Mass Spectrometry-Based Structural Proteomics for Metal Ion/Protein Binding Studies

Metal ions are critical for the biological and physiological functions of many proteins. Mass spectrometry (MS)-based structural proteomics is an ever-growing field that has been adopted to study protein and metal ion interactions. Native MS offers information on metal binding and its stoichiometry. Footprinting approaches coupled with MS, including hydrogen/deuterium exchange (HDX), “fast photochemical oxidation of proteins” (FPOP) and targeted amino-acid labeling, identify binding sites and regions undergoing conformational changes. MS-based titration methods, including “protein–ligand interactions by mass spectrometry, titration and HD exchange” (PLIMSTEX) and “ligand titration, fast photochemical oxidation of proteins and mass spectrometry” (LITPOMS), afford binding stoichiometry, binding affinity, and binding order. These MS-based structural proteomics approaches, their applications to answer questions regarding metal ion protein interactions, their limitations, and recent and potential improvements are discussed here. This review serves as a demonstration of the capabilities of these tools and as an introduction to wider applications to solve other questions.

Identification of a phage display-derived peptide interacting with the N-terminal region of Factor VII activating protease (FSAP) enables characterization of zymogen activation.

Increased Factor VII activating protease (FSAP) activity has a protective effect in diverse disease conditions as inferred from studies in FSAP-/- mice and humans deficient in FSAP activity due to a single nucleotide polymorphism. The activation of FSAP zymogen in plasma is mediated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear if this activation mechanism is specific and amenable to manipulation. Using a phage display approach we have identified a peptide, NNKC9/41, that activates pro-FSAP in plasma. Other commonly found zymogens in the plasma were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. Blocking the contact pathway of coagulation did not influence pro-FSAP activation by the peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30-35) in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41 and this was reversed by MA-FSAP-38C7 demonstrating the utility of this peptide. Identification of this peptide, and the corresponding interaction site, provides proof of principle that it is possible to activate a single protease zymogen in blood in a specific manner. Peptide NNKC/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP in more detail, elucidate its biological role.

Synthesis, Characterization and in vitro Anticancer Studies of New Co(II), Ni(II), Cu(II) and Zn(II) Complexes of (E)-4-((Quinoline-8-ylimino)methyl)benzene-1,2,3-triol Ligand

Herein, a new tridentate (NNO) Schiff base ligand, (E)-4-[(quinoline-8-ylimino)methyl]benzene-1,2,3- triol derived from the condensation of 8-aminoquinoline with 2,3,4-trihydroxy benzaldehyde is reported. The ligand was complexed with certain metal ions like Co(II) (1), Ni(II) (2), Cu(II) (3), Zn(II) (4) and were characterized by various spectroscopic and analytical techniques such as FT-IR, UV-Vis, 1H NMR, 13C NMR, ESI-Mass, ESR, elemental analysis and magnetic susceptibility. Spectral data revealed octahedral geometry for cobalt(II), nickel(II), copper(II) complexes and tetrahedral geometry for zinc(II) complex. All the metal(II) complexes along with the Schiff base ligand were screened for their anticancer activities. The CT-DNA binding studies revealed high binding propensity for metal complexes with Kb values 1.50 × 104 M-1 for 1; 3.62 × 104 M-1 for 2; 2.53 × 104 M-1 for 3 and 1.8 × 104 M-1 for 4, respectively. Anticancer studies against A549 & MCF-7 demonstrated excellent antiproliferative activity with IC50 values in the range 17.62-48.82 μM. A standard drug cisplatin was employed to compare the activity of metal complexes. The complexes exhibited remarkable antitumour activity due to their high binding ability with DNA. It is interesting to observe that the complexes did not produce any cytotoxicity towards the normal cell lines.

Novel interaction between neurotrophic factor-α1/carboxypeptidase E and serotonin receptor, 5-HTR1E, protects human neurons against oxidative/neuroexcitotoxic stress via β-arrestin/ERK signaling

Protecting neurons from death during oxidative and neuroexcitotoxic stress is key for preventing cognitive dysfunction. We uncovered a novel neuroprotective mechanism involving interaction between neurotrophic factor-α1 (NF-α1/carboxypeptidase E, CPE) and human 5-HTR1E, a G protein-coupled serotonin receptor with no previously known neurological function. Co-immunoprecipitation and pull-down assays confirmed interaction between NFα1/CPE and 5-HTR1E and 125I NF-α1/CPE-binding studies demonstrated saturable, high-affinity binding to 5-HTR1E in stably transfected HEK293 cells (Kd = 13.82 nM). Treatment of 5-HTR1E stable cells with NF-α1/CPE increased pERK 1/2 and pCREB levels which prevented a decrease in pro-survival protein, BCL2, during H2O2-induced oxidative stress. Cell survival assay in β-arrestin Knockout HEK293 cells showed that the NF-α1/CPE-5-HTR1E-mediated protection against oxidative stress was β-arrestin-dependent. Molecular dynamics studies revealed that NF-α1/CPE interacts with 5-HTR1E via 3 salt bridges, stabilized by several hydrogen bonds, independent of the serotonin pocket. Furthermore, after phosphorylating the C-terminal tail and intracellular loop 3 (ICL3) of NF-α1/CPE-5-HTR1E, it recruited β-arrestin1 by forming numerous salt bridges and hydrogen bonds to ICL2 and ICL3, leading to activation of β-arrestin1. Immunofluorescence studies showed 5-HTR1E and NF-α1/CPE are highly expressed and co-localized on cell surface of human hippocampal neurons. Importantly, knock-down of 5-HTR1E in human primary neurons diminished the NF-α1/CPE-mediated protection of these neurons against oxidative stress and glutamate neurotoxicity-induced cell death. Thus, NF-α1/CPE uniquely interacts with serotonin receptor 5-HTR1E to activate the β-arrestin/ERK/CREB/BCL2 pathway to mediate stress-induced neuroprotection.

