Identification of a critical ankyrin-binding loop on the cytoplasmic domain of erythrocyte membrane band 3 by crystal structure analysis and site-directed mutagenesis

The cytoplasmic domain of erythrocyte membrane band 3 (cdb3) serves as a center of membrane organization, interacting with such proteins as ankyrin, protein 4.1, protein 4.2, hemoglobin, several glycolytic enzymes, a tyrosine phosphatase, and a tyrosine kinase, p72(syk). The crystallographic structure of the cdb3 dimer has revealed that residues 175-185 assume a beta-hairpin loop similar to a putative ankyrin-binding motif at the cytoplasmic surface of the Na(+)/K(+)-ATPase. To test whether this hairpin loop constitutes an ankyrin-binding site on cdb3, we have deleted amino acids 175-185 and substituted the 11-residue loop with a Gly-Gly dipeptide that bridges the deletion without introducing strain into the structure. Although the deletion mutant undergoes the same native conformational changes exhibited by wild type cdb3 and binds other peripheral proteins normally, the mutant exhibits no affinity for ankyrin. This suggests that the exposed beta-hairpin turn indeed constitutes a major ankyrin-binding site on cdb3. Other biochemical studies suggest that ankyrin also docks at the NH(2) terminus of band 3. Thus, antibodies to the NH(2) terminus of cdb3 block ankyrin binding to the cdb3, and ankyrin binding to cdb3 prevents p72(syk) phosphorylation of cdb3 at its NH(2) terminus (predominantly at Tyr-8). However, a truncation mutant of cdb3 lacking the NH(2)-terminal 50 residues displays the same binding affinity as wild type cdb3. These data thus suggest that the NH(2) terminus of cdb3 is proximal to but not required for the cdb3-ankyrin interaction.     

Localization of the protein 4.1-binding site on the cytoplasmic domain of erythrocyte membrane band 3.

Of the several proteins that bind along the cytoplasmic domain of erythrocyte membrane band 3, only the sites of interaction of proteins 4.1 and 4.2 remain to be at least partially localized. Using five independent techniques, we have undertaken to map and characterize the binding site of band 4.1 on band 3. First, transfer of a radioactive cross-linker (125I-2-(p-azido-salicylamido)ethyl-1-3-dithiopropionate) from purified band 4.1 to its binding sites on stripped inside-out erythrocyte membrane vesicles (stripped IOVs) revealed major labeling of band 3, glycophorin C, and glycophorin A. Proteolytic mapping of the stripped IOVs then demonstrated that the label on band 3 was confined largely to a fragment comprising residues 1-201. Second, competitive binding experiments with Fab fragments of monoclonal and peptide-specific polyclonal antibodies to numerous epitopes along the cytoplasmic domain of band 3 displayed stoichiometric competition only with Fabs to epitopes between residues 1 and 91 of band 3. Weak competition was also observed with Fabs to a sequence of the cytoplasmic domain directly adjacent to the membrane-spanning domain, but only at 50-100-fold excess of Fab. Third, band 4.1 protected band 3 from chymotryptic hydrolysis at tyrosine 46 and to a much lesser extent at a site within the junctional peptide connecting the membrane-spanning and cytoplasmic domains of band 3. Fourth, ankyrin, which has been previously shown to interact with band 3 both near a putative central hinge and at the N terminus competed with band 4.1 for band 3 in stripped IOVs. Since band 4.1 does not associate with band 3 near the flexible central hinge, the competition with ankyrin can be assumed to derive from a mutual association with the N terminus. Finally, a synthetic peptide corresponding to residues 1-15 of band 3 was found to mildly inhibit band 4.1 binding to stripped IOVs. Taken together, these data suggest that band 4.1 binds band 3 predominantly near the N terminus, with a possible secondary site near the junction of the cytoplasmic domain and the membrane.     

