Crystal structures of carbamate kinase from Giardia lamblia bound with citric acid and AMP-PNP.

The parasite Giardia lamblia utilizes the L-arginine dihydrolase pathway to generate ATP from L-arginine. Carbamate kinase (CK) catalyzes the last step in this pathway, converting ADP and carbamoyl phosphate to ATP and ammonium carbamate. Because the L-arginine pathway is essential for G. lamblia survival and absent in high eukaryotes including humans, the enzyme is a potential target for drug development. We have determined two crystal structures of G. lamblia CK (glCK) with bound ligands. One structure, in complex with a nonhydrolyzable ATP analog, adenosine 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP), was determined at 2.6 Å resolution. The second structure, in complex with citric acid bound in the postulated carbamoyl phosphate binding site, was determined in two slightly different states at 2.1 and 2.4 Å resolution. These structures reveal conformational flexibility of an auxiliary domain (amino acid residues 123-170), which exhibits open or closed conformations or structural disorder, depending on the bound ligand. The structures also reveal a smaller conformational change in a region associated the AMP-PNP adenine binding site. The protein residues involved in binding, together with a model of the transition state, suggest that catalysis follows an in-line, predominantly dissociative, phosphotransfer reaction mechanism, and that closure of the flexible auxiliary domain is required to protect the transition state from bulk solvent

A model of the transition state.

<p>(A) Stereoscopic representation showing the key protein and Mg²⁺ interactions with the transition state components. The apical axis between the donor and acceptor oxygen atoms is shown as dashed lines in magenta and the distances from the transferred phosphorous to the donor and acceptor oxygen atoms are 2.9 Å. Atoms are colored as follows: Carbon – gray, Oxygen – red, Nitrogen – blue, Magnesium – magenta. The carbamate is labeled CM (B) The relationship between the transition state and Arg163 and Asp157 on the auxiliary domain (colored in yellow), as defined by the closed conformation of <i>gl</i>CK. The side chains of Arg163 and Asp157 project towards the transition state. However, the distances (listed in Å) are too far, allowing bulk solvent access into the active site. Protection of the transition state and prevention of phosphate hydrolysis requires a further closure of the auxiliary domain.</p

Ribbon depiction of dimeric <b><i>gl</i></b><b>CK structures.</b>

<p>The flexible auxiliary domains and the core α/β domains are highlighted in different colors. Bound ligands are shown as stick models. (A) One homodimer of the <i>gl</i>CK-citrateL structure contains one auxiliary domain in an open conformation and the second in a closed conformation. (B) The second homodimer of the <i>gl</i>CK-citrateL structure and both homodimers of the <i>gl</i>CK-citrateS structure exhibit the auxiliary domains only in the closed conformation. (C) The homodimer in the <i>gl</i>CK-AMPPNP crystal asymmetric unit that exhibits the open conformation. The auxiliary domains of the second homodimer are disordered, which is not shown. (D) Amino acid sequence conservation of <i>gl</i>CK based on multiple alignment of the top 100 sequences identified using the BLAST protein sequence homology search <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064004#pone.0064004-Altschul1" target="_blank">[28]</a>. Multiple sequence alignment was performed with ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064004#pone.0064004-Thompson1" target="_blank">[29]</a>. Invariant residues are colored in red. The N-terminus of two CKs in the non-redundant sequence database are truncated, thus only 98 sequences were used to define the first three invariant residues. Secondary structure units are boxed. β-strands and α-helices are show in red and blue colors, respectively, and in addition, the auxiliary domain is boxed in magenta color.</p

Ligands bound to the <i>gl</i>CK active site and their key interactions.

<p>(A) Citric acid. (B) AMP-PNP. The auxiliary domain (colored yellow) is in the closed conformation in (A) and in the open conformation in (B). Note the conformational differences of the loops 244–250 and 267–275 (colored yellow) with and without the bound AMP-PNP, leading to the stacking of Tyr245 above the adenine ring of AMP-PNP. The difference Fourier maps with the coefficients <i>F</i>_o−<i>F</i>_c, omitting the ligand from the model, are contoured at 2.5σ level. (C) Schemes and atom numbering of the ligands and their interactions with the protein. The figure was generated using the program LIGPLOT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064004#pone.0064004-Wallace1" target="_blank">[30]</a>.</p

The reaction catalyzed by carbamate kinase.

<p>The reaction catalyzed by carbamate kinase.</p

X-ray data collection and structure refinement statistics.

a<p>The values in parentheses are for the highest resolution shell, 2.40–2.49 Å for <i>gl</i>CK-citrateL, 2.10–2.15 Å for <i>gl</i>CK-citrateS, and 2.60–2.73 Å for <i>gl</i>CK-AMPPNP.</p>b<p><i>R_merge = </i>∑<i>_hkl</i> [(∑<i>_j</i> | <i>I_j</i> –<<i>I</i>>| )/∑<i>_j</i> | <i>I_j</i> | ].</p>c<p><i>R</i>_cryst = ∑<i>_hkl</i> | |<i>F_o</i>| – |<i>F_c</i>| |/∑<i>_hkl</i> |<i>F_o</i>|, where <i>F_o</i> and <i>F_c</i> are the observed and calculated structure factors, respectively.</p>d<p><i>R</i>_free is computed using 2,009 randomly selected reflections omitted from the refinement for <i>gl</i>CK-citrateL, 2,670 for <i>gl</i>CK-citrateS, and 1,616 for <i>gl</i>CK-AMPPNP.</p>e<p>Ramachandran plot categories are most favored, allowed, generously allowed, and disallowed.</p

Investigation of Mechanical Engineering Academicians’ Use of Distance Education Technologies

The aim of this study is to determine the use of distance education technologies, conditions of use and how often they use various computer applications, and also to investigate the use of these applications by academicians who teach in the Department of Mechanical Engineering. In the research carried out with the scanning model, there were 370 volunteers from various universities in Russia, consisting of academicians who teach in the field of mechanical engineering. The research was carried out in the spring term of 2020–2021; before the research, a 6-week online training was given to mechanical engineer academicians. In the study, the ‘distance education technologies’ measurement tool developed by the researchers and compiled by experts in the field was used. The measurement tool was delivered to the academicians via the online method and collected. The analysis of the data was carried out by using the SPSS programme, frequency analysis, t-test and ANOVA test, and the results were added to the research with tables. Accord-ing to the results obtained from the research, although the distance education technologies of the academicians who teach in the field of mechanical engineering are satisfactory, the rate of academicians never using new technologies in the teaching process is quite low and the rate of using them very often is quite high