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.     

Structure and ligand binding of carbohydrate-binding module CsCBM6-3 reveals similarities with fucose-specific lectins and "galactose-binding" domains

Carbohydrate-binding polypeptides, including carbohydrate-binding modules (CBMs) from polysaccharidases, and lectins, are widespread in nature. Whilst CBMs are classically considered distinct from lectins, in that they are found appended to polysaccharide-degrading enzymes, this distinction is blurring. The crystal structure of CsCBM6-3, a "sequence-family 6" CBM in a xylanase from Clostridium stercorarium, at 2.3 A reveals a similar, all beta-sheet fold to that from MvX56, a module found in a family 33 glycoside hydrolase sialidase from Micromonospora viridifaciens, and the lectin AAA from Anguilla anguilla. Sequence analysis leads to the classification of MvX56 and AAA into a family distinct from that containing CsCBM6-3. Whilst these polypeptides are similar in structure they have quite different carbohydrate-binding specificities. AAA is known to bind fucose; CsCBM6-3 binds cellulose, xylan and other beta-glucans. Here we demonstrate that MvX56 binds galactose, lactose and sialic acid. Crystal structures of CsCBM6-3 in complex with xylotriose, cellobiose, and laminaribiose, 2.0 A, 1.35 A, and 1.0 A resolution, respectively, reveal that the binding site of CsCBM6-3 resides on the same polypeptide face as for MvX56 and AAA. Subtle differences in the ligand-binding surface give rise to the different specificities and biological activities, further blurring the distinction between classical lectins and CBMs.     

Evidence for multisite ligand binding and stretching of filamin by integrin and migfilin

Filamin, a large cytoskeletal adaptor, connects plasma membrane to cytoskeleton by binding to transmembrane receptor integrin and actin. Seven of 24 filamin immunoglobulin repeats have conserved integrin binding sites, of which repeats 19 and 21 were shown to be autoinhibited by their adjacent repeats 18 and 20, respectively. Here we show using nuclear magnetic resonance spectroscopy that the autoinhibition can be relieved by integrin or integrin regulator migfilin. We further demonstrate that repeats 19 and 21 can simultaneously engage ligands. The data suggest that filamin is mechanically stretched by integrin or migfilin via a multisite binding mechanism for regulating cytoskeleton and integrin-mediated cell adhesion.     

Structure of the DNA repair helicase hel308 reveals DNA binding and autoinhibitory domains

Hel308 is a superfamily 2 helicase conserved in eukaryotes and archaea. It is thought to function in the early stages of recombination following replication fork arrest and has a specificity for removal of the lagging strand in model replication forks. A homologous helicase constitutes the N-terminal domain of human DNA polymerase Q. The Drosophila homologue mus301 is implicated in double strand break repair and meiotic recombination. We have solved the high resolution crystal structure of Hel308 from the crenarchaeon Sulfolobus solfataricus, revealing a five-domain structure with a central pore lined with essential DNA binding residues. The fifth domain is shown to act as an autoinhibitory domain or molecular brake, clamping the single-stranded DNA extruded through the central pore of the helicase structure to limit the helicase activity of the enzyme. This provides an elegant mechanism to tune the processivity of the enzyme to its functional role. Hel308 can displace streptavidin from a biotinylated DNA molecule, and this activity is only partially inhibited when the DNA is pre-bound with abundant DNA-binding proteins RPA or Alba1, whereas pre-binding with the recombinase RadA has no effect on activity. These data suggest that one function of the enzyme may be in the removal of bound proteins at stalled replication forks and recombination intermediates.     

The three recombinant domains of human serum albumin. Structural characterization and ligand binding properties.

In an attempt to systematically dissect the ligand binding properties of human serum albumin (HSA), the gene segments encoding each of its three domains were defined based on their conserved homologous structural motifs and separately cloned into a secretion vector for Pichia pastoris. We were able to establish a generally applicable purification protocol based on Cibacron Blue affinity chromatography, suggesting that each of the three domains carries a binding site specific for this ligand. Proteins were characterized by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, gel filtration, N-terminal sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, as well as near- and far-UV CD. In addition to the affinity chromatography ligand Cibacron Blue, binding properties toward hemin, warfarin, and diazepam, each of which represents a standard ligand for HSA, respectively, were investigated by the measurement of induced circular dichroism. Clear experimental evidence is provided here for the location of the primary hemin binding site to be on domain I of HSA, and for the primary diazepam binding site to be on domain III. Further, secondary binding sites were found for hemin to be located on domains II and III, and for diazepam on domain I. The warfarin binding site was located primarily on domain II, while on domain I, a secondary binding site and/or parts of the primary binding site were found.     