Tyrosine phosphorylation of band 3 inhibits peripheral protein binding

The cytoplasmic domain of band 3 (cdb3) of the human erythrocyte membrane is a good substrate of endogenous and exogenous protein-tyrosine kinases. Because one site of tyrosine phosphorylation is within the glycolytic enzyme/hemoglobin-binding region at the N terminus of the polypeptide, we have investigated whether tyrosine phosphorylation of cdb3 might influence its interaction with the above peripheral proteins. Using p40, a protein-tyrosine kinase isolated from bovine thymus, we demonstrate that aldolase binding to cdb3 linked to Affi-Gel 15 is significantly inhibited by phosphorylation of the immobilized band 3. Importantly, upon dephosphorylation of the gel with acid phosphatase, aldolase binding returns to prephosphorylated values. Similarly, cdb3 phosphorylation was found to inhibit glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, and hemoglobin binding to immobilized cdb3. In the converse experiment, untreated soluble cdb3 was shown to bind to immobilized aldolase, whereas phosphorylated cdb3 (approximately equal to 1.8 mol of Pi/mol of cdb3) did not. Furthermore, phosphorylated cdb3 was unable to inhibit aldolase catalysis, whereas untreated cdb3, as shown previously by others, was a potent inhibitor. Taken together, these results demonstrate that phosphorylation of cdb3 on tyrosine residues inhibits peripheral protein binding at the polypeptide's N terminus. In view of the known effect of glycolytic enzyme binding to band 3 on catalytic activity, tyrosine phosphorylation of band 3 may modulate glycolysis in vivo.     

Mapping the ankyrin-binding site of the human erythrocyte anion exchanger

This report describes initial efforts to map the ankyrin-binding site of the cytoplasmic domain of the human erythrocyte anion exchanger. The conclusions are that this site is likely to involve a fairly extended sequence in the midregion of the cytoplasmic domain and requires interactions that are not provided by isolated peptides. The region of the sequence involving residues 174-186 is likely to participate in the ankyrin-binding site based on several experiments. Limited tryptic cleavage in the midregion of the cytoplasmic domain (residues 174 and/or 181) nearly abolished the ability of the cytoplasmic domain to inhibit binding of ankyrin to the anion exchanger. Ankyrin protected the cytoplasmic domain from tryptic digestion. Finally, peptide-specific antibodies against the sequence encompassing the site(s) of tryptic cleavage (residues 174-186) blocked binding of ankyrin to the anion exchanger. However, the sequence comprising the tryptic site is not sufficient for high affinity binding of ankyrin. A 39-amino acid peptide (residues 161-200) that includes the tryptic cleavage site(s) was inactive in inhibiting binding of ankyrin to the anion exchanger. Further evidence for a complex ankyrin-binding site is that peptide-specific antibodies against two different, noncontiguous regions (residues 118-162 and 174-186) both inhibited binding of ankyrin to the anion exchanger and were only 10-20% as effective as antibody against the entire cytoplasmic domain. Finally, the ankyrin-binding site of the anion exchanger did not renature following sodium dodecyl sulfate electrophoresis and transfer to nitrocellulose paper even though spectrin did recover ability to bind ankyrin under the same conditions. Thus, the ankyrin-binding site is not defined by a short continuous sequence. A simple consensus sequence for ankyrin-binding regions in other proteins is not likely.     

Characterization of the reversible conformational equilibrium in the cytoplasmic domain of human erythrocyte membrane band 3

The cytoplasmic domain of erythrocyte membrane band 3 (cdb3) serves as a center of membrane organization, interacting with such proteins as ankyrin, protein 4.1, protein 4.2, hemoglobin, several glycolytic enzymes, and a tyrosine kinase, p72syk. cdb3 exists in a reversible, pH-dependent conformational equilibrium characterized by large changes in Stokes radius (11 A) and intrinsic fluorescence (2-fold). Based on the crystallographic structure of the cdb3 dimer, we hypothesized that the above conformational equilibrium might involve the movement of flanking peripheral protein binding domains away from a shared dimerization domain. To test this hypothesis, we have mutated both donor (W105L) and acceptor (D316A) residues of a prominent H bond that bridges the above two domains and have examined the effect on the resulting conformational equilibrium. Analysis of the intrinsic fluorescence, Stokes radius, thermal stability, urea stability, and segmental mobility of these mutants reveals that the above H bond is indeed present in the low pH conformation of cdb3 and broken in a higher pH conformation. The data further reveal that cdb3 exists in three native pH-dependent conformations and that rupture of the aforementioned H bond occurs only during conversion of the low pH conformation to the mid-pH conformation. Conversion of the mid-pH conformation to the high pH conformation would now appear to involve structural changes primarily in the peripheral protein binding domain. Because ankyrin associates avidly with the low pH conformation of cdb3, ankyrin occupancy should strongly influence this structural equilibrium and thereby affect band 3 and perhaps global membrane properties.     

