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Cross-linkers play a critical role in capturing protein dynamics in chemical cross-linking mass spectrometry techniques. Various types of cross-linkers with different backbone features are widely used in the study of proteins. However, it is still not clear how the cross-linkers' backbone affect their own structure and their interactions with proteins. In this study, we systematically characterized and compared methylene backbone and polyethylene glycol (PEG) backbone cross-linkers in terms of capturing protein structure and dynamics. The results indicate the cross-linker with PEG backbone have a better ability to capture the inter-domain dynamics of calmodulin, adenylate kinase, maltodextrin binding protein and dual-specificity protein phosphatase. We further conducted quantum chemical calculations and all-atom molecular dynamics simulations to analyze thermodynamic and kinetic properties of PEG backbone and methylene backbone cross-linkers. Solution nuclear magnetic resonance was employed to validate the interaction interface between proteins and cross-linkers. Our findings suggest that the polarity distribution of PEG backbone enhances the accessibility of the cross-linker to the protein surface, facilitating the capture of sites located in dynamic regions. By comprehensively benchmarking with disuccinimidyl suberate (DSS)/bis-sulfosuccinimidyl-suberate(BS3), bis-succinimidyl-(PEG)2 revealed superior advantages in protein dynamic conformation analysis in vitro and in vivo , enabling the capture of a greater number of cross-linking sites and better modeling of protein dynamics. Furthermore, our study provides valuable guidance for the development and application of PEG backbone cross-linkers. [ABSTRACT FROM AUTHOR]

SHIP2 is a multi-domain inositol 5-phosphatase binding to a variety of phosphotyrosine (pY)-containing proteins through its SH2 domain, so as to regulate various cell signaling pathways by modulating the phosphatidylinositol level in the plasma membrane. Unfavorably, Helicobacter pylori can hijack SHIP2 through the CagA protein to induce gastric cell carcinogenesis. To date, the interaction between SHIP2 and CagA was not analyzed from a structural point of view. Here, the binding of SHIP2-SH2 with Tyr-phosphorylated peptides from four EPIYA motifs (A/B/C/D) in CagA was studied using NMR spectroscopy. The results showed that EPIYA-C and -D bind to a similar interface of SHIP2-SH2, including a pY-binding pocket and a hydrophobic pocket, to achieve high affinity, while EPIYA-A and -B bind to a smaller interface of SHIP2-SH2 with weak affinity. By summarizing the interface and affinity of SHIP2-SH2 for CagA EPIYA-A/B/C/D, c-MET and FcgR2B ITIM, it was proposed that, potentially, SHIP2-SH2 has a selective preference for L > I > V for the aliphatic residues at the pY+3 position in its ligand. This study reveals the rule of the ligand sequence bound by SHIP2-SH2 and the mechanism by which CagA protein hijacks SHIP2, which will help design a peptide inhibitor against SHIP2-SH2. [ABSTRACT FROM AUTHOR]

Protein diffusion in living cells might differ significantly from that measured in vitro. Little is known about the effect of globular protein size on rotational diffusion in cells because each protein has distinct surface properties, which result in different interactions with cellular components. To overcome this problem, the B1 domain of protein G (GB1) and several concatemers of the protein were labeled with 5-fluorotryptophan and studied by F NMR in Escherichia coli cells, Xenopus laevis oocytes, and in aqueous solutions crowded with glycerol, or Ficoll70™ and lysozyme. Relaxation data show that the size dependence of protein rotation in cells is due to weak interactions of the target protein with cellular components, but the effect of these interactions decreases as protein size increases. The results provide valuable information for interpreting protein diffusion data acquired in living cells. [ABSTRACT FROM AUTHOR]

Conformational change of cytochrome c (cyt c) caused by interaction with cardiolipin (CL) is an important step during apoptosis, but the underlying mechanism is controversial. To comprehensively clarify the structural transformations of cyt c upon interaction with CL and avoid the unpredictable alias that might come from protein labeling or mutations, the conformation of purified yeast iso–1 cyt c with natural isotopic abundance in different contents of CL was measured by using NMR spectroscopy, in which the trimethylated group of the protein was used as a natural probe. The data demonstrate that cyt c has two partially unfolded conformations when interacted with CL: one with Fe–His33 coordination and the other with a penta–coordination heme. The Fe–His33 coordination conformation can be converted into a penta–coordination heme conformation in high content of CL. The structure of cyt c becomes partially unfolded with more exposed heme upon interaction with CL, suggesting that cyt c prefers a high peroxidase activity state in the mitochondria, which, in turn, makes CL easy to be oxidized, and causes the release of cyt c into the cytoplasm as a trigger in apoptosis. [ABSTRACT FROM AUTHOR]

The two-component system (TCS) is a significant signal transduction system for bacteria to adapt to complicated and variable environments, and thus has recently been regarded as a novel target for developing antibacterial agents. The natural product luteolin (Lut) can inhibit the autophosphorylation activity of the typical histidine kinase (HK) HK853 from Thermotoga maritime, but the inhibition mechanism is not known. Herein, we report on the binding mechanism of a typical flavone with HK853 by using solution NMR spectroscopy, isothermal titration calorimetry (ITC), and molecular docking. We show that luteolin inhibits the activity of HK853 by occupying the binding pocket of adenosine diphosphate (ADP) through hydrogen bonds and π-π stacking interaction structurally. Our results reveal a detailed mechanism for the inhibition of flavones and observe the conformational and dynamics changes of HK. These results should provide a feasible approach for antibacterial agent design from the view of the histidine kinases. [ABSTRACT FROM AUTHOR]