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Original Russian Text © K.M. Boyko, V.I. Timofeev, V.R. Samygina, I.P. Kuranova, V.O. Popov, M.V. Koval'chuk, 2016, published in Kristallografiya, 2016, Vol.

ISSN 1063-7745, Crystallography Reports, 2016, Vol. 61, No. 5, pp. 718–729. © Pleiades Publishing, Inc., 2016. Original Russian Text © K.M. Boyko, V.I. Timofeev, V.R. Samygina, I.P. Kuranova, V.O. Popov, M.V. Koval’chuk, 2016, published in Kristallografiya, 2016, Vol. 61, No. 5, pp. 691–702.

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Protein Crystallization under Microgravity Conditions. Analysis of the Results of Russian Experiments Performed on the International Space Station in 2005−2015 K. M. Boykoa,b,*, V. I. Timofeeva,c, V. R. Samyginaa,c, I. P. Kuranovaa,c, V. O. Popova,b, and M. V. Koval’chuka,c aNational

Research Centre “Kurchatov Institute”, pl. Akademika Kurchatova 1, Moscow, 123098 Russia Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 119071 Russia c Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics,” Russian Academy of Sciences, Leninskii pr. 59, Moscow, 119333 Russia *e-mail: [email protected] b

Received May 6, 2016

Abstract—Conditions of mass transport to growing crystals have a considerable effect on the crystal size and quality. The reduction of convective transport can help improve the quality of crystals for X-ray crystallography. One approach to minimizing convective transport is crystallization in a microgravity environment, in particular, in space. The data obtained by our research team in protein crystallization experiments on the International Space Station are surveyed and analyzed. DOI: 10.1134/S1063774516050059

CONTENTS Introduction 1. Russian Experiments on Protein Crystal Growth under Microgravity Conditions 1.1. Module 1 1.2. JAXA Crystallization Box 2. The Most Remarkable Results Obtained in the Framework of Russian−Japanese Experiments Conclusions INTRODUCTION Structural biology is one of the most important branches of modern physicochemical biology. Knowledge of the three-dimensional structures of biological macromolecules (proteins and nucleic acids) is of great importance for understanding molecular mechanisms of the action of cells and their components [1]. The determination of the three-dimensional structure of the target protein is essential for the search for new promising molecules that can serve as the basis for drugs [2, 3], as well as for the design and optimization of new biocatalysts for different fields of biotechnology [4]. X-ray crystallography is currently the most favored technique for investigating the three-dimensional structures of macromolecules. As of April 2016, there

are more than 117 000 different structures in the Protein Data Base (www.rcsb.org), almost 90% of which were determined by this technique. The main limitation of X-ray crystallography is that it involves the crystallization of samples as a preliminary step, which often constitutes a bottleneck [5, 6]. The quality of grown crystals is responsible for the resolution of X-ray diffraction data and, finally, for the accuracy and precision of structural information. The general principle of crystallization of macromolecules from solution is to reduce their solubility until the supersaturation is reached by varying such parameters as pH, temperature, precipitant concentration, etc. [6, 7]. During crystal growth, the mass transport to the growing crystal occurs through two mechanisms─diffusion and convection [8, 9]. The proportion between these two processes has an effect on the crystal size and quality. During space flight, the absence of gravity leads to the disappearance of convective flows in a crystallization cell and the mass transport to a growing crystal occurs exclusively by diffusion, which can result in the formation of more perfect crystals [10]. The first experiments under microgravity conditions were carried out by W. Littke’s research group in 1984 for lysozyme and β-galactosidase [11]. The crystals grown in these experiments were larger than Earth grown crystals. From 1986 to 2000, extensive experi-