Kinetic analysis of interaction of BRCA1 tandem breast cancer c-terminal domains with phosphorylated peptides reveals two binding conformations

Tandem breast cancer C-terminal (BRCT) domains, present in many DNA repair and cell cycle checkpoint signaling proteins, are phosphoprotein binding modules. The best-characterized tandem BRCT domains to date are from the protein BRCA1 (BRCA1-BRCT), an E3 ubiquitin ligase that has been linked to breast and ovarian cancer. While X-ray crystallography and NMR spectroscopy studies have uncovered the structural determinants of specificity of BRCA1-BRCT for phosphorylated peptides, a detailed kinetic and thermodynamic characterization of the interaction is also required to understand how structure and dynamics are connected and therefore better probe the mechanism of phosphopeptide recognition by BRCT domains. Through a global analysis of binding kinetics data obtained from surface plasmon resonance (SPR) and stopped-flow fluorescence spectroscopy, we show that the recognition mechanism is complex and best modeled by two equilibrium conformations of BRCA1-BRCT in the free state that both interact with a phosphopeptide, with dissociation constants ( K d) in the micromolar range. We show that the apparent global dissociation constant derived from this kinetic analysis is similar to the K d values measured using steady-state SPR, isothermal titration calorimetry, and fluorescence anisotropy. The dynamic nature of BRCA1-BRCT may facilitate the binding of BRCA1 to different phosphorylated protein targets.     

Starch-synthase III family encodes a tandem of three starch-binding domains

The starch-synthase III (SSIII), with a total of 1025 residues, is one of the enzymes involved in plants starch synthesis. SSIII from Arabidopsis thaliana contains a putative N-terminal transit peptide followed by a 557-amino acid SSIII-specific domain (SSIII-SD) with three internal repeats and a C-terminal catalytic domain of 450 amino acids. Here, using computational characterization techniques, we show that each of the three internal repeats encodes a starch-binding domain (SBD). Although the SSIII from A. thaliana and its close homologous proteins show no detectable sequence similarity with characterized SBD sequences, the amino acid residues known to be involved in starch binding are well conserved.     

Structure of FBP11 WW1-PL ligand complex reveals the mechanism of proline-rich ligand recognition by group II/III WW domains

FBP11/HYPA is a mammalian homologue of yeast splicing factor Prp40. The first WW domain of FBP11/HYPA (FBP11 WW1) is essential for preventing severe neurological diseases such as Huntington disease and Rett syndrome and strongly resembles the WW domain of FCA, the essential regulator for flowering time control. We have solved the structure of FBP11 WW1 and a Pro-Pro-Leu-Pro ligand complex, and demonstrated the binding mechanism with mutational analysis using surface plasmon resonance. The overall structure of FBP11 WW1 in the complex form is quite similar to the structures of WW domains from Group I and IV in complexes. In addition, conformation of FBP11 WW1 does not change much upon ligand binding. The binding orientation of the ligand against FBP11 WW1 is the same as that of the Group IV WW domain-ligand complex, but opposite to that of the Group I complex. The ligand interacts with two grooves formed by surface aromatic residues. The Pro and Leu residues in the ligand interact with the grooves and the Loop I region of FBP11 WW1, respectively, which are necessary interactions for binding the ligand. Interestingly, the two aromatic grooves recognize the Pro residues in entirely different manners, which allows FBP11 WW1 to recognize shorter sequences than the SH3 domain. Combined with homology models of other WW domains, the present report shows the detailed mechanism of ligand binding by Group II/III WW domains, and provides information useful in designing drugs to treat neurodegenerative diseases.     

Multimerization of the ligand binding domains of cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels comprise four subunits and are activated by the direct binding of cyclic nucleotide to an intracellular domain on each subunit. This ligand binding domain is thought to contain a beta roll followed by two alpha helices, designated the B and C helices. To examine the quaternary structure of CNG channels and how it changes during ion channel gating, we introduced single cysteines along the C helix of each subunit in an otherwise cysteineless channel. We found that cysteines on the C helices could form intersubunit disulfide bonds, even between diagonal subunits. Disulfide bond formation occurred primarily in closed channels and inhibited channel opening. These data suggest that the C helices from all four channel subunits are in close proximity in the closed state and move apart during channel opening.     

