A high‐throughput genetically directed protein crosslinking analysis reveals the physiological relevance of the ATP synthase ‘inserted’ state

Abstract

ATP synthase, a highly conserved protein complex that has a subunit composition of a 3 b 3 cdeab 2 c 8-15 for the bacterial enzyme, is a key player in supplying energy to living organisms. This protein complex consists of a peripheral F 1 sector (a 3 b 3 cde) and a membrane-integrated F o sector (ab 2 c 8-15 ). Structural analyses of the isolated protein components revealed that, remarkably, the C-terminal domain of its e-subunit seems to adopt two dramatically different structures, but the physiological relevance of this conformational change remains largely unknown. In an attempt to decipher this, we developed a high-throughput in vivo protein photo-cross-linking analysis pipeline based on the introduction of the unnatural amino acid into the target protein via the scarless genome-targeted site-directed mutagenesis technique, and probing the cross-linked products via the highthroughput polyacrylamide gel electrophoresis technique. Employing this pipeline, we examined the interactions involving the C-terminal helix of the e-subunit in cells living under a variety of experimental conditions. These studies enabled us to uncover that the bacterial ATP synthase exists as an equilibrium between the 'inserted' and 'noninserted' state in cells, maintaining a moderate but significant level of net ATP synthesis when shifting to the former upon exposing to unfavorable energetically stressful conditions. Such a mechanism allows the bacterial ATP synthases to proportionally and instantly switch between two reversible functional states in responding to changing environmental conditions. Importantly, this high-throughput approach could allow us to decipher the physiological relevance of protein-protein interactions identified under in vitro conditions or to unveil novel physiological context-dependent protein-protein interactions that are unknown before.

Regulation of bacterial ATP synthase activity: A gear‐shifting or a pawl–ratchet mechanism?

“…Further research is needed to elucidate the molecular mechanism that regulates the ATP synthase complex, but in vivo approaches as the one reported by Liu et al . [1] are essential to fully understand the protein–protein interactions established in physiological conditions. Irrespective of whether the proposed gear‐shifting or pawl–ratchet mechanism accounts for the regulatory role of the ε‐subunit, the authors have made a major contribution evaluating the in vivo associations established by this subunit and have provided novel, important insights on how bacterial ATP synthase may be regulated under different physiological conditions.…”

“…Therefore, the relative quantities of observed cross‐linked products in the work of Liu et al . [1] could reflect the state of each individual complex among different cells rather than a functional intermediate conformer of the ε‐subunit.…”

“…The work of Liu et al . published in this issue [1] describes an unbiased, robust experimental strategy that provides important insights into the regulatory role of the ε‐subunit of the bacterial F 1 F o ‐ATP synthase in suppressing the ATP hydrolytic activity in vivo . Escherichia coli variants carrying the UV‐reactive unnatural amino acid p ‐benzoyl‐ l ‐phenylalanine at many positions in the ε‐subunit were tested in several growth conditions, and the cross‐link products formed upon exposure to UV irradiation were analyzed.…”

“…The work by Liu et al . [1] suggests the existence of a fine‐tuning mechanism that interconverts ‘inserted’ (i.e., ‘up’ or ‘extended’) to ‘noninserted’ (i.e., ‘down’ or ‘compact’) states of the ε‐subunit, and compares it to a gear‐shifting mechanism in the power transmission system of an automobile. This proposed new regulatory mechanism represents a strategy of the cell to supply as much energy as possible when exposed to unfavorable environmental conditions.…”

Exploring proteins in living cells

“…The schematic shows how an unnatural amino acid, in this case Bpa, is introduced via site-directed mutagenesis at the endogenous target gene in the genome [21,22] . [21,22] 将基因组上的内源性目标基因特定三联体碱基替换为 TAG(图 2) [25] 。这样的引 入了非天然氨基酸的目标蛋白质的表达特征(如，表达的时空条件和水平等)与 内源性蛋白质高度接近，这就使得实验结果更能反映真实情况。 3.4 建立了一种单块胶可分析 384 个蛋白质样品的高通量聚丙烯酰胺凝胶 电泳技术 [25] [25] 。 Figure 3. A representative high-throughput polyacrylamide gel electrophoresis immunoblot image showing 384 protein samples analyzed on a single gel [25] .…”