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ments on the protein crystallization in microgravity were performed in the United States, some EU countries, the Soviet Union (and then Russia), Japan, and China on the Russian Space Station Mir and on the International Space Station (ISS) [12, 13]. In 1988, crystals of Thermus thermophilus 30S ribosomal subunit and Penicillium vitale catalase were grown during a space mission on the Foton (Photon) spacecraft of the Soviet Union [14, 15]. National and international collaborative projects on the protein crystal growth in a microgravity environment have been implemented and are currently run by Russia, the United States, EU countries, China, Canada, Japan, and other countries. As a pioneer in space exploration, Russia is actively involved in research projects conducted on the ISS [16]. The available experimental data show that crystals grown in microgravity are, in many cases, larger and have a more ordered internal structure, reduced twinning, and lower mosaicity; as a consequence, they give higher resolution diffraction patterns compared with Earth grown crystals of the same proteins [16–22]. An improvement in diffraction characteristics of the crystals was observed in approximately 30% of cases [23]. However, the desired improvement in crystal quality of some proteins was not achieved in a microgravity environment [18]. In Russia, the first experiments on the crystallization of macromolecules in space were performed in 2005 on the ISS. The present review systematically summarizes experimental data on the protein crystal growth in microgravity obtained in 2005–2009 during the implementation of the Russian program and in the framework of the ongoing Russian−Japanese collaborative project that started in 2009. These experimental data are consistent with those published in the literature concerning the improvement of characteristics of microgravity-grown crystals (crystal size, diffraction limit, mosaicity, etc.) compared to equivalent crystals grown on Earth. This review gives examples of the three-dimensional structures of proteins promising for the solution of problems in medicine and biotechnology, which were determined using microgravitygrown crystals that yielded higher quality diffraction patterns compared to those obtained from Earth grown crystals. 1. RUSSIAN EXPERIMENTS ON PROTEIN CRYSTAL GROWTH UNDER MICROGRAVITY CONDITIONS Various known techniques are used to grow crystal in microgravity: vapor diffusion [24, 25], counter-diffusion [26, 27], dialysis [28, 29], and batch crystallization. The equipment for microgravity crystallization experiments is substantially upgraded and, in some cases, specially designed taking into account the specific features of space experiments [30]. The vapor diffusion is the most commonly employed method of protein crystallization. However, the equipment for CRYSTALLOGRAPHY REPORTS

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the implementation of this method in space is most technologically sophisticated. Therefore, the batch crystallization and counter-diffusion techniques are methods of choice for the growth of protein single crystals in a microgravity environment. The Module 1 protein crystallization apparatus was designed for the batch crystallization in the Shubnikov Institute of Crystallography of the Russian Academy of Sciences. Crystallization by the counter-diffusion technique is performed using the JAXA Crystallization Box as a crystallization device. 1.1. Module 1 During the period between 2005 and 2009, the specialized Module 1 protein crystallization apparatus [31] designed in the Shubnikov Institute of Crystallography of the Russian Academy of Sciences was used for protein crystal growth under microgravity conditions. The Module 1 (Fig. 1) is a revolving-type apparatus consisting of two parts tightly ground together, each part having eight cylindrical cells. The cells in the glass part are filled with a protein solution, and the cells placed in the Teflon part are filled with a reservoir solution. The cells of the two parts are connected together by turning one of the parts, which is performed manually by an astronaut directly aboard the ISS. The cells containing the protein solution and those containing the reservoir solution have equal volumes of 70 μL. The diameter of the cells is 2–4 mm; the lengths are 10 mm. The batch crystallization in the Module 1 begins once the parts are connected. Below are the most remarkable examples of protein crystallization in the Module 1 apparatus, resulting in crystal quality improvement. Formate dehydrogenase (FDH) from Arabidopsis thaliana Formate dehydrogenase catalyzes the oxidation of the formate ion to carbon dioxide coupled with the reduction of NAD+ to NADH. This enzyme is of interest for fine organic synthesis as a component of the so-called NADH cofactor regeneration system. Plant FDHs act as stress proteins in plants and protect them against stress by providing cells with energy as a result of NADH synthesis via the oxidation of toxic formate. The crystallization of Arabidopsis thaliana FDH in the Module 1 gave crystals with a size of up to 1 mm (Fig. 1), which is an absolute record for this class of plant enzymes that was never reached on Earth. In addition, as opposed to Earth grown crystals, the crystals grown in microgravity belong to another space group (P43212). An X-ray diffraction data set was collected from one crystal to 1.22 Å resolution [32], which is the best resolution for this protein.

