Pengju Jiang

Structure of three tandem filamin domains reveals auto-inhibition of ligand binding

Human filamins are large actin‐crosslinking proteins composed of an N‐terminal actin‐binding domain followed by 24 Ig‐like domains (IgFLNs), which interact with numerous transmembrane receptors and cytosolic signaling proteins. Here we report the 2.5 Å resolution structure of a three‐domain fragment of human filamin A (IgFLNa19–21). The structure reveals an unexpected domain arrangement, with IgFLNa20 partially unfolded bringing IgFLNa21 into close proximity to IgFLNa19. Notably the N‐terminus of IgFLNa20 forms a β‐strand that associates with the CD face of IgFLNa21 and occupies the binding site for integrin adhesion receptors. Disruption of this IgFLNa20–IgFLNa21 interaction enhances filamin binding to integrin β‐tails. Structural and functional analysis of other IgFLN domains suggests that auto‐inhibition by adjacent IgFLN domains may be a general mechanism controlling filamin–ligand interactions. This can explain the increased integrin binding of filamin splice variants and provides a mechanism by which ligand binding might impact filamin structure.

Structural Basis of the Migfilin-Filamin Interaction and Competition with Integrin β Tails

A link between sites of cell adhesion and the cytoskeleton is essential for regulation of cell shape, motility, and signaling. Migfilin is a recently identified adaptor protein that localizes at cell-cell and cell-extracellular matrix adhesion sites, where it is thought to provide a link to the cytoskeleton by interacting with the actin cross-linking protein filamin. Here we have used x-ray crystallography, NMR spectroscopy, and protein-protein interaction studies to investigate the molecular basis of migfilin binding to filamin. We report that the N-terminal portion of migfilin can bind all three human filamins (FLNa, -b, or -c) and that there are multiple migfilin-binding sites in FLNa. Human filamins are composed of an N-terminal actin-binding domain followed by 24 immunoglobulin-like (IgFLN) domains and we find that migfilin binds preferentially to IgFLNa21 and more weakly to IgFLNa19 and -22. The filamin-binding site in migfilin is localized between Pro5 and Pro19 and binds to the CD face of the IgFLNa21 β-sandwich. This interaction is similar to the previously characterized β7 integrin-IgFLNa21 interaction and migfilin and integrin β tails can compete with one another for binding to IgFLNa21. This suggests that competition between filamin ligands for common binding sites on IgFLN domains may provide a general means of modulating filamin interactions and signaling. In this specific case, displacement of integrin tails from filamin by migfilin may provide a mechanism for switching between different integrin-cytoskeleton linkages.

Novel zinc fluorescent probe bearing dansyl and aminoquinoline groupsElectronic supplementary information (ESI) available: NMR spectra and assignment, UV titration details, crystal structure and competitive fluorescent experiments of L. See http://www.rsc.org/suppdata/cc/b2/b202976f/

A novel fluorescent chemosensor (L) demonstrates a remarkable selectivity and sensitivity for zinc(II) ion as evidenced from the solution characterisations and in vitro experiments using Hela cell lines.

Fast Self-Assembly Kinetics of Quantum Dots and a Dendrimeric Peptide Ligand

Engineered peptide ligands with exceptionally high affinity for metal can self-assemble with nanoparticles in biological fluids. A high-affinity dendrimeric peptide ligand for CdSe-ZnS quantum dots (QDs) exhibited very fast association kinetics with QDs and reached equilibrium within 2 s. Here, we have combined a droplet-based microfluidic device with fluorescence detection based on Förster resonance energy transfer (FRET) to provide subsecond resolution in dissecting this fast self-assembly kinetics in solution. This work represents the first application of microfluidic devices to ligand-particle assembly for the measurement of fast assembly kinetics in solution.

Crystal structure and superoxide dismutase activity of a six-coordinate manganese(III) complex

Abstract A six-coordinate manganese(III) complex [Mn(bpb)(H2O)Cl], where H2bpb is N,N′-(1,2-phenylene)bis(pyridine-2-carboxaminde), was prepared and characterized by X-ray crystallography. It crystallizes in the monoclinic system, space group P 2(1)/c with a=6.735(1) A, b=22.910(4) A, c=11.647(2) A, β=102.20(1)°, Z=4 and V =1756.5(5) A 3 . Mn (III) in the complex adopts an octahedral coordination geometry. The cyclic voltammetry of the complex recorded in DMF indicates a reversible one-electron redox reaction. The complex has been shown to catalyze effectively the dismutation of superoxide (O2−) in the riboflavin–methionine–nitro blue tetrazolium assay with an IC50 value of 2.93 μM.

The C-terminal rod 2 fragment of filamin A forms a compact structure that can be extended

Filamins are large proteins that cross-link actin filaments and connect to other cellular components. The C-terminal rod 2 region of FLNa (filamin A) mediates dimerization and interacts with several transmembrane receptors and intracellular signalling adaptors. SAXS (small-angle X-ray scattering) experiments were used to make a model of a six immunoglobulin-like domain fragment of the FLNa rod 2 (domains 16-21). This fragment had a surprising three-branched structural arrangement, where each branch was made of a tightly packed two-domain pair. Peptides derived from transmembrane receptors and intracellular signalling proteins induced a more open structure of the six domain fragment. Mutagenesis studies suggested that these changes are caused by peptides binding to the CD faces on domains 19 and 21 which displace the preceding domain A-strands (18 and 20 respectively), thus opening the individual domain pairs. A single particle cryo-EM map of a nine domain rod 2 fragment (domains 16-24), showed a relatively compact dimeric particle and confirmed the three-branched arrangement as well as the peptide-induced conformation changes. These findings reveal features of filamin structure that are important for its interactions and mechanical properties.

