Unidirectional Mechanistic Valved Mechanisms for Ammonia Transport in GatCAB

Glutamine amidotransferase CAB (GatCAB), a crucial enzyme involved in translational fidelity, catalyzes three reactions: (i) the glutaminase reaction to yield ammonia (NH₃ or NH₄⁺) from glutamine, (ii) the phosphorylation of Glu-tRNA^Gln, and (iii) the transamidase reaction to convert the phosphorylated Glu-tRNA^Gln to Gln-tRNA^Gln. In the crystal structure of GatCAB, the two catalytic centers are far apart, and the presence of a hydrophilic channel to transport the molecules produced by the reaction (i) was proposed. We investigated the transport mechanisms of GatCAB by molecular dynamics (MD) simulations and free energy (PMF) calculations. In the MD simulations (in total ∼1.1 μs), the entrance of the previously proposed channel is closed, as observed in the crystal structure. Instead, a novel hydrophobic channel has been identified in this study: Since the newly identified entrance opened and closed repeatedly in the MD simulations, it may act as a gate. The calculated free energy difference revealed the significant preference of the newly identified gate/channel for NH₃ transport (∼10⁴-fold). In contrast, with respect to NH₄⁺, the free energy barriers are significantly increased for both channels due to tight hydrogen-bonding with hydrophilic residues, which hinders efficient transport. The opening of the newly identified gate is modulated by Phe206, which acts as a “valve”. For the backward flow of NH₃, our PMF calculation revealed that the opening of the gate is hindered by Ala207, which acts as a mechanistic “stopper” against the motion of the “valve” (Phe206). This is the first report to elucidate the detailed mechanisms of unidirectional mechanistic valved transport inside proteins

Graphs of the Fluctuation of the P-score Classified into 4 Groups.

<p>A: Upward type, B: Downward type, C: Repetition type, D: Unspecified type The solid lines represent the results of the ASP group and the broken lines that of the controls. The X axis represents the number of trial and the Y axis represents the P-score. Representative results of each group are shown in this figure. The difference in the depth of the color was given to make it easier for readers to distinguish each line.</p

The Results of the PD Game.

<p>TFT: Tit for tat. The results are shown as mean ± S.D. The p values were obtained by Student’s t-test and considered significant when they were <0.05.</p

Canonical 2×2 PD Payoff Matrix.

<p>The 2×2 matrix based on the combination of both players’ selection makes four different patterns of scores. In this matrix, R stands for Reward for mutual cooperation, P for Punishment for mutual defection, T for Temptation to defect and S for Sucker’s payoff. In these conditions, when the matrix fulfills the formula 2R > S+T and T > R >P> S, the game can be defined as Prisoner’s Dilemma.</p

The Payoff Matrix of This Study.

<p>This 2×2 matrix indicates the score given to each player depending on the combination of their choices. The P-score, which goes to both players when they cooperate, at the beginning of the game was set as −4. The P-score changed according to the results of the previous trial. Scores in <i>italics</i> are given to player A and scores in <b>bold</b> are given to player B.</p

Subject Characteristics.

<p>IQ: Intelligence Quotient, FSIQ: full-scale IQ on Wechsler Adult Intelligence Scale (WAIS)-III, VIQ: Verbal IQ on WAIS-III, PIQ: Performance IQ on WAIS-III.</p><p>The p values were obtained by Student’s t-test. No significant differences were found between the 2 groups. The age of each group was indicated as the mean ± S.D.</p

Rates of work-life balance and environmental satisfaction.

<p>A: Are you experiencing difficulties in maintaining your work-life balance? (1: not at all, 5: very much) B: Are you satisfied with your work environment? (1: not satisfied at all, 5: very much satisfied).</p

Means MBI scores and burnout rates.

<p>a Chi-squared test; the results of three burnout subsets were compared.</p

Factors associated with burnout and stress: multivariate linear regression analysis (n = 704).

<p>β: standardised regression coefficient.</p><p>VIF: variance inflation factor.</p>a<p>Coded as: 0 =  Male; 1 =  Female.</p>b<p>Coded as: 1 =  Professor or Associate Professor; 2 =  Assistant Professor; 3 =  Psychiatric Resident; 4 =  Postgraduate Student.</p>c<p>Coded as: 0 =  Married; 1 =  Not married.</p>d<p>Coded as: 0 =  Have children; 1 =  Do not have children.</p>e<p>Coded as: 1 =  Less than 40 hours; 2 = 40 to less than 50 hours; 3 = 50 hours or more.</p>f<p>Coded as: 1 =  None; 2 =  Less than 4 times; 3 = 5 to 9 times; 4 = 10 times or more.</p>g<p>Possible range: 1–5. Higher scores indicate higher satisfaction levels.</p>*<p>Statistically significant variables.</p

Baseline characteristics of psychiatrists in the sample group.

<p>a Percentage of the total number of valid values for each variable.</p