Antigen–Antibody Interactions and Structural Flexibility of a Femtomolar-Affinity Antibody

The femtomolar-affinity mutant antibody (4M5.3) generated by directed evolution is interesting because of the potential of antibody engineering. In this study, the mutant and its wild type (4-4-20) were compared in terms of antigen–antibody interactions and structural flexibility to elucidate the effects of directed evolution. For this purpose, multiple steered molecular dynamics (SMD) simulations were performed. The pulling forces of SMD simulations elucidated the regions that form strong attractive interactions in the binding pocket. Structural analysis in these regions showed two important mutations for improving attractive interactions. First, mutation of Tyr102­(H) to Ser (sequence numbering of Protein Data Bank entry 1FLR) played a role in resolving the steric hindrance on the pathway of the antigen in the binding pocket. Second, mutation of Asp31­(H) to His played a role in resolving electrostatic repulsion. Potentials of mean force (PMFs) of both the wild type and the mutant showed landscapes that do not include obvious intermediate states and go directly to the bound state. These landscapes were regarded as funnel-like binding free energy landscapes. Furthermore, the structural flexibility based on the fluctuations of the positions of atoms was analyzed. It was shown that the fluctuations in the positions of the antigen and residues in contact with antigen tend to be smaller in the mutant than in the wild type. This result suggested that structural flexibility decreases as affinity is improved by directed evolution. This suggestion is similar to the relationship between affinity and flexibility for in vivo affinity maturation, which was suggested by Romesberg and co-workers [Jimenez, R., et al. (2003) <i>Proc. Natl. Acad. Sci. U.S.A.</i> <i>100</i>, 92–97]. Consequently, the relationship was found to be applicable up to femotomolar affinity levels

A QM/MM study of nitric oxide reductase-catalysed N₂O formation

<div><p>Nitrous oxide (N₂O), with a greenhouse effect 300 times that of CO₂, is increasingly eliminated into the atmosphere. Using a hybrid quantum mechanics/molecular mechanics (QM/MM) method, we examined nitric oxide reductase-catalysed N₂O formation, which includes two important chemical reactions of N–N bond formation and N–O bond cleavage. The N–N bond formation has no activation barrier, but N–O bond cleavage exhibits an activation barrier of 20.9 kcal·mol⁻¹ at the QM/MM level. We show that the N–O bond cleavage occurs via a hyponitrous intermediate (Fe_B (II; <i>s</i> = 4/2)/N₂O₂ (−1; <i>s</i> = 1/2)/ (III; <i>s</i> = −1/2)), with bidentate coordination between Glu211 and a non-heme iron atom. The Glu211 coordination decreases the N–O bond cleavage energy barrier by inhibiting the formation of stable, five-membered ring intermediate (Fe_B–O¹–N¹–N²–O²–).</p></div

Substrate-mediated proton relay mechanism for the religation reaction in topoisomerase II

<div><p>The DNA religation reaction of yeast type II topoisomerase (topo II) was investigated to elucidate its metal-dependent general acid/base catalysis. Quantum mechanical/molecular mechanical calculations were performed for the topo II religation reaction, and the proton transfer pathway was examined. We found a substrate-mediated proton transfer of the topo II religation reaction, which involves the 3′ OH nucleophile, the reactive phosphate, water, Arg781, and Tyr782. Metal A stabilizes the transition states, which is consistent with a two-metal mechanism in topo II. This pathway may be required for the cleavage/religation reaction of topo IA and II and will provide a general explanation for the catalytic mechanism in the topo IA and II.</p></div

Synchronous Multiple Lung Adenocarcinomas: Estrogen Concentration in Peripheral Lung