Monitoring the Reactions of the Anticancer Drug Carboplatin with the Chemopreventive Agent Selenomethionine by Electrospray Mass Spectrometry and [1H,15N] HSQC NMR Spectroscopy

The reactions of the second-generation platinum anticancer drug carboplatin with L-selenomethionine (L-Se-MetH) and L-methionine (L-MetH) (abbreviations used: L-Se-MetH: L-selenomethionine; L-Se-Met: deprotonated L-selenomethionine; L-MetH: L-methionine; L-Met: deprotonated L-methionine) were studied by ESMS and 2D [1H,15N] HSQC NMR spectroscopy. The combination of the two techniques provided the unambiguous assignment of [Pt(NH3)2(CBDCA-O)(L-Se-MetH-Se)] (1), the long-lived ring-opened adduct, [Pt(NH3)(CBDCA-O)(L-Se-Met-Se,N)] (2), [Pt(CBDCA-O,O)(L-Se-MetH-Se,N)] (3) and [Pt(L-Se-Met-Se,N)2] (4). The reaction of carboplatin with L-MetH is very similar to that with L-Se-MetH, except for a slower reaction rate. Interestingly, we observed the dimer and polymer forms of carboplatin in solution by electrospray mass spectrometry. This work demonstrates that carboplatin differs from cisplatin in that both reactions with L-Se-MetH and L-MetH form [Pt(L-Se-Met-Se,N)2] or [Pt(L-Met-S,N)2] as the dominant adducts. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

Model of a six immunoglobulin-like domain fragment of filamin A (16-21) built using residual dipolar couplings.

Filamins are actin-binding proteins that participate in a wide range of cell functions, including cell morphology, locomotion, membrane protein localization, and intracellular signaling. The three filamin isoforms found in humans, filamins A, B, and C, are highly homologous, and their roles are partly complementary. In addition to actin, filamins interact with dozens of other proteins that have roles as membrane receptors and channels, enzymes, signaling intermediates, and transcription factors. Filamins are composed of an N-terminal actin-binding domain and 24 filamin-type immunoglobulin-like domains (FLN) that form tail-to-tail dimers with their C-terminal FLN domain. Many of the filamin interactions including those for glycoprotein Ibα and integrins have been mapped to the region comprising FLN domains 16-21. Traditionally, FLN domains have been viewed as independent folding units, arranged in a linear chain joined with flexible linkers. Recent structural findings have shown that consecutive FLNs form more intricate superstructures. The crystal structure of filamin A domains 19-21 (FLNa19-21) revealed that domains 20 and 21 fold together and that the domain interaction can be autoregulatory. The solution structure of domains 18-19 showed a similar domain interaction, whereas domain pair 16-17 has a completely different domain packing mode. In this study, we characterize the domain organization of the FLNa domain sextet 16-21 using NMR spectroscopy. A structure model of this 60-kDa protein has been built using residual dipolar coupling restraints. RDCs and (15)N relaxation data have been used to characterize interdomain motions.

Assembly of a Filamin Four-domain Fragment and the Influence of Splicing Variant-1 on the Structure

Filamins are scaffold proteins that bind to various proteins, including the actin cytoskeleton, integrin adhesion receptors, and adaptor proteins such as migfilin. Alternative splicing of filamin, largely constructed from 24 Ig-like domains, is thought to have a role in regulating its interactions with other proteins. The filamin A splice variant-1 (FLNa var-1) lacks 41 amino acids, including the last β-strand of domain 19, FLNa(19), and the first β-strand of FLNa(20) that was previously shown to mask a key binding site on FLNa(21). Here, we present a structural characterization of domains 18–21, FLNa(18–21), in the FLNa var-1 as well as its nonspliced counterpart. A model of nonspliced FLNa(18–21), obtained from small angle x-ray scattering data, shows that these four domains form an L-shaped structure, with one arm composed of a pair of domains. NMR spectroscopy reveals that in the splice variant, FLNa(19) is unstructured whereas the other domains retain the same fold as in their canonical counterparts. The maximum dimensions predicted by small angle x-ray scattering data are increased upon migfilin binding in the FLNa(18–21) but not in the splice variant, suggesting that migfilin binding is able to displace the masking β-strand and cause a rearrangement of the structure. Possible function roles for the spliced variants are discussed.

Integrin Binding Immunoglobulin Type Filamin Domains Have Variable Stability

Filamin, a large modular protein composed mainly of many immunoglobulin-like domains, is a potent cross-linker of actin filaments. The region containing immunoglobulin type modules 19-21 makes up the binding site for the cytoplasmic tails of the integrin adhesion receptors. Here we investigate the stability of the Ig-like filamin domains using NMR studies over a range of pH and temperature. We show that the 21st Ig-like module (FLNa21) is partly unfolded even under physiological conditions and when attached to FLNa20. It is, however, appreciably stabilized upon binding to integrins. FLNa21 is noticeably less stable than neighboring homologous modules, such as FLNa19 and FLNa17. This variability in stability could be related to the known sensitivity of filamin to cell-mediated mechanical forces.