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(b)

(c)

Fig. 1. (a) Crystallization in microgravity in the Module 1 apparatus, (b) lysozyme crystals in cells of the Module 1 apparatus, and (c) crystals of Arabidopsis thaliana FDH.

Pyrophosphatase (PPase) from V. cholerae Pyrophosphatase from Vibrio cholerae is a metaldependent enzyme belonging to family I PPases. It catalyzes the hydrolysis of pyrophosphate to two phosphate ions. The importance of this class of enzymes is that pyrophosphate is a by-product of many biosynthetic reactions. PPase is of interest as a protein isolated from a pathogenic organism (potential target). Due to certain properties, family I PPases are readily crystallizable enzymes [33]. However, perfect crystals are grown in the presence of high concenctrations of ammonium sulfate or phosphate [33]. The fact that sulfate or phosphate binds to the active site of the enzyme makes it impossible to study the apo form of the enzyme and its complexes with pyrophosphate or its analogues in the active site, which is essential for understanding the details of the functioning of this enzyme. Crystals of V. cholerae PPase were grown from a specimen produced in the Belozersky Institute of Physico-Chemical Biology at Moscow State University. Most of the Earth grown crystals were thin triangular plates. This crystal morphology is not wellsuited for X-ray diffraction data collection [34]. The crystallization in microgravity in the Module 1 apparatus made it possible to obtain crystals of the apo form having a more isometric morphology with sizes of 0.2 × 0.3 × 0.2 mm. The crystals belong to sp. gr. P3 and were used to collect an X-ray diffraction data set to 2.0 Å resolution [35]. Recombinant Human Insulin Insulin-dependent diabetes is a socially significant disease that is a frequent reason for a decline in quality of life and premature death among the population. Insulin is a polypeptide hormone that regulates carbohydrate metabolism in the body, in particular, the glucose level in the blood. Hence, insulin has long been successfully used in the therapy of insulin-dependent diabetes. Currently, there are genetically engineered types of insulin with a different time of onset and duration of action. Taking into account the importance of

diabetes treatment, new versions of this drug are always in demand, and investigating the highly variable three-dimensional structure of genetically engineered insulin is still relevant. The experiments using the Module 1 apparatus demonstrated that insulin crystals grown in microgravity, although being similar in size (0.3–0.5 mm) to Earth grown crystals, are characterized by higher X-ray diffraction quality, which is critical for the elucidation of the structural details essential for enzyme functioning. The X-ray diffraction data set was collected from a microgravitygrown insulin crystal to 1.6 Å resolution, whereas Earth grown crystals were diffracted at 1.85 Å resolution [36]. Because of the design limitations of the Module 1 apparatus (the rather large weight of the apparatus and small number of cells per module) and the short time of program implementation using the Module 1 apparatus, relatively few proteins were crystallized. The counter-diffusion technique proved more promising from the standpoint of the development of experimental equipment. 1.2. JAXA Crystallization Box In 2002 the European Space Agency successfully tested a device for protein crystallization by counterdiffusion techniques (Granada Crystallization Box) [37]. Later on, Japanese researchers improved this technique to simplify the sample preparation for experiments and reduce the sample consumption [27]. This approach provides the possibility of screening a number of crystallization conditions in one capillary due to a gradual change in the precipitant concentration as the precipitant solution moves along the capillary containing the protein solution, thus substituting for several wells in the vapor-diffusion techniques. The method was successfully employed by Russian researchers in the framework of the Russian−Japanese High Quality Protein Crystal Growth Experiment (JAXA PCG) project that started in 2009 under the guidance of the Russian Federal Space Agency and the Japan Aerospace Exploration Agency (JAXA). The

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Fig. 2. JAXA crystallization box (JCB) for microgravity crystallization experiments; the JCB device contains six assembled cells (capillaries), thus making it possible to try six different crystallization conditions in one JCB. Each capillary contains a protein solution connected to the reservoir solution (the reservoir in the left side of the figure) through a gel tube. The modified figure is taken from [7].