<div><p>Background</p><p>The detection rate of synchronous multiple lung adenocarcinomas (SMLA), which display multiple ground glass opacity nodules in the peripheral lung, is increasing due to advances in high resolution computed tomography. The backgrounds of multicentric development of adenocarcinoma are unknown. In this study, we quantitated estrogen concentration in the peripheral lungs of postmenopausal female patients with SMLA.</p><p>Methods</p><p>The tissue concentration of estrogens (estrone [E1] and estdadiol [E2]) in the noncancerous peripheral lung were measured with liquid chromatography/electrospray tandem mass spectrometry in postmenopausal female patients with lung adenocarcinoma. The expression levels of <i>CYP19A1</i> in the normal lung were also quantitated with real-time PCR. Thirty patients with SMLA and 79 cases of control patients with single lung adenocarcinoma were analyzed.</p><p>Results</p><p>The concentrations of E1 and E2 in the noncancerous tissue were significantly higher in SMLA cases than control cases (P = 0.004 and P = 0.02, respectively). The minor allele (A) of single nucleotide polymorphism rs3764221 were significantly associated with higher concentration of E1 and E2 (P = 0.002 and P = 0.01, respectively) and higher CYP19A1 mRNA expression (P = 0.03).</p><p>Conclusion</p><p>The tissue estrogen concentration of peripheral lung was significantly higher in SMLA than control cases. The high concentration of estrogen may be one of the causes of multicentric development of peripheral lung adenocarcinomas.</p></div

Tissue concentration (pg/g) of Estrone and Estradiol in the noncancerous peripheral lung of SMLA cases and control cases using LC–MS/MS analysis.

<p>Data are represented as box and whisker plots. The median value was represented by a horizontal line in the box pot, and gray box denoted the 75th (upper margin) and 25th percentiles of the values (lower margin), respectively. The upper and lower bars indicated the 90th and 10th percentiles, respectively. The statistical analysis was performed using a Mann-Whitney test. P-value less than 0.05 was considered significant.</p

Distribution of relative expression of <i>CYP19A1</i> messenger RNA using real-time PCR in noncancerous tissues of lung cancer patients divided by the major allele (guanine) versus homozygosity and heterozygosity of the minor allele (adenine) of a rs3764221 in <i>CYP19A1</i>.

<p>Data are represented as box and whisker plots. The median value was represented by a horizontal line in the box pot, and gray box denoted the 75th (upper margin) and 25th percentiles of the values (lower margin), respectively. The upper and lower bars indicated the 90th and 10th percentiles, respectively. The statistical analysis was performed using a Mann-Whitney test.</p

Tissue concentration (pg/g) of Estrone and Estradiol in the noncancerous peripheral lung, according to the minor allele (adenine) of a SNP rs3764221 of <i>CYP19A1</i> using LC–MS/MS analysis.

<p>Data are represented as box and whisker plots. Data are represented as box and whisker plots. The median value was represented by a horizontal line in the box pot, and gray box denoted the 75th (upper margin) and 25th percentiles of the values (lower margin), respectively. The upper and lower bars indicated the 90th and 10th percentiles, respectively. The statistical analysis was performed using a Mann-Whitney test.</p

A QM/MM Study of the l‑Threonine Formation Reaction of Threonine Synthase: Implications into the Mechanism of the Reaction Specificity

Threonine synthase catalyzes the most complex reaction among the pyridoxal-5′-phosphate (PLP)-dependent enzymes. The important step is the addition of a water molecule to the Cβ–Cα double bond of the PLP−α-aminocrotonate aldimine intermediate. Transaldimination of this intermediate with Lys61 as a side reaction to form α-ketobutyrate competes with the normal addition reaction. We previously found that the phosphate ion released from the <i>O</i>-phospho-l-homoserine substrate plays a critical role in specifically promoting the normal reaction. In order to elucidate the detailed mechanism of this “product-assisted catalysis”, we performed comparative QM/MM calculations with an exhaustive search for the lowest-energy-barrier reaction pathways starting from PLP−α-aminocrotonate aldimine intermediate. Satisfactory agreements with the experiment were obtained for the free energy profile and the UV/vis spectra when the PLP pyridine N1 was unprotonated and the phosphate ion was monoprotonated. Contrary to an earlier proposal, the base that abstracts a proton from the attacking water was the ε-amino group of Lys61 rather than the phosphate ion. Nevertheless, the phosphate ion is important for stabilizing the transition state of the normal transaldimination to form l-threonine by making a hydrogen bond with the hydroxy group of the l-threonine moiety. The absence of this interaction may account for the higher energy barrier of the side reaction, and explains the mechanism of the reaction specificity afforded by the phosphate ion product. Additionally, a new mechanism, in which a proton temporarily resides at the phenolate O3′ of PLP, was proposed for the transaldimination process, a prerequisite step for the catalysis of all the PLP enzymes