Mechanistic basis for the recognition of laminin-511 by α6β1 integrin

Mamoru Takizawa, Takao Arimori, Yukimasa Taniguchi, Yu Kitago, Erika Yamashita, Junichi Takagi and Kiyotoshi Sekiguchi, "Mechanistic basis for the recognition of laminin-511 by α6β1 integrin", Science Advances, Vol. 3, No. 9, e1701497, AAAS, 201

Engineered fast-dissociating antibody fragments for multiplexed super-resolution microscopy

モノクローナル抗体の用途を広げる革新技術 --多重超解像可視化プローブへの迅速変換法--. 京都大学プレスリリース. 2022-09-21.Image reconstruction by integrating exchangeable single-molecule localization (IRIS) achieves multiplexed super-resolution imaging by high-density labeling with fast exchangeable fluorescent probes. However, previous methods to develop probes for individual targets required a great amount of time and effort. Here, we introduce a method for generating recombinant IRIS probes with a new mutagenesis strategy that can be widely applied to existing antibody sequences. Several conserved tyrosine residues at the base of complementarity-determining regions were identified as candidate sites for site-directed mutagenesis. With a high probability, mutations at candidate sites accelerated the off rate of recombinant antibody-based probes without compromising specific binding. We were able to develop IRIS probes from five monoclonal antibodies and three single-domain antibodies. We demonstrate multiplexed localization of endogenous proteins in primary neurons that visualizes small synaptic connections with high binding density. It is now practically feasible to generate fast-dissociating fluorescent probes for multitarget super-resolution imaging

NUDT5 hydrolyzes oxidized deoxyribonucleoside diphosphates with broad substrate specificity

The human NUDT5 protein catalyzes the hydrolysis of 8-hydroxy-dGDP. To examine its substrate specificity, four oxidized deoxyribonucleotides (2-hydroxy-dADP, 8-hydroxy-dADP, 5-formyl-dUDP, and 5-hydroxy-dCDP) were incubated with the NUDT5 protein. Interestingly, all of the nucleotides, except for 5-hydroxy-dCDP, were hydrolyzed with various efficiencies. The kinetic parameters indicated that 8-hydroxy-dADP was hydrolyzed as efficiently as 8-hydroxy-dGDP. The hydrolyzing activities for their triphosphate counterparts were quite weak. These results suggest that the NUDT5 protein eliminates various oxidized deoxyribonucleoside diphosphates from the nucleotide pool and prevents their toxic effects

突然変異抑制酵素NUDT5 の幅広い基質特異性発現機構

DNA は生物にとって最も重要な遺伝情報を担う物質でありながら，塩基の酸化，メチル化，チミンダイマーの形成，鎖の切断など，様々な損傷を受けることが知られている．そのため，生物はこれらの損傷に対する多様な防御機構を備えている．DNA の塩基部位の酸化は代表的な損傷の一つであり，特に国内においては酸化損傷およびその修復機構に関する研究が精力的に進められている．DNA を構成する4 種類の塩基（アデニン，グアニン，シトシン，チミン）の中でも，グアニンは最も酸化損傷を受けやすく，その中でも最も生成され易い8-オキソグアニン（8-oxo-7,8,-dihydroguanine，8-oxoG）は，シトシンだけでなくアデニンともミス塩基対を形成してしまうため，DNA のトランスバージョン変異を引き起こす要因となる（図１)1)~3)．その結果，8-oxoG はアルツハイマー病4)，心疾患5)，ガンなど6)，多くの疾患へ関与することも報告されている．ヒトNUDT5 は元来，ADPリボースなどのADP-sugar と呼ばれる一連の修飾ヌクレオチドの加水分解酵素として同定されたが7)8)，その後の研究で酸化損傷に対する防御機構としても働くことが発見された酵素である．後に詳述するが，DNA の酸化に対する防御機構としては，DNA鎖上の酸化塩基の除去だけでなく，DNA 前駆体であるヌクレオチドレベルでの防御機構も重要であり，NUDT5 は，ヒトの細胞内の酸化損傷ヌクレオチドの除去において中心的な役割を担っている酵素の一つと考えられている．また，多くの酸化損傷ヌクレオチドおよびADP-sugar を加水分解することから，幅広く細胞内の浄化に寄与している酵素ともいえる．最近われわれは，NUDT5 と酸化ヌクレオチドとの複合体の立体構造解析の結果などから，NUDT5 が酵素学的にも大変興味深い非常にユニークな機構により様々な基質を認識し，加水分解していることを明らかにした9)．本稿では，その立体構造解析の結果を中心に，NUDT5 による基質認識機構および加水分解反応機構を概説したい

Structural mechanism of laminin recognition by integrin

Recognition of laminin by integrin receptors is central to the epithelial cell adhesion to basement membrane, but the structural background of this molecular interaction remained elusive. Here, we report the structures of the prototypic laminin receptor α6β1 integrin alone and in complex with three-chain laminin-511 fragment determined via crystallography and cryo-electron microscopy, respectively. The laminin-integrin interface is made up of several binding sites located on all five subunits, with the laminin γ1 chain C-terminal portion providing focal interaction using two carboxylate anchor points to bridge metal-ion dependent adhesion site of integrin β1 subunit and Asn189 of integrin α6 subunit. Laminin α5 chain also contributes to the affinity and specificity by making electrostatic interactions with large surface on the β-propeller domain of α6, part of which comprises an alternatively spliced X1 region. The propeller sheet corresponding to this region shows unusually high mobility, suggesting its unique role in ligand capture.JSPS KAKENHI grant number JP18H02389 from Japan Society for the Promotion of Science to T.A., and by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Innovative Drug Discovery and Life Science Research (BINDS)) funded by Japan Agency for Medical Research and Development (AMED) under Grant Number JP19am0101075 to J.T

doi:10.1093/nar/gkr575 Diverse substrate recognition and hydrolysis mechanisms of human NUDT5

Human NUDT5 (hNUDT5) hydrolyzes various modified nucleoside diphosphates including 8-oxo-dGDP, 8-oxo-dADP and ADP-ribose (ADPR). However, the structural basis of the broad substrate specificity remains unknown. Here, we report the crystal structures of hNUDT5 complexed with 8-oxo-dGDP and 8-oxo-dADP. These structures reveal an unusually different substrate-binding mode. In particular, the positions of two phosphates (a and b phosphates) of substrate in the 8-oxo-dGDP and 8-oxo-dADP complexes are completely inverted compared with those in the previously reported hNUDT5–ADPR complex structure. This result suggests that the nucleophilic substitution sites of the substrates involved in hydrolysis reactions differ despite the similarities in the chemical structures of the substrates and products. To clarify this hypothesis, we employed the isotope-labeling method and revealed that 8-oxo-dGDP is attacked by nucleophilic water at Pb, whereas ADPR is attacked at Pa. This observation reveals that the broad substrate specificity of hNUDT5 is achieved by a diversity of not only substrate recognition, but also hydrolysis mechanisms and leads to a novel aspect that enzymes do not always catalyze the reaction of substrates with similar chemical structures by using the chemically equivalent reaction site