JAXA Crystallization Box (JCB) is used in these experiments (Fig. 2) [38].

Earth. The configuration of experiments was such that at least two capillaries were used for crystallization of each protein both in microgravity and on Earth. A total of 72 proteins were investigated in experiments accomplished in the framework of this project, the crystals being obtained for 58 proteins. The analysis showed that some crystals grown in microgravity have better quality than Earth grown crystals (Table 1).

Since the beginning of the project in 2009, nine crystallization experiments were performed aboard the ISS. To evaluate the effect of microgravity on the quality of the grown crystals, each crystallization experiment on the ISS was repeated in parallel on

Table 1. Statistical summary of selected characteristics of the crystals grown in JAXA PCG experiments during the period between 2009 and 2015 Maximum crystal Best resolusize, μm tion, Å

Protein

Mosaicity

Twinning

Purine nucleoside phosphorylase from E. coli

300 (200)

0.99 (1.50)

0.1 (0.3)

No (Yes)

Phosphopantetheine adenylyltransferase from M. tuberculosis

400 (400)

1.59 (2.10)

0.8 (1.6)

No (No)

Thymidine phosphorylase

300 (300)

1.50 (2.20)

0.4 (0.6)

No (No)

Phosphoribosyl pyrophosphate synthase

100 (100)

2.71 (>4)

1.8 (–)

No (No)

Carboxypeptidase B

200 (50)

1.25 (1.60)

0.2 (0.7)

No (Yes)

Carboxypeptidase T

300 (300)

1.29 (2.10)

0.3 (0.5)

No (No)

Uridine phosphorylase from S. oneidensis

500 (300)

0.93 (1.6)

0.3 (0.5)

Yes (Yes)*

BTB domain of the CP190 protein

300 (200)

1.4 (2.2)

0.35 (1.6)

No (No)

Cytochrome c-nitrite reductase from Tv. paradixus

500 (500)

1.6 (1.6)

0.07 (0.1)

No (Yes)

NAD-Dependent formate dehydrogenase from S. aureus

100 (100)

2.0 (2.2)

0.4 (0.2)

No (No)

Histone-like protein HU from M. gallicepticum

500 (400)

4.0 (>5.0)

1.1 (no data) No (No)

Aldehyde dehydrogenase from Pyrobacullum sp 1147

300 (300)

1.9 (2.2)

0.15 (0.5)

No (No)

Xaa-Pro aminopeptidase from T. sibiricus 0821

100 (200)

2.3 (2.6)

0.4 (0.8)

No (No)

Protein DJ-1 from H. sapiens

300 (400)

1.2 (1.2)

0.2 (0.3)

No (No)

Protein of unknown function 2Q02 from S. typhimurium

100 (100)

1.6 (1.7)

0.4 (0.7)

No (No)

Beta-glycosidase from A. sacharovoronas

400 (300)

1.80 (2.0)

0.9 (1.7)

No (No)

Pyrophosphatase from M. tuberculosis

300 (200)

1.4(1.6)

0.25(0.4)

No (No)

For the sake of brevity, the table includes data only for the proteins whose microgravity-grown crystals have improved characteristics. The data obtained in the control experiments on Earth are given in parentheses. * For the crystals of this enzyme, the degree of twinning in microgravity was lower compared to the experiment on Earth. CRYSTALLOGRAPHY REPORTS

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Fig. 3. Electron density for the residue Trp184 of uridine phosphorylase. The 2Fo−Fc electron density is contoured at the 1σ level; (a) structure determined using a microgravity-grown crystal (0.93 Å resolution), hydrogen atoms are shown; (b) structure determined using an Earth-grown crystal (1.6 Å resolution).

