Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells1

Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL−1 CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL−1, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control.

Binding of the cellulose-binding domain of exoglucanase Cex from Cellulomonas fimi to insoluble microcrystalline cellulose is entropically driven.

Isothermal titration microcalorimetry is combined with solution-depletion isotherm data to analyze the thermodynamics of binding of the cellulose-binding domain (CBD) from the beta-1,4-(exo)glucanase Cex of Cellulomonas fimi to insoluble bacterial microcrystalline cellulose. Analysis of isothermal titration microcalorimetry data against two putative binding models indicates that the bacterial microcrystalline cellulose surface presents two independent classes of binding sites, with the predominant high-affinity site being characterized by a Langmuir-type Ka of 6.3 (+/-1.4) x 10(7) M-1 and the low-affinity site by a Ka of 1.1 (+/-0.6) x 10(6) M-1. CBDCex binding to either site is exothermic, but is mainly driven by a large positive change in entropy. This differs from protein binding to soluble carbohydrates, which is usually driven by a relatively large exothermic standard enthalpy change for binding. Differential heat capacity changes are large and negative, indicating that sorbent and protein dehydration effects make a dominant contribution to the driving force for binding.

The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose.

Cellulose-binding domains (CBDs) bind specifically to cellulose, and form distinct domains of most cellulose degrading enzymes. The CBD-mediated binding of the enzyme has a fundamental role in the hydrolysis of the solid cellulose substrate. In this work we have investigated the reversibility and kinetics of the binding of the CBD from Trichoderma reesei cellobiohydrolase I on microcrystalline cellulose. The CBD was produced in Escherichia coli, purified, and radioactively labeled by reductive alkylation with 3H. Sensitive detection of the labeled CBD allowed more detailed analysis of its behavior than has been possible before, and important novel features were resolved. Binding of the CBD was found to be temperature sensitive, with an increased affinity at lower temperatures. The interaction of the CBD with cellulose was shown to be fully reversible and the CBD could be eluted from cellulose by simple dilution. The rate of exchange measured for the CBD-cellulose interaction compares well with the hydrolysis rate of cellobiohydrolase I, which is consistent with its proposed mode of action as a processive exoglucanase.

Minimizing a binding domain from protein A.

We present a systematic approach to minimizing the Z-domain of protein A, a three-helix bundle (59 residues total) that binds tightly (Kd = 10 nM) to the Fc portion of an immunoglobin IgG1. Despite the fact that all the contacts seen in the x-ray structure of the complex with the IgG are derived from residues in the first two helices, when helix 3 is deleted, binding affinity is reduced > 10(5)-fold (Kd > 1 mM). By using structure-based design and phage display methods, we have iteratively improved the stability and binding affinity for a two-helix derivative, 33 residues in length, such that it binds IgG1, with a Kd of 43 nM. This was accomplished by stepwise selection of random mutations from three regions of the truncated Z-peptide: the 4 hydrophobic residues from helix 1 and helix 2 that contacted helix 3 (the exoface), followed by 5 residues between helix 1 and helix 2 (the intraface), and lastly by 19 residues at or near the interface that interacts with Fc (the interface). As selected mutations from each region were compiled (12 in total), they led to progressive increases in affinity for IgG, and concomitant increases in alpha-helical content reflecting increased stabilization of the two-helix scaffold. Thus, by sequential increases in the stability of the structure and improvements in the quality of the intermolecular contacts, one can reduce larger binding domains to smaller ones. Such mini-protein binding domains are more amenable to synthetic chemistry and thus may be useful starting points for the design of smaller organic mimics. Smaller binding motifs also provide simplified and more tractable models for understanding determinants of protein function and stability.

Evidence for a role for the phosphotyrosine-binding domain of Shc in interleukin 2 signaling.