On the biological activity of cytokinin free bases and their ribosides

Main conclusion The free bases of cytokinins are the biologically active forms of the hormone while cytokinin ribosides become active only upon removal of the ribose residue. Abstract Cytokinins (CKs) belong to the classical plant hormones. They were discovered more than 65 years ago, but which molecular forms possess genuine CK activity is still matter of debate. Numerous studies support the view that only the free bases are the biologically active molecules. This standpoint has been challenged in a recent review (Nguyen et al. in Planta 254: 45, 2021) proposing that also CK ribosides may have genuine own CK activity. Here we critically discuss the pros and cons of this viewpoint considering the results of biological assays, CK binding studies, 3D structural data of CK-receptor interaction and mutant analyses. It is concluded that all types of study provide clear and convincing evidence only for biological activity of free bases and not ribosides; the latter are rather a transport form of the hormone without their own biological activity.

PAR recognition by multiple reader domains of PARP1 allosterically regulates the DNA-dependent activities and independently stimulates the catalytic activity of PARP1

Poly(ADP-ribosyl)ation is a post translational modification, predominantly catalyzed by Poly(ADP-ribose) polymerase 1 (PARP1) in response to DNA damage, mediating the DNA repair process to maintain genomic integrity. Single strand (SSB) and double strand (DSB) DNA breaks are bonafide stimulators of PARP1 activity. We identified that, in addition, single strand (ss) DNA also binds and stimulates the PARP1 activity. Poly(ADP-ribose) (PAR) is chemically similar to ssDNA. However, PAR mediated PARP1 regulation remains unexplored. Here, we report ZnF3, BRCT and WGR, hitherto uncharacterized, as PAR-specific reader domains of PARP1. Surprisingly, these domains recognize PARylated protein with a higher affinity compared to PAR, but do not bind to DNA. Conversely, N-terminal domains, ZnF1 and ZnF2, of PARP1 recognize DNA but not PAR. Further competition binding studies suggest that PAR binding, allosterically releases DNA from PARP1. Unexpectedly, PAR showed catalytic stimulation of PARP1 but hampers the DNA dependent stimulation. Altogether, our work discovers dedicated PAR and DNA reader domains of the PARP1, and uncovers a novel mechanism of allosteric stimulation of the catalytic activity of PARP1 but retardation of DNA-dependent activities of PARP1 by its catalytic product PAR.

Crystal structures and functional analysis of the ZnF5-WWE1-WWE2 region of PARP13/ZAP define a new mode of engaging poly(ADP-ribose)

PARP13/ZAP acts against multiple viruses through recognizing and promoting degradation of cytoplasmic viral mRNA. PARP13 has four N-terminal Zn-finger motifs that bind CG-rich nucleotide sequences, and a C-terminal ADP ribosyltransferase fold similar to other PARPs. A central region predicted to contain a fifth Zn-finger and two tandem WWE domains is implicated in binding poly(ADP-ribose); however, there are limited insights into the structure and function of this PARP13 region (ZnF5-WWE1-WWE2). Here, we present crystal structures of ZnF5-WWE1-WWE2 from mouse PARP13 in complex with ADP-ribose and with ATP. ZnF5-WWE1-WWE2 crystallized as a dimer with major contacts formed between WWE1 and WWE2 originating from different monomers, indicative of a more compact monomeric arrangement of the tandem WWE domains. Solution scattering experiments and biophysical analysis indicated a monomer in solution, suggesting that the crystal dimer represents domain swapping that could potentially represent a PARP13 conformation assumed when signaling viral RNA detection. The crystal structure and binding studies demonstrate that WWE2 interacts with ADP-ribose and ATP, whereas WWE1 does not have a functional binding site. The shape of the WWE2 binding pocket disfavors interaction with the ribose-ribose linkage of poly(ADP-ribose). Binding studies with poly(ADP-ribose) ligands indicate that WWE2 serves as an anchor for preferential binding to the terminal end of poly(ADP-ribose), and the composite structure of ZnF5-WWE1-WWE2 forms an extended surface to engage polymer chains of ADP-ribose. This model represents a novel mode of poly(ADP-ribose) recognition and provides a structural framework for investigating poly(ADP-ribose) impact on PARP13 function.