Visualization of neural cell adhesion molecule by electron microscopy

The 130- and 160-kD polypeptide forms of the neural cell adhesion molecule (NCAM) were analyzed by electron microscopy after low angle rotary shadowing and freeze replication. Individual NCAM molecules appeared as uniformly thick rods, with a distinct bend or hinge region near their middle. Aggregates were also present, containing two to six rods in a pinwheel-like configuration without measurable overlap between rods. The 130- and 160-kD NCAM forms had lengths of 38 and 51 nm, respectively, with a difference in arm length distal to the bend, but not toward the center of the pinwheel. Although enzymatic removal of the polysialic acid moiety on NCAM did not alter the appearance of individual molecules, it did increase the average number of arms per aggregate. Monoclonal antibodies that recognize defined regions of the NCAM polypeptide were used to provide landmarks on the observed molecular figures. Two antibodies specific for cytoplasmic epitopes near the COOH terminus were clustered at the distal tip of aggregated arms. Two other antibodies that react with epitopes near the NH2 terminus and the middle of the molecule bound to sites more centrally located on the pinwheel structure. Together, these results suggest that the observed aggregates represent an association of molecules near their NH2-terminal homophilic binding site, and have led to several predictions about the nature of an NCAM-mediated cell-cell bond.     

Determination of structural models of the complex between the cytoplasmic domain of erythrocyte band 3 and ankyrin-R repeats 13-24

The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18-20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.     

Characterization of the reversible conformational equilibrium of the cytoplasmic domain of erythrocyte membrane band 3

The cytoplasmic domain of the erythrocyte membrane protein, band 3, contains binding sites for hemoglobin, several glycolytic enzymes, and ankyrin, the linkage to the cytoskeleton. In an earlier study, we found evidence which suggested that band 3 might undergo a native conformational change. We demonstrate here that the cytoplasmic domain of band 3 does exist in a reversible, pH-dependent conformational equilibrium among 3 native states. At physiological salt concentrations this equilibrium is characterized by apparent pKa values of 7.2 and 9.2; however, these apparent pKa values change if the domain's sulfhydryl groups are modified. A major component of the structural change appears to involve the pivoting of two subdomains of the cytoplasmic domain at a central hinge, as evidenced by both hydrodynamic and fluorescence energy transfer measurements. The probable site of this hinge is between residues 176 and 191, a region highly accessible to proteases and also rich in proline. These structural rearrangements also apparently extend to the cluster of tryptophan residues near the N terminus, since the domain's intrinsic fluorescence more than doubles between pH 6.5 and 9.5. No measurable change in band 3 secondary or quaternary structure could be detected during the conformational transitions. A structural model of the cytoplasmic domain of band 3 is presented to show the possible spatial relationships between the regions of conformational change and the sites of peripheral protein binding.     

Molecular regions controlling the activity of CNG channels

The alpha subunits of CNG channels of retinal photoreceptors (rod) and olfactory neurons (olf) are proteins that consist of a cytoplasmic NH(2) terminus, a transmembrane core region (including the segments S1-S6), and a cytoplasmic COOH terminus. The COOH terminus contains a cyclic nucleotide monophosphate binding domain NBD) that is linked by the C-linker (CL) to the core region. The binding of cyclic nucleotides to the NBD promotes channel opening by an allosteric mechanism. We examined why the sensitivity to cGMP is 22 times higher in olf than in rod by constructing chimeric channels and determining the [cGMP] causing half maximum channel activity (EC(50)). The characteristic difference in the EC(50) value between rod and olf was introduced by the NH(2) terminus and the core-CL region, whereas the NBD showed a paradoxical effect. The difference of the free energy difference Delta(DeltaG) was determined for each of these three regions with all possible combinations of the other two regions. For rod regions with respect to corresponding olf regions, the open channel conformation was destabilized by the NH(2) terminus (Delta(DeltaG) = -1.0 to -2.0 RT) and the core-CL region (Delta(DeltaG) = -2.0 to -2.9 RT), whereas it was stabilized by the NBD (Delta(DeltaG) = 0.3 to 1.1 RT). The NH(2) terminus deletion mutants of rod and olf differed by Delta(DeltaG) of only 0.9 RT, whereas the wild-type channels differed by the much larger value of 3.1 RT. The results show that in rod and olf, the NH(2) terminus, the core-CL region, and the NBD differ by characteristic Delta(DeltaG) values that do not depend on the specific composition of the other two regions and that the NH(2) terminus generates the main portion of Delta(DeltaG) between the wild-type channels.     