2. THE MOST REMARKABLE RESULTS OBTAINED IN THE FRAMEWORK OF RUSSIAN−JAPANESE EXPERIMENTS An analysis of the results obtained during the implementation of Russian−Japanese experiments on the crystal growth under microgravity conditions shows that in some cases the microgravity conditions resulted in higher quality crystals. For some proteins, a substantial improvement in the crystal quality was achieved, which can make a significant contribution to the quality of the structural data obtained from these crystals in X-ray diffraction experiments. The following are some of the most interesting results obtained by the Russian consortium in the framework of the JAXA PCG project in 2009−2015. Uridine Phosphorylase from the Bacterium Shewanella oneidensis Uridine phosphorylases belong to pyrimidine nucleoside phosphorylases. It is known that uridine phosphorylase exhibits an increased level of activity in cancer cells. Therefore, one approach to cancer treatment in worldwide clinical practice is the use of modern drugs based on modified nucleosides acting as uridine phosphorylase inhibitors [39, 40]. This class of enzymes is also of interest due to their potential application as biocatalysts for the synthesis of nucleosides, in particular, modified nucleotides having antiviral and anticancer properties. An examination of the Earth grown crystals of uridine phosphorylase showed that they diffracted to 1.6 Å resolution (Table 1). Meanwhile, the crystals

grown in microgravity gave atomic resolution (0.93 Å), which made it possible to reveal fine details of the three-dimensional structure of the enzyme with high accuracy (Fig. 3) [41, 42]. It should be noted that the Earth grown crystals of uridine phosphorylase are characterized by twinning (three potential domains with fractions of 0.5, 0.3, and 0.2). The crystals of the enzyme grown in microgravity were also twins, but they differ in the fraction ratio (0.80, 0.10, and 0.10). BTB Domain of the CP190 Protein from Drosophila melanogaster The spatial organization of the genome plays an essential role in the regulation of its activity and cell differentiation. Genome elements spaced far apart are involved in the regulation of transcription in eukaryotes. According to current concepts, there is a specific class of proteins, including known insulator proteins (insulators are genome elements that compartmentalize the genome into chromatin domains), that are involved in the formation and stabilization of the three-dimensional structure of the chromosome, as well as in the control of local enhancer−promoter interactions. The CP190 protein (centrosomal protein) is a universal component of many insulator protein complexes. This protein contains the BTB (brica-brac, tramtrack, broad-complex) domain essential for its activity [43]. The BTB domains belong to one type of conserved domains, which mediate multimerization [44] and are involved in various protein−protein interactions. However, their structure is poorly known.

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Fig. 4. Electron density for the residue Tyr89 of the BTB domain of the CP190 protein. The 2Fo−Fc electron density is contoured at the 1σ level; (a) structure determined using a microgravity-grown crystal (1.4 Å resolution); (b) structure determined using an Earth-grown crystal (2.2 Å resolution).

Crystals of the BTB domain of the CP190 protein were grown both in microgravity and in the control experiment on Earth (Table 1). A comparison of the crystals demonstrated that the microgravity-grown crystals were up to 300 μm in size and diffracted to 1.4 Å resolution versus 200 μm and 2.2 Å for the Earth grown crystals. It should be noted that the Earth grown crystals had a much higher mosaic spread (Table 1). The difference in the electron density for the three-dimensional structures determined using two types of crystals is presented in Fig. 4. Purine Nucleoside Phosphorylase and Thymidine Phosphorylase from E. coli Purine nucleoside phosphorylase and thymidine phosphorylase (PNP and TP, respectively) are key enzymes involved in nucleotide metabolism. In the presence of phosphate ions, both enzymes catalyze phosphorolysis of the glycosidic bond in nucleosides, but they have different quaternary structures and specificity [45–47]. Both enzymes catalyze the transglycosylation reaction, i.e., the transfer of a carbohydrate moiety from one base to another. Transglycosylation is a key reaction in the salvage pathway of nucleoside biosynthesis. Therefore, both enzymes are widely used in the combined chemical−enzymatic synthesis of nucleoside derivatives, many of which serve as antiviral and anticaner drugs [48]. CRYSTALLOGRAPHY REPORTS