Actin-binding and dimerization domains of HeLa cell filamin

HeLa cell filamin is a linear, bivalent, homodimer of high molecular weight subunits (Mr 250,000 that may cross-link actin filaments in vivo into supramolecular structures such as networks and bundles. We used millimolar Ca protease from chicken breast muscle to cleave the subunit into smaller fragments that we mapped with respect to the overall structure of the dimer. The enzyme cleaves HeLa filamin into a larger (Mr 192,000) and a smaller (Mr 104,000) fragment; the smaller fragment is the precursor of a still smaller (Mr 92,000) fragment. Only the larger fragment bound to actin in a cosedimentation test, suggesting that it contains the actin-binding region of the subunit. Digestion of HeLa filamin that had been cross-linked with dimethyl adipimidate produced a good yield of the Mr 192,000 fragment but a poor yield of the Mr 104,000/92,000 fragments. Since native filamins are head-to-head dimers, it was expected that cross-linking would proceed most readily at the dimerization site and, therefore, it appears that the Mr 192,000 fragment is cleaved from cross-linked filamin because it is distal to the dimerization region, while the Mr 104,000/92,000 fragments are absent because they lie at the dimerization region and were cross-linked to a form that was not identifiable by sodium dodecyl sulfate electrophoresis.     

From crystal clear ligand binding to designer I domains

A long awaited crystal structure of an integrin I domain in complex with a peptide derived from collagen has revealed the ligand-bound conformation of this domain and suggests a mechanism for allosteric control of integrin function by ligand binding. Also, a computational protein design approach has allowed the creation of stable, high affinity forms of the I domain for the first time.     

Alternative modes of binding of proteins with tandem SH2 domains

The issue of specificity in tyrosine kinase intracellular signaling mediated by src homology 2 (SH2) domains has great importance in the understanding how individual signals maintain their mutual exclusivity and affect downstream responses. Several proteins contain tandem SH2 domains that, on interacting with their ligand, provide a higher level of specificity than can be afforded by the interaction of a single SH2 domain. In this study, we focus on the comparison of two proteins ZAP70 and the p85 subunit of PI 3-kinase, which although distinctly different in function and general structure, possess tandem SH2 domains separated by a linker region and which bind to phosphorylated receptor molecules localized to the cell membrane. Binding studies using isothermal titration calorimetry show that these two proteins interact with peptides mimicking their physiological ligands in very different ways. In the case of the SH2 domains from ZAP70, they interact with a stoichiometry of unity, while p85 is able to make two distinct interactions, one with a stoichiometry of 1:1 and the other with two p85 molecules interacting with one receptor. The observation of two different modes of binding of p85 might be important in providing different cellular responses based on fluctuating intracellular concentration regimes of this protein. Thermodynamic data on both proteins suggest that a conformational change occurs on binding. On investigation of this structural change using a truncated form of p85 (including just the two SH2 domains and the inter-SH2 region), both NMR and circular dichroism spectroscopic studies failed to show significant changes in secondary structure. This suggests that any conformational change associated with binding is small and potentially limited to loop regions of the protein.     

Structural features and ligand binding properties of tandem WW domains from YAP and TAZ, nuclear effectors of the Hippo pathway

The paralogous multifunctional adaptor proteins YAP and TAZ are the nuclear effectors of the Hippo pathway, a central mechanism of organ size control and stem cell self-renewal. WW domains, mediators of protein-protein interactions, are essential for YAP and TAZ function, enabling interactions with PPxY motifs of numerous partner proteins. YAP has single and double WW domain isoforms (YAP1 and YAP2) whereas only a single WW domain isoform of TAZ has been described to date. Here we identify the first example of a double WW domain isoform of TAZ. Using NMR, we have characterized conformational features and peptide binding of YAP and TAZ tandem WW domains (WW1-WW2). The solution structure of YAP WW2 confirms that it has a canonical three-stranded antiparallel β-sheet WW domain fold. While chemical shift-based analysis indicates that the WW domains in the tandem WW pairs retain the characteristic WW domain fold, 15N relaxation data show that, within the respective WW pairs, YAP WW1 and both WW1 and WW2 of TAZ undergo conformational exchange. 15N relaxation data also indicate that the linker between the WW domains is flexible in both YAP and TAZ. Within both YAP and TAZ tandem WW pairs, WW1 and WW2 bind single PPxY-containing peptide ligand concurrently and noncooperatively with sub-mM affinity. YAP and TAZ WW1-WW2 bind a dual PPxY-containing peptide with approximately 6-fold higher affinity. Our results indicate that both WW domains in YAP and TAZ are functional and capable of enhanced affinity binding to multi-PPxY partner proteins such as LATS1, ErbB4, and AMOT.     