The structural basis of ankyrin function. II. Identification of two functional domains

Human erythrocyte ankyrin was cleaved by restricted proteolysis at 0 degrees C into two distinct chemical domains. The site on ankyrin that binds spectrin was found to be within a 55,000-dalton domain by spectrin affinity chromatography and co-sedimentation with spectrin in a sucrose gradient. A 32,000-dalton fragment of this domain was prepared (tryptic digest, 0 degrees C, 24 h), separated by gel filtration, and shown to inhibit spectrin binding to the membrane. By comparison with previous two-dimensional peptide maps, the spectrin-binding site was located within this 32,000-dalton fragment near the end of the molecule. The band 3-binding site was identified within an 82,000-dalton domain by binding to a band 3 affinity column. Gel electrophoresis in the absence of detergents confirmed these results and demonstrated that a peptide from the cytoplasmic portion of band 3 retained the capacity to bind the 82,000-dalton domain. The binding properties of the structural domains of ankyrin were correlated with a determination of the affinity constant of the intact molecule. Ankyrin bound with a high affinity to the cytoplasmic portion of band 3 (KD = 8 X 10(-8) M) and to spectrin tetramer (KD = 1 X 10(-7) M) but less so to spectrin dimer (KD = 1 X 10(-6) M). These findings are summarized in a preliminary structural and functional model of ankyrin's role in linking spectrin to the membrane.     

Structural Requirements for Outside-In and Inside-Out Signaling by Drosophila Neuroglian,a Member of the L1 Family of Cell Adhesion Molecules

Expression of the Drosophila cell adhesion molecule neuroglian in S2 cells leads to cell aggregation and the intracellular recruitment of ankyrin to cell contact sites. We localized the region of neuroglian that interacts with ankyrin and investigated the mechanism that limits this interaction to cell contact sites. Yeast two-hybrid analysis and expression of neuroglian deletion constructs in S2 cells identified a conserved 36-amino acid sequence that is required for ankyrin binding. Mutation of a conserved tyrosine residue within this region reduced ankyrin binding and extracellular adhesion. However, residual recruitment of ankyrin by this mutant neuroglian molecule was still limited to cell contacts, indicating that the lack of ankyrin binding at noncontact sites is not caused by tyrosine phosphorylation. A chimeric molecule, in which the extracellular domain of neuroglian was replaced with the corresponding domain from the adhesion molecule fasciclin II, also selectively recruited ankyrin to cell contacts. Thus, outside-in signaling by neuroglian in S2 cells depends on extracellular adhesion, but does not depend on any unique property of its extracellular domain. We propose that the recruitment of ankyrin to cell contact sites depends on a physical rearrangement of neuroglian in response to cell adhesion, and that ankyrin binding plays a reciprocal role in stabilizing the adhesive interaction.     

A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1.

Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown.     

Use of expression mutants and monoclonal antibodies to map the erythrocyte Ca2+ pump.

Deletion and truncation mutants of the human erythrocyte Ca2+ pump (hPMCA4b) were expressed in COS-1 cells. The reactivity patterns of these mutants with seven monoclonal antibodies were examined. Of the seven, six (JA9, JA3, 1G4, 4A4, 3E10 and 5F10) react from the cytoplasmic side. JA9 and JA3 reacted near the NH2 terminus and the COOH terminus of the molecule, respectively. 5F10 and 3E10 recognized portions of the large hydrophilic region in the middle of the protein. The epitopes of 1G4 and 4A4 were discontinuous and included residues from the long hydrophilic domain and residues between the proposed transmembrane domains M2 and M3. Antibody 1B10, which reacts from the extracellular side, recognized the COOH-terminal half of the molecule. These results show that the NH2 terminus, the COOH terminus, the region between M2 and M3, and the large hydrophilic region are all on the cytoplasmic side. This means that there are an even number of membrane crossings in both the NH2-terminal and the COOH-terminal halves. Between residues 75 and 300 there must be at least two membrane crossings, and there are at least two membrane crossings in the COOH-terminal half of the molecule.     