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Purine nucleoside phosphorylases are essential for the normal development of mammalian T lymphocytes. The loss of PNP activity causes serious immunodeficiency. Hence, PNP inhibitors are used for creation of selective T-cell immunodeficiency in a human body necessary for the organ and tissue transplantation and chemotherapy of some pathologies. Purine nucleoside phosphorylase from E. coli has a good medicinal potential for cancer treatment. The transfection of the PNP gene into tumor cells activates nucleoside analogs to their cytotoxic form [49–52]. Crystals of PNP grown in microgravity diffracted to 0.99 Å resolution, which made it possible to obtain detailed information on the three-dimensional structure of the enzyme [53] (Fig. 5). The atomic coordinates of the model determined with high accuracy were deposited in the Protein Data Bank (www.rcsb.org). They can serve as a reliable structural basis for the construction of inhibitors of the enzyme by the drug design method and are also suitable for the computer modeling of the enzyme-catalyzed reaction. The hexameric molecule of the enzyme is shown in Fig. 6. Mammalian TP homologous to the bacterial enzyme is involved in angiogenesis, which occurs continuously and intensely in tumor cells, where the major (de novo) synthetic pathway for nucleosides is absent and the level of TP that catalyzes the salvage biosynthetic pathway is substantially enhanced.

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Fig. 5. Fragment of the electron density map for the PNP molecule at 0.99 Å resolution contoured at the 1σ level.

Therefore, the inhibition of TP activity may serve as a plausible therapeutic strategy for the cancer treatment and the search for new inhibitors of the enzyme is a challenge. Two compounds exhibiting a therapeutic potential─azidothymidine (AZT) and 3'-fl uoro-2',3'dideoxyuridine (3'F-2',3'-ddU)─were found to be not cleaved by the enzyme and to be reversible inhibitors of TP [54]. Crystals of free TP and its complexes with AZT and 3'F-2',3'-ddU were grown in microgravity. The threedimensional structure of apoTP was determined at higher resolution (1.55 Å) than that achieved earlier (2.38 Å). The grown crystals were used to establish the three-dimensional structures of both TP complexes [55]. The position of the ligands in the nucleosidebinding subsite of the active site was determined. A comparison of the arrangement of both ligands with the position of the substrate (thymidine) in homologous S. aureus TP showed that, although both ligands bind to the nucleoside-binding subsite of the active site, their position only partially overlaps with the position of the thymidine serving as the substrate [55]. It was shown that the binding of nucleosides containing the 3'-azido group is accompanied by the formation of an additional hydrophobic pocket for this group in the active site of the enzyme. Phosphopantetheine Adenylyltransferase from Mycobacterium tuberculosis Phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis (РРАТ Mt) catalyzes the formation of dephosphocoenzyme A─a key step in

coenzyme A biosynthesis essential for the survival of mycobacteria. Therefore, РРАТ Mt is a promising target for antituberculosis agents. Crystals of the enzyme in the apo form and in complexes with functional

Fig. 6. Hexameric molecule of purine nucleoside phosphorylase.

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Fig. 7. Electron density for CoA in the active site of PPAT Mt. contoured at the 1σ level; (a) structure determined using a microgravity-grown crystal (2.10 Å resolution); (b) structure determined using an Earth-grown crystal (1.59 Å resolution).

ligands (АTP, coenzyme А, dephosphocoenzyme А) were grown in a microgravity environment. These crystals were used to determine the three-dimensional structures at a resolution from 1.59 to 1.70 Å. The ligands bound to the active site were located in electron difference maps with nearly full occupancy. The crystals of the РРАТ Mt/СоА complex grown earlier on Earth diffracted to 2.1 Å resolution, which was insufficient for the reliable determination of the position of the coenzyme molecule [56] (Fig. 7). The РРАТ molecule is a hexamer composed of identical subunits with a solvent-filled central channel (Fig. 8). Six active sites of the enzyme are located inside the channel in the vicinity of the interface between two trimers comprising the hexamer. Each of the threedimensional structures (of the apo enzyme, the РРАТ/АTP complex with the substrate, and the РРАТ/dPCoA complex with the product) characterizes the conformation of the РРАТ molecule in successive steps of the enzyme-catalyzed reaction. A comparison of these structures revealed conformational changes that occur in the course of the catalyzed reaction, and the molecular mechanism of the observed changes was described at the atomic level [57]. Carboxypeptidase T (CPT) from Тhermoactinomyces vulgaris Proteins of the metallocarboxypeptidase family catalyze the removal of a C-terminal amino-acid resiCRYSTALLOGRAPHY REPORTS