Stimulation via the T-cell growth factor interleukin 2 (IL-2) leads to tyrosine phosphorylation of Shc, the interaction of Shc with Grb2, and the Ras GTP/GDP exchange factor, mSOS. Shc also coprecipitates with the IL-2 receptor (IL-2R), and therefore, may link IL-2R to Ras activation. We have further characterized the Shc-IL-2R interaction and have made the following observations. (i) Among the two phosphotyrosine-interaction domains present in Shc, the phosphotyrosine-binding (PTB) domain, rather than its SH2 domain, interacts with the tyrosine-phosphorylated IL-2R beta chain. Moreover, the Shc-PTB domain binds a phosphopeptide derived from the IL-2R beta chain (corresponding to residues surrounding Y338, SCFTNQGpYFF) with high affinity. (ii) In vivo, mutant IL-2R beta chains lacking the acidic region of IL-2Rbeta (which contains Y338) fail to phosphorylate Shc. Furthermore, when wild type or mutant Shc proteins that lack the PTB domain were expressed in the IL-2-dependent CTLL-20 cell line, an intact Shc-PTB domain was required for Shc phosphorylation by the IL-2R, which provides further support for a Shc-PTB-IL-2R interaction in vivo. (iii) PTB and SH2 domains of Shc associate with different proteins in IL-2- and T-cell-receptor-stimulated lysates, suggesting that Shc, through the concurrent use of its two different phosphotyrosine-binding domains, could assemble multiple protein complexes. Taken together, our in vivo and in vitro observations suggest that the PTB domain of Shc interacts with Y338 of the IL-2R and provide evidence for a functional role for the Shc-PTB domain in IL-2 signaling.

An inhalational anesthetic binding domain in the nicotinic acetylcholine receptor.

To determine inhalational anesthetic binding domains on a ligand-gated ion channel, I used halothane direct photoaffinity labeling of the nicotinic acetylcholine receptor (nAChR) in native Torpedo membranes. [14C]Halothane photoaffinity labeling of both the native Torpedo membranes and the isolated nAChR was saturable, with Kd values within the clinically relevant range. All phospholipids were labeled, with greater than 95% of the label in the acyl chain region. Electrophoresis of labeled nAChR demonstrated no significant subunit selectivity for halothane incorporation. Within the alpha-subunit, greater than 90% of label was found in the endoprotease Glu-C digestion fragments which contain the four transmembrane regions, and the pattern was different from that reported for photoactivatable phospholipid binding to the nAChR. Unlabeled halothane reduced labeling more than did isoflurane, suggesting differences in the binding domains for inhalational anesthetics in the nAChR. These data suggest multiple similar binding domains for halothane in the transmembrane region of the nAChR.

Identification of residues that control specific binding of the Shc phosphotyrosine-binding domain to phosphotyrosine sites.

The Shc adaptor protein contains two phosphotyrosine [Tyr(P)]binding modules--an N-terminal Tyr(P) binding (PTB) domain and a C-terminal Src homology 2 (SH2) domain. We have compared the ability of the Shc PTB domain to bind the receptors for nerve growth factor and insulin, both of which contain juxtamembrane Asn-Pro-Xaa-Tyr(P) motifs implicated in PTB binding. The Shc PTB domain binds with high affinity to a phosphopeptide corresponding to the nerve growth factor receptor Tyr-490 autophosphorylation site. Analysis of individual residues within this motif indicates that the Asn at position -3 [with respect to Tyr(P)], in addition to Tyr(P), is critical for PTB binding, while the Pro at position -2 plays a less significant role. A hydrophobic amino acid 5 residues N-terminal to the Tyr(P) is also essential for high-affinity binding. In contrast, the Shc PTB domain does not bind stably to the Asn-Pro-Xaa-Tyr(P) site at Tyr-960 in the activated insulin receptor, which has a polar residue (Ser) at position -5. Substitution of this Ser at position -5 with Ile markedly increased binding of the insulin receptor Tyr-960 phosphopeptide to the PTB domain. These results suggest that while the Shc PTB domain recognizes a core sequence of Asn-Pro-Xaa-Tyr(P), its binding affinity is modulated by more N-terminal residues in the ligand, which therefore contribute to the specificity of PTB-receptor interactions. An analysis of residues in the Shc PTB domain required for binding to Tyr(P) sites identified a specific and evolutionarily conserved Arg (Arg-175) that is uniquely important for ligand binding and is potentially involved in Tyr(P) recognition.

The wing of the enhancer-binding domain of Mu phage transposase is flexible and is essential for efficient transposition.

