Organometallics act through specific biomolecular targets or tumor homeostasis perturbation to induce various cell death pathways.

We show that the three conformational states of integrin α ₅ β ₁ have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000‐fold higher affinity of the extended‐open state than the bent‐closed and extended‐closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact α ₅ β ₁ . On the surface of K562 cells, α ₅ β ₁ is 99.8% bent‐closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N‐glycans and leg domains in each subunit that connect the ligand‐binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single‐span transmembrane domains.

Significance: β ₁ integrins form the largest and most diverse integrin subfamily, with 12 distinct αβ heterodimers. Together, the seven other integrin β-subunits form an equal number of 12 heterodimers. Despite speculation that different integrin subfamilies may differ in activation mechanism or conformation, we show that α ₅ β ₁ , similar to β ₂ and β ₃ integrins, can adopt the bent-closed, extended-closed, and extended-open overall global conformational states. We characterize nine function-perturbing antibodies for the subunit and domain they bind and the conformation they stabilize. Only the extended-open α ₅ β ₁ conformation mediates adhesion to fibronectin. Our results enable many cell biological studies on the 12 β ₁ integrins to be interpreted in terms of the integrin conformational states on cell surfaces that are stabilized.

Significance: Three metal ions are at the heart of the ligand binding site in integrin β-subunits. Furthermore, motions at many domain–domain junctions in integrins regulate ligand binding affinity. High-resolution crystal structures and highly accurate solution ligand binding assays are required to understand the mechanisms by which integrins transmit conformational change in their complex dodecadomain ectodomains. Here we provide information fundamental to understanding these processes in the integrin that assembles fibronectin into fibrils in matrices, α ₅ β ₁ .

Despite the broad-spectrum antimicrobial activities of silver, its internal usage is restricted, owing to the toxicity. Strategies to enhance its efficacy are highly desirable but rely heavily on the understanding of its molecular mechanism of action. However, up to now, no direct silver-targeting proteins have been mined at a proteome-wide scale, which hinders systemic studies on the biological pathways interrupted by silver. Herein, we build up a unique system, namely liquid chromatography gel electrophoresis inductively coupled plasma mass spectrometry (LC-GE-ICP-MS), allowing 34 proteins directly bound by silver ions to be identified in Escherichia coli. By using integrated omic approaches, including metalloproteomics, metabolomics, bioinformatics, and systemic biology, we delineated the first dynamic antimicrobial actions of silver (Ag⁺) in E. coli, i.e., it primarily damages multiple enzymes in glycolysis and tricarboxylic acid (TCA) cycle, leading to the stalling of the oxidative branch of the TCA cycle and an adaptive metabolic divergence to the reductive glyoxylate pathway. It then further damages the adaptive glyoxylate pathway and suppresses the cellular oxidative stress responses, causing systemic damages and death of the bacterium. To harness these novel findings, we coadministrated metabolites involved in the Krebs cycles with Ag⁺ and found that they can significantly potentiate the efficacy of silver both in vitro and in an animal model. Our study reveals the comprehensive and dynamic mechanisms of Ag⁺ toxicity in E. coli cells and offers a novel and general approach for deciphering molecular mechanisms of metallodrugs in various pathogens and cells to facilitate the development of new therapeutics.

Bismuth drugs, despite being clinically used for decades, surprisingly remain in use and effective for the treatment of Helicobacter pylori infection, even for resistant strains when co-administrated with antibiotics. However, the molecular mechanisms underlying the clinically sustained susceptibility of H. pylori to bismuth drugs remain elusive. Herein, we report that integration of in-house metalloproteomics and quantitative proteomics allows comprehensive uncovering of the bismuth-associated proteomes, including 63 bismuth-binding and 119 bismuth-regulated proteins from Helicobacter pylori, with over 60% being annotated with catalytic functions. Through bioinformatics analysis in combination with bioassays, we demonstrated that bismuth drugs disrupted multiple essential pathways in the pathogen, including ROS defence and pH buffering, by binding and functional perturbation of a number of key enzymes. Moreover, we discovered that HpDnaK may serve as a new target of bismuth drugs to inhibit bacterium-host cell adhesion. The integrative approach we report, herein, provides a novel strategy to unveil the molecular mechanisms of antimicrobial metals against pathogens in general. This study sheds light on the design of new types of antimicrobial agents with multiple targets to tackle the current crisis of antimicrobial resistance.