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due in proteins and peptides and have a variety of functions in living organisms. Carboxypeptidases in blood plasma are involved in a cascade of reactions leading to the thrombus formation and the development of myocardial infarction [58]. Inhibitors of these carboxypeptidases are useful in the treatment of heart attacks. Regulatory carboxypeptidases mediate the processing of neuropeptides, neurotransmitters, and prohormones; regulate the peptide hormone activity; and play a role in the regulation of protein−protein and protein−cell interactions [59]. The three-dimensional structure of carboxypeptidase T and the structure of its active site are similar to those of carboxypeptidases А and В. However, carboxypeptidase T substantially differs from the latter two enzymes in the character of specificity and catalyzes the removal of both hydrophobic and positively charged С-terminal amino-acid residues. Due to broad specificity, carboxypeptidase is a convenient model to search for the structural determinants of carboxypeptidase specificity. The three-dimensional structure of the apo form of carboxypeptidase has been determined earlier [60]. To locate the active-site regions of the enzyme responsible for the binding of substrates of different natures (hydrophobic and positively charged), crystals of bacterial CPT and crystals of CPT in complexes with substrate analogs, reaction products, and selective inhibitors of the enzyme were grown in microgravity [61–64].

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Fig. 8. Hexameric molecule of PPAT Mt.

In the three-dimensional structures of the CPT complexes, the amino-acid residues involved in the binding of hydrophobic and positively charged groups of the substrates, as well as in the catalysis, were identified. A comparison of the positions of different inhibitors in the active site of CPT showed that hydrophobic and charged side chains of the ligands are bound to spatially separated regions (Fig. 9). Comparison of the Module 1 and the JAXA Crystallization Box (JCB) and Prospects of Improving Equipment for Protein Crystallization in Space Each type of equipment used in microgravity experiments, whether it is the Module 1 or the JAXA Crystallization Box (JCB), has advantages and drawbacks summarized in Table 2. These types of equipment utilize different crystallization techniques, such as batch crystallization and counter diffusion. The Module 1 requires a large amount of protein per cell (about 70 μL), as opposed to the JCB, which uses substantially smaller amounts (10 μL) because it is equipped with smaller inner-diameter capillaries (0.5 mm). The weight of the Module 1 together with a metallic container is, on average, 0.400–0.450 kg. By contrast, the JCB is a lightweight portable device that enables the simultaneous screening for the conditions of protein crystallization for a larger number of proteins, which is essential taking into account the limited payload capability aboard the ISS. The crystallization in the Module 1 is initiated by connecting two parts of

the container made up of materials of different kinds. Therefore, there are risks of cell leakage, which can adversely affect the results of crystallization. Meanwhile, the protein crystallization in the Module 1 is a controlled process, because the crystallization is initiated directly by an astronaut at a specified time. In the JCB device, the onset of crystallization can be controlled only by carefully adjusting the precipitant/protein concentration ratio and the length of the gel tube through which the diffusion occurs. Such a number of factors that have an effect on the crystal nucleation may result in crystallization starting already on Earth, which is why the influence of microgravity on the crystal growth is difficult to evaluate. In general, the counter-diffusion technique is advantageous over the batch crystallization because, as was mentioned above, each capillary provides mini screening for different protein and precipitant concentrations. This simplifies the protein crystallization and in some cases results in the formation of different crystal forms of a protein in one capillary [65]. Experiments demonstrated that the JCB device has the following advantages: higher effectiveness of crystallization experiments and efficient use of the area aboard the spacecraft. A shortcoming of the JCB method is a small inner diameter of capillaries, which limits the maximum size of growing crystals. Despite the development of a new generation of synchrotron radiation sources and the use of microfocusing beamlines, which enable researchers to collect X-ray diffraction data from tiny protein crystals (

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