Structure-function analysis of human interleukin-2. Identification of amino acid residues required for biological activity

To locate functional domains of the interleukin-2 (IL-2) protein, a cDNA clone encoding biologically active human IL-2 was mutagenized using synthetic oligonucleotides to incorporate defined amino acid substitutions and deletions in the mature protein. The IL-2 analogs were then produced in Escherichia coli and assayed for the ability to induce proliferation of IL-2-dependent cells and the ability to compete for binding to the IL-2 receptor. Our analysis of over 50 different mutations demonstrated that the integrity of at least three regions of the IL-2 molecule is required for full biological activity: the NH2 terminus (residues 1-20), the COOH terminus (residues 121-133), and 2 of the 3 cysteine residues (58 and 105). Deletion of the NH2-terminal 20 amino acids or the COOH-terminal 10 amino acids resulted in the loss of greater than 99% of bioactivity and binding. Amino acid substitutions at specific positions in these regions also resulted in proteins which retained less than 1% activity. The NH2 terminus and an adjacent internal region were recognized by neutralizing anti-IL-2 antibodies. In combination with the results from epitope competition analysis with neutralizing antibodies, these data are consistent with the IL-2 protein being folded such that the NH2 terminus, the COOH terminus, and the internal 30- to 60-region are juxtaposed to form the binding site recognized by the IL-2 receptor.     

Interaction of the Nav1.2a subunit of the voltage-dependent sodium channel with nodal ankyrinG. In vitro mapping of the interacting domains and association in synaptosomes

Voltage-dependant sodium channels at the axon initial segment and nodes of Ranvier colocalize with the nodal isoforms of ankyrin(G) (Ank(G) node). Using fusion proteins derived from the intracellular regions of the Nav1.2a subunit and the Ank repeat domain of Ank(G) node, we mapped a major interaction site in the intracellular loop separating alpha subunit domains I-II. This 57-amino acid region binds the Ank repeat region with a K(D) value of 69 nm. We identified another site in intracellular loop III-IV, and we mapped both Nav1.2a binding sites on the ankyrin repeat domain to the region encompassing repeats 12-22. The ankyrin repeat domain did not bind the beta(1) and beta(2) subunit cytoplasmic regions. We showed that in cultured embryonic motoneurons, expression of the beta(2) subunit is not necessary for the colocalization of Ank(G) node with functional sodium channels at the axon initial segment. Antibodies directed against the beta(1) subunit intracellular region, alpha subunit loop III-IV, and Ank(G) node could not co-immunoprecipitate Ank(G) node and sodium channels from Triton X-100 solubilisates of rat brain synaptosomes. Co-immunoprecipitation of sodium channel alpha subunit and of the 270- and 480-kDa AnkG node isoforms was obtained when solubilization conditions that maximize membrane protein extraction were used. However, we could not find conditions that allowed for co-immunoprecipitation of ankyrin with the sodium channel beta(1) subunit.     

Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in Escherichia coli.

The cytoplasmic domain of the human erythrocyte membrane protein, band 3 (cdb3), contains binding sites for hemoglobin, several glycolytic enzymes, band 4.1, band 4.2, and ankyrin, and constitutes the major linkage between the membrane skeleton and the membrane. Although erythrocyte cdb3 has been partially purified from proteolyzed red blood cells, further separation of the water-soluble 43-kDa and 41-kDa proteolytic fragments has never been achieved. In order to obtain pure cdb3 for crystallization and site-directed mutagenesis studies, we constructed an expression plasmid that has a tandemly linked T7 promoter placed upstream of the N-terminal 379 amino acids of the erythrocyte band 3 gene. Comparison of several Escherichia coli strains led to the selection of the BL21 (DE3) strain containing the pLysS plasmid as the best host for efficient production of cdb3. About 10 mg of recombinant cdb3 can be easily purified from 4 L of E. coli culture in two simple steps. Comparison of cdb3 released from the red blood cell by proteolysis with recombinant cdb3 reveals that both have the same N-terminal sequence, secondary structure, and pH-dependent conformational change. The purified recombinant cdb3 is also a soluble stable dimer with the same Stokes radius as erythrocyte cdb3. The affinities of the two forms of cdb3 for ankyrin are essentially identical; however, recombinant cdb3 with its unblocked N-terminus exhibits a slightly lower affinity for aldolase.     

Associations of human erythrocyte band 4.2. Binding to ankyrin and to the cytoplasmic domain of band 3