A tetramer of the Mu transposase (MuA) pairs the recombination sites, cleaves the donor DNA, and joins these ends to a target DNA by strand transfer. Juxtaposition of the recombination sites is accomplished by the assembly of a stable synaptic complex of MuA protein and Mu DNA. This initial critical step is facilitated by the transient binding of the N-terminal domain of MuA to an enhancer DNA element within the Mu genome (called the internal activation sequence, IAS). Recently we solved the three-dimensional solution structure of the enhancer-binding domain of Mu phage transposase (residues 1-76, MuA76) and proposed a model for its interaction with the IAS element. Site-directed mutagenesis coupled with an in vitro transposition assay has been used to assess the validity of the model. We have identified five residues on the surface of MuA that are crucial for stable synaptic complex formation but dispensable for subsequent events in transposition. These mutations are located in the loop (wing) structure and recognition helix of the MuA76 domain of the transposase and do not seriously perturb the structure of the domain. Furthermore, in order to understand the dynamic behavior of the MuA76 domain prior to stable synaptic complex formation, we have measured heteronuclear 15N relaxation rates for the unbound MuA76 domain. In the DNA free state the backbone atoms of the helix-turn-helix motif are generally immobilized whereas the residues in the wing are highly flexible on the pico- to nanosecond time scale. Together these studies define the surface of MuA required for enhancement of transposition in vitro and suggest that a flexible loop in the MuA protein required for DNA recognition may become structurally ordered only upon DNA binding.

Specific mutations in the ligand binding domain selectively abolish the silencing function of human thyroid hormone receptor beta.

Although most nuclear hormone receptors are ligand-dependent transcriptional activators, certain members of this superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR), are involved in transcriptional repression. The silencing function of these receptors has been localized to the ligand binding domain (LBD). Previously, we demonstrated that overexpression of either the entire LBD or only the N-terminal region of the LBD (amino acids 168-259) is able to inhibit the silencing activity of TR. From this result we postulated the existence of a limiting factor (corepressor) that is necessary for TR silencing activity. To support this hypothesis, we identified amino acids in the N-terminal region of the LBD of TR that are important for the corepressor interaction and for the silencing function of TR. The silencing activity of TR was unaffected by overexpression of the LBD of mutant TR (V174A/D177A), suggesting that valine at position 174 and/or aspartic acid at position 177 are important for corepressor interaction. This mutant receptor protein, V174/D177, also lost the ability to silence target genes, suggesting that these amino acids are important for silencing function. Control experiments indicate that this mutant TR maintains its wild-type hormone binding and transactivation functions. These findings further strengthen the idea that the N-terminal region of the LBD of TR interacts with a putative corepressor protein(s) to achieve silencing of basal gene transcription.

Two subsites in the binding domain of the acetylcholine receptor: an aromatic subsite and a proline subsite.

The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is localized in the alpha-subunit within a domain containing the tandem Cys-192 and -193. By analyzing the binding-site region of AcChoR from animal species that are resistant to alpha-neurotoxins, we have previously shown that four residues in this region, at positions 187, 189, 194, and 197, differ between animals sensitive (e.g., mouse) and resistant (e.g., mongoose and snake) to alpha-bungarotoxin (alpha-BTX). In the present study, we performed site-directed mutagenesis on a fragment of the mongoose AcChoR alpha-subunit (residues 122-205) and exchanged residues 187, 189, 194, and 197, either alone or in combination, with those present in the mouse alpha-subunit sequence. Only the mongoose fragment in which all four residues were mutated to the mouse ones exhibited alpha-BTX binding similar to that of the mouse fragment. The mongoose double mutation in which Leu-194 and His-197 were replaced with proline residues, which are present at these positions in the mouse AcChoR and in all other toxin binders, bound alpha-BTX to approximately 60% of the level of binding exhibited by the mouse fragment. In addition, replacement of either Pro-194 or -197 in the mouse fragment with serine and histidine, respectively, markedly decreased alpha-BTX binding. All other mutations resulted in no or just a small increase in alpha-BTX binding. These results have led us to propose two subsites in the binding domain for alpha-BTX: the proline subsite, which includes Pro-194 and -197 and is critical for alpha-BTX binding, and the aromatic subsite, which includes amino acid residues 187 and 189 and determines the extent of alpha-BTX binding.

A protein-binding domain, EH, identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution.

