution [5]. Unexpectedly, no NADP⁺ was observed in the fusion protein structure although NADP⁺ was contained in the crystallization droplets and NADP⁺ was observed in the previous complex structure of ΔTGEE and heme–HO-1. Because the structural features of the NADP⁺-free form of CPR were also observed in the fusion protein structure, the fusion protein structure reflects the NADP⁺-free form of ΔTGEE–heme–HO-1 complex. Structural comparison of the NADP⁺-bound ΔTGEE–heme–HO-1 complex and the NADP⁺-free fusion protein suggests that NADP⁺/NADPH binding regulates the conformation change of the FAD-binding domain of CPR, which may control the efficiency of the electron transfer from FMN to heme–HO-1.

[1] Iyanagi, T., Xia, C. & Kim, J. J. P. (2012) Arch. Biochem. Biophys. 528, 72.

[2] Sugishima, M., Sato, H., Higashimoto, Y., Harada, J., Wada, K., Fukuyama, K. & Noguchi, M. (2014) Proc. Natl. Acad. Sci. USA 111, 2524.

[3] Sugishima, M., Taira, J., Sagara, T., Nakao, R., Sato, H., Noguchi, M., Fukuyama, K., Yamamoto, K., Yasunaga, T. & Sakamoto, H. (2020) Antioxidants 9, 673.

[4] Iijima, M., Ohnuki, J., Sato, T., Sugishima, M. & Takano, M. (2019) Sci. Rep. 9, 9341.

[5] Sugishima, M., Sato, H., Wada, K. & Yamamoto, K. (2019) FEBS Lett. 593, 868.

Keywords: Heme metabolism; Electron transfer; Domain motion; Cofactor binding

We acknowledged Mr. Sagara and Ms. Takemoto of Kyushu Inst. Tech., and Dr. Ohnuki and Dr. Sato of Waseda Univ. for analytical centrifugation and MD experiments, respectively. We also acknowledged beamline staffs of BL44XU, SPring-8 for crystallographic data collection. This work was partially supported by Kakenhi Grant numbers 25840026, 16K07280, and 19K06515 from JSPS, and grants from Takeda Science Foundation and Protein Research Foundation.

Succinyl-CoA synthetase (SCS), the enzyme that catalyzes the only substrate-level phosphorylation in the citrate cycle, is the prototype for a family of ADP- or GDP-forming acyl-CoA synthetases that includes ATP-citrate lyase (ACLY) [1]. These enzymes catalyze the formation of a thioester bond between an organic acid and CoA, using the energy of nucleotide triphosphate (NTP) and in the presence of magnesium ions. A histidine residue is transiently phosphorylated during catalysis [2], leading to the proposed catalytic mechanism:

E + NTP ⇌ E–PO₃ + NDP (1)

E–PO₃ + carboxylate ⇌ E∙carboxyl–phosphate (2)

E∙carboxyl–phosphate + CoA ⇌ E + carboxyl–CoA + P_i (3)

where E represents the enzyme; –, a covalent bond; and ∙ , noncovalent interactions. For SCS, the carboxylate is succinate; for ACLY, it is citrate and there is fourth step in which citryl-CoA is cleaved to form acetyl-CoA and oxaloacetate.

A glutamate residue of ACLY, E599, was proposed to play a role in the cleavage of citryl-CoA [3]. This glutamate residue is conserved not only in ACLYs but also in SCSs (Fig. 1). The structures of SCSs and ACLYs found in the Protein Data Bank [4] are used to investigate the role of this conserved glutamate residue.