Dynamic stabilization of nuclear receptor ligand binding domains by hormone or corepressor binding

We have developed a novel assembly assay to examine structural changes in the ligand binding domain (LBD) of the thyroid hormone receptor (TR). Fragments including the first helix of the TR LBD interact only weakly with the remainder of the LBD in the absence of hormone, but this interaction is strongly enhanced by the addition of either hormone or the corepressor NCoR. Since neither the ligand nor the corepressor shows direct interaction with this helix, we propose that both exert their effects by stabilizing the overall structure of the LBD. Current models of activation of nuclear hormone receptors focus on a ligand-induced allosteric shift in the position of the C-terminal helix 12 that generates the coactivator binding site. Our results suggest that ligand binding also has more global effects that dynamically alter the structure of the receptor LBD.     

Differential polyubiquitin recognition by tandem ubiquitin binding domains of Rabex-5

The biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) is a Rieske-type oxygenase that catalyzes the stereospecific oxygenation of many heterocyclic aromatics including dibenzofuran. In a previous work, we evolved BphAE(LB400) and obtained BphAE(RR41). This variant metabolizes dibenzofuran and 2-chlorodibenzofuran more efficiently than BphAE(LB400). However, the regiospecificity of BphAE(RR41) toward these substrates differs. Dibenzofuran is metabolized principally through a lateral dioxygenation whereas 2-chlorodibenzofuran is metabolized principally through an angular dioxygenation. In order to explain this difference, we examined the crystal structures of both substrate-bound forms of BphAE(RR41) obtained under anaerobic conditions. This structure analysis, in combination with biochemical data for a Ser283Gly mutant provided evidences that the substrate is compelled to move after oxygen-binding in BphAE(RR41):dibenzofuran. In BphAE(RR41):2-chlorodibenzofuran, the chlorine atom is close to the side chain of Ser283. This contact is missing in the BphAE(RR41):dibenzofuran, and strong enough in the BphAE(RR41):2-chlorodibenzofuran to help prevent substrate movement during the catalytic reaction.     

Structure of tandem RNA recognition motifs from polypyrimidine tract binding protein reveals novel features of the RRM fold

Polypyrimidine tract binding protein (PTB), an RNA binding protein containing four RNA recognition motifs (RRMs), is involved in both pre-mRNA splicing and translation initiation directed by picornaviral internal ribosome entry sites. Sequence comparisons previously indicated that PTB is a non-canonical RRM protein. The solution structure of a PTB fragment containing RRMs 3 and 4 shows that the protein consists of two domains connected by a long, flexible linker. The two domains tumble independently in solution, having no fixed relative orientation. In addition to the betaalphabetabetaalphabeta topology, which is characteristic of RRM domains, the C-terminal extension of PTB RRM-3 incorporates an unanticipated fifth beta-strand, which extends the RNA binding surface. The long, disordered polypeptide connecting beta4 and beta5 in RRM-3 is poised above the RNA binding surface and is likely to contribute to RNA recognition. Mutational analyses show that both RRM-3 and RRM-4 contribute to RNA binding specificity and that, despite its unusual sequence, PTB binds RNA in a manner akin to that of other RRM proteins.     

Mapping ligand binding domains in chimeric fibroblast growth factor receptor molecules. Multiple regions determine ligand binding specificity

Fibroblast growth factors (FGFs) mediate essential cellular functions by activating one of four alternatively spliced FGF receptors (FGFRs). To determine the mechanism regulating ligand binding affinity and specificity, soluble FGFR1 and FGFR3 binding domains were compared for activity. FGFR1 bound well to FGF2 but poorly to FGF8 and FGF9. In contrast, FGFR3 bound well to FGF8 and FGF9 but poorly to FGF2. The differential ligand binding specificity of these two receptors was exploited to map specific ligand binding regions in mutant and chimeric receptor molecules. Deletion of immunoglobulin-like (Ig) domain I did not effect ligand binding, thus localizing the binding region(s) to the distal two Ig domains. Mapping studies identified two regions that contribute to FGF binding. Additionally, FGF2 binding showed positive cooperativity, suggesting the presence of two binding sites on a single FGFR or two interacting sites on an FGFR dimer. Analysis of FGF8 and FGF9 binding to chimeric receptors showed that a broad region spanning Ig domain II and sequences further N-terminal determines binding specificity for these ligands. These data demonstrate that multiple regions of the FGFR regulate ligand binding specificity and that these regions are distinct with respect to different members of the FGF family.     