We have examined the associations of purified red cell band 4.2 with red cell membrane and membrane skeletal proteins using in vitro binding assays. Band 4.2 bound to the purified cytoplasmic domain of band 3 with a Kd between 2 and 8 X 10(-7) M. Binding was saturable and slow, requiring 2-4 h to reach equilibrium. This finding confirms previous work suggesting that the principal membrane-binding site for band 4.2 lies within the 43-kDa cytoplasmic domain of band 3 (Korsgren, C., and Cohen, C. M. (1986) J. Biol. Chem. 261, 5536-5543). Band 4.2 also bound to purified ankyrin in solution with a Kd between 1 and 3.5 X 10(-7) M. As with the cytoplasmic domain of band 3, binding was saturable and required 4-5 h to reach equilibrium. Reconstitution with ankyrin of inside-out vesicles stripped of all peripheral proteins had no effect upon band 4.2 binding to membranes; similarly, reconstitution with band 4.2 had no effect upon ankyrin binding. This shows that ankyrin and band 4.2 bind to distinct loci within the 43-kDa band 3 cytoplasmic domain. Coincubation of ankyrin and band 4.2 in solution partially blocked the binding of both proteins to the membrane. Similarly, coincubation of bands 4.1 and 4.2 in solution partially blocked binding of both to membranes. In all cases, the data suggest the possibility that domains on each of these proteins responsible for low affinity membrane binding are principally affected. The data also provide evidence for an association of band 4.2 with band 4.1. Our results show that band 4.2 can form multiple associations with red cell membrane proteins and may therefore play an as yet unrecognized structural role on the membrane.     

Structure of the discoidin domain receptor 1 extracellular region bound to an inhibitory Fab fragment reveals features important for signaling

The discoidin domain receptors, DDR1 and DDR2, are constitutively dimeric receptor tyrosine kinases that are activated by triple-helical collagen. Aberrant DDR signaling contributes to several human pathologies, including many cancers. We have generated monoclonal antibodies (mAbs) that inhibit DDR1 signaling without interfering with collagen binding. The crystal structure of the monomeric DDR1 extracellular region bound to the Fab fragment of mAb 3E3 reveals that the collagen-binding discoidin (DS) domain is tightly associated with the following DS-like domain, which contains the epitopes of all mAbs. A conserved surface patch in the DS domain outside the collagen-binding site is shown to be required for signaling. Thus, the active conformation of the DDR1 dimer involves collagen-induced contacts between the DS domains, in addition to the previously identified association of transmembrane helices. The mAbs likely inhibit signaling by sterically blocking the extracellular association of DDR1 subunits.     

The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. Characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments

The glycoprotein Ib (GPIb), a two-chain integral platelet membrane protein, acts as a receptor for von Willebrand factor. In order to obtain information on the domain involved in this function, as well as on the structural organization of GPIb, the protein has been purified and submitted to limited proteolysis using three different enzymes. The resulting fragments were topographically oriented by means of partial NH2-terminal sequence analysis and immunological identification using monoclonal antibodies. One of these antibodies (LJ-Ib1) inhibited the von Willebrand factor-GPIb interaction completely, one (LJ-P3) partially, and one (LJ-Ib10) had no inhibitory effect. Three distinct fragments, the 38-kDa fragment produced by Serratia marcescens protease as well as the 45- and 35-kDa fragments produced by trypsin, had the same NH2 terminus as the intact GPIb alpha-chain (apparent molecular mass = 140 kDa). These fragments and the alpha-chain reacted with the inhibitory antibodies. On the other hand, three fragments produced by Staphylococcus aureus V8 protease, one of 92 kDa similar to the previously described "macroglycopeptide" and two others of 52 and 45 kDa, had NH2-terminal sequences different from that of the GPIb alpha-chain and reacted only with the noninhibitor monoclonal antibody LJIb10. Thus, the binding domain for von Willebrand factor resides near the NH2 terminus of the GPIb alpha-chain, whereas the carbohydrate-rich region is part of the innermost portion of GPIb and does not appear to be involved in the von Willebrand factor binding function.     

Biochemical and structural studies of ASPP proteins reveal differential binding to p53, p63, and p73

ASPP1 and ASPP2 are activators of p53-dependent apoptosis, whereas iASPP is an inhibitor of p53. Binding assays showed differential binding for C-terminal domains of iASPP and ASPP2 to the core domains of p53 family members p53, p63, and p73. We also determined a high-resolution crystal structure for the C terminus of iASPP, comprised of four ankyrin repeats and an SH3 domain. The crystal lattice revealed an interaction between eight sequential residues in one iASPP molecule and the p53-binding site of a neighboring molecule. ITC confirmed that a peptide corresponding to the crystallographic interaction shows specific binding to iASPP. The contributions of ankyrin repeat residues, in addition to those of the SH3 domain, generate distinctive architecture at the p53-binding site suitable for inhibition by small molecules. These results suggest that the binding properties of iASPP render it a target for antitumor therapeutics and provide a peptide-based template for compound design.