Human ACLY IRTIAIIAEGIPEALTRKLIKKA-DQKGVTIIGPATVGGIKPGCFKIGNTGGMLDNILASKLYR

Chlorobium limicola ACLY IQLVSMITEGVPEKDAKRLKKLA-QKLGKMLNGPSSIGIMSAGECRLGVIGGEFKNLKLCNLYR
Human GTPSCS α-subunit IPLVVCITEGIPQQDMVRVKHKLLRQEKTRLIGPNCPGVINPGECKIGIMPG--------HIHK
Escherichia coli α-subunit IKLIITITEGIPTLDMLTVKVKL-DEAGVRMIGPNCPGVITPGECKIGIQPG--------HIHK
Thermus aquaticus α-subunit IPLIVLITEGIPTLDMVRAVEEI-KALGSRLIGGNCPGIISAEETKIGIMPG--------HVFK

Figure 1. Alignment of portions of the sequences of ACLYs and SCSs. The alignment shows conservation of a glutamate residue, E599 in human ACLY, E112 in the A-subunit of Chlorobium limicola ACLY, E105a of human GTPSCS, E98a of E. coli SCS, and E97a of Thermus aquaticus GTPSCS.

[1] Sánchez, L. B., Galperin, M. Y. & Müller, M. (2000). J. Biol. Chem. 275, 5794. [2] Kreil, G. & Boyer, P. D. (1964). Biochem. Biophys. Res. Commun. 16, 551.

[3] Wei, X., Schultz, K., Bazilevsky, G. A., Vogt, A. & Marmorstein, R. (2020). Nat. Struct. Mol. Biol. 27, 33.

[4] Berman, H. M. et al. (2000). Nucleic Acids Res. 28, 235.

Obtaining well-diffracting crystals is often a bottelneck of biocrystallographic studies. It is increasingly important in serial crystallography which requires a reproducible production of microcrystals that are homogneous in size and diffraction quality. In order to gain a better control over the crystallization process, we used an instrument called the XtalController. This recent technology gives access to the full monitoring of crystallization assays using dynamic light scattering and videomicroscopy, and integrates a crystallization chamber with temperature and humidity regulation, as well as piezo injectors that allow the modification of the mother liquor composition during the experiment [1]. We exploited this technology to study the crystallization of two enzymes, the CCA-adding enzyme of Planococcus halocryophilus, a cold-adapted bacterium from the permafrost, and the hen egg white lysozyme in the presence of a synthetic chemical nucleant, the crystallophore Tb-Xo4. Using the XtalController, we were able to detect early nucleation events and drive the crystallization system toward growth conditions yielding crystals with excellent diffraction properties using cycles of dissolution/crystallization [2]. This work illustrates the potential of XtalController technology for the rational production of samples for crystallography, ranging from nanocrystals for electron diffraction, microcrystals for serial or conventional X-ray diffraction, to larger crystals for neutron diffraction.

​[1] Meyer, A., Dierks, K., Hilterhaus, D., Klupsch, T., Mühlig, P., Kleesiek, J., Schöpflin, R., Einspahr, H., Hilgenfeld, R. & Betzel, C. (2012). Acta Cryst. F, 68, 994.

​[2] de Wijn, R., Rollet, K., Engilberge, S., McEwen, A.G., Hennig, O., Betat, H., Mörl, M., Riobé, F., Maury, O., Girard, E., Bénas, P., Lorber, B. & Sauter, C. (2020). Crystals, 10, 65.

The water oxidation process in Photosystem II, a Bio-machinery that evolved nearly three billion years ago, fascinates us with its capabilities of harvesting solar energy and storing it in a chemical form. The X-ray Free Electron Lasers enabled us to study this phenomenal protein in ways that were not possible before. In the current manuscript, we introduce new approaches for XFEL data to understand better the catalytic activity, not only for PSII, via deeper analysis of the water network around the active site. Using a high-resolution 1.89 Å room temperature crystal structure of PS II and the re-processed crystallography data at various time points between the S2 to S3 transition of Kok's cycle, we identified the substrate water intake channel extends starting near O1 of the OEC to the lumenal side of the membrane. Three main well-coordinated structural events during the S2 to S3 transition occurred within the water channels, resulting in substrate insertion and proton egress. In particular, the rotation of D1-E65 and the appearance of new water before the substrate insertion likely facilitate proton removal through the Cl 1channel. While the arrival of new water near D1-E329 after the substrate insertion probably indicates the delivery via the O1 channel.