TrkA immunoglobulin-like ligand binding domains inhibit spontaneous activation of the receptor

The extracellular region of the nerve growth factor (NGF) receptor, TrkA, contains two immunoglobulin (Ig)-like domains that are required for specific ligand binding. We have investigated the possible role of these two Ig-like domains in receptor dimerization and activation by using different mutants of the TrkA extracellular region. Deletions of each Ig-like domain, of both, and of the entire extracellular region were made. To probe the structural constraints on ligand-independent receptor dimerization, chimeric receptors were generated by swapping the Ig-like domains of the TrkA receptor for the third or fourth Ig-like domain of c-Kit. We also introduced single-amino-acid changes in conserved residues within the Ig-like domains of TrkA. Most of these TrkA variants did not bind NGF, and their expression in PC12nnr5 cells, which lack endogenous TrkA, promoted ligand-independent neurite outgrowth. Some TrkA mutant receptors induced malignant transformation of Rat-1 cells, as assessed by measuring proliferation in the absence of serum, anchorage-independent growth, and tumorigenesis in nude mice. These mutants exhibited constitutive phosphorylation and spontaneous dimerization consistent with their biological activities. Our data suggest that spontaneous dimerization of TrkA occurs when the structure of the Ig-like domains is altered, implying that the intact domains inhibit receptor dimerization in the absence of NGF.     

Collisional unfolding of multiprotein complexes reveals cooperative stabilization upon ligand binding

Cooperative binding mechanisms are a common feature in biology, enabling a diverse range of protein-based molecular machines to regulate activities ranging from oxygen uptake to cellular membrane transport. Much, however, is not known about such cooperative binding mechanisms, including how such events typically add to the overall stability of such protein systems. Measurements of such cooperative stabilization events are challenging, as they require the separation and resolution of individual protein complex bound states within a mixture of potential stoichiometries to individually assess protein stabilities. Here, we report ion mobility-mass spectrometry results for the concanavalin A tetramer bound to a range of polysaccharide ligands. We use collision induced unfolding, a relatively new methodology that functions as a gas-phase analog of calorimetry experiments in solution, to individually assess the stabilities of concanavalin A bound states. By comparing the differences in activation voltage required to unfold different concanavalin A–ligand stoichiometries, we find evidence suggesting a cooperative stabilization of concanavalin A occurs upon binding most carbohydrate ligands. We critically evaluate this observation by assessing a broad range of ligands, evaluating the unfolding properties of multiple protein charge states, and by comparing our gas-phase results with those obtained from calorimetry experiments carried out in solution.     

Disparity between expression of transferrin receptor ligand binding and non-ligand binding domains on human lymphocytes

We compared transferrin receptor (TfR) expression on human peripheral blood lymphocytes (PBL) activated by phorbol myristate acetate (PMA) or L-phytohemagglutinin (LPHA) using two techniques: (1) 125I-iron-saturated transferrin (FeTf) binding, (2) reactivity with monoclonal anti-TfR antibodies--OKT9 and B3/25. These monoclonal antibodies do not block FeTf binding, and therefore bind to TfR domains separate from the ligand binding site. Unstimulated PBL bound fewer than 1,000 molecules of 125I-FeTf per cell, and less than 5% of cells expressed TfR antigens detected by OKT9 or B3/25. 125I-FeTf binding and antibody binding increased in parallel on LPHA-activated PBL. After exposure to LPHA for 72 hr, 125I-FeTf binding increased 100-fold to 10(5) molecules per cell and greater than 50% of cells expressed TfR antigens. By contrast, PMA activation of PBL markedly increased binding of OKT9 and B3/25 but not the binding of 125I-FeTf. Cell surface expression of TfR antigens seen by OKT9 and B3/25 did not differ between LPHA- and PMA-activated PBL. However, after 72 hr with PMA, 125I-FeTf binding increased only 6-fold and consistently remained at less than 10(4) molecules per cell. Therefore, PMA induced a disparity between expression of TfR ligand binding domains and immunological domains at the cell surface. Cell proliferation assessed by fluorescent DNA analysis was similar in cultures stimulated by LPHA or PMA. These data indicate that lymphoid cells may possess a mechanism for modulating TfR expression in which down-regulation of FeTf binding occurs without receptor internalization. Alternatively, it is possible that this observation may reflect a membrane perturbation effect of PMA.