In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heterogeneous proteins of yeast and nematode. The EH domain spans about 70 amino acids and shows approximately 60% overall amino acid conservation. We demonstrated the ability of the EH domain to specifically bind cytosolic proteins in normal and malignant cells of mesenchymal, epithelial, and hematopoietic origin. These observations prompted our search for additional EH-containing proteins in mammalian cells. Using an EH domain-specific probe derived from the eps15 cDNA, we cloned and characterized a cDNA encoding an EH-containing protein with overall similarity to Eps15; we designated this protein Eps15r (for Eps15-related). Structural comparison of Eps15 and Eps15r defines a family of signal transducers possessing extensive networking abilities including EH-mediated binding and association with Src homology 3-containing proteins.

Four p53 DNA-binding domain peptides bind natural p53-response elements and bend the DNA.

Recent structural studies of the minimal core DNA-binding domain of p53 (p53DBD) complexed to a single consensus pentamer sequence and of the isolated p53 tetramerization domain have provided valuable insights into their functions, but many questions about their interacting roles and synergism remain unanswered. To better understand these relationships, we have examined the binding of the p53DBD to two biologically important full-response elements (the WAF1 and ribosomal gene cluster sites) by using DNA circularization and analytical ultracentrifugation. We show that the p53DBD binds DNA strongly and cooperatively with p53DBD to DNA binding stoichiometries of 4:1. For the WAF1 element, the mean apparent Kd is (8.3 +/- 1.4) x 10(-8) M, and no intermediate species of lower stoichiometries can be detected. We show further that complex formation induces an axial bend of at least 60 degrees in both response elements. These results, taken collectively, demonstrate that p53DBD possesses the ability to direct the formation of a tight nucleoprotein complex having the same 4:1 DNA-binding stoichiometry as wild-type p53 which is accompanied by a substantial conformational change in the response-element DNA. This suggests that the p53DBD may play a role in the tetramerization function of p53. A possible role in this regard is proposed.

Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue.

Complexed with its intracellular receptor, FKBP12, the natural product rapamycin inhibits G1 progression of the cell cycle in a variety of mammalian cell lines and in the yeast Saccharomyces cerevisae. Previously, a mammalian protein that directly associates with FKBP12-rapamycin has been identified and its encoding gene has been cloned from both human (designated FRAP) [Brown, E.J., Albers, M.W., Shin, T.B., Ichikawa, K., Keith, C.T., Lane, W.S. & Schreiber, S.L. (1994) Nature (London) 369, 756-758] and rat (designated RAFT) [Sabatini, D.M., Erdjument-Bromage, H., Lui, M., Tempst, P. & Snyder, S.H. (1994) Cell 78, 35-43]. The full-length FRAP is a 289-kDa protein containing a putative phosphatidylinositol kinase domain. Using an in vitro transcription/translation assay method coupled with proteolysis studies, we have identified an 11-kDa FKBP12-rapamycin-binding domain within FRAP. This minimal binding domain lies N-terminal to the kinase domain and spans residues 2025-2114. In addition, we have carried out mutagenesis studies to investigate the role of Ser2035, a potential phosphorylation site for protein kinase C within this domain. We now show that the FRAP Ser2035-->Ala mutant displays similar binding affinity when compared with the wild-type protein, whereas all other mutations at this site, including mimics of phosphoserine, abolish binding, presumably due to either unfavorable steric interactions or induced conformational changes.

Transplantation of a 17-amino acid alpha-helical DNA-binding domain into an antibody molecule confers sequence-dependent DNA recognition.

Recombinant antibodies capable of sequence-specific interactions with nucleic acids represent a class of DNA- and RNA-binding proteins with potential for broad application in basic research and medicine. We describe the rational design of a DNA-binding antibody, Fab-Ebox, by replacing a variable segment of the immunoglobulin heavy chain with a 17-amino acid domain derived from TFEB, a class B basic helix-loop-helix protein. DNA-binding activity was studied by electrophoretic mobility-shift assays in which Fab-Ebox was shown to form a specific complex with DNA containing the TFEB recognition motif (CACGTG). Similarities were found in the abilities of TFEB and Fab-Ebox to discriminate between oligodeoxyribonucleotides containing altered recognition sequences. Comparable interference of binding by methylation of cytosine residues indicated that Fab-Ebox and TFEB both contact DNA through interactions along the major groove of double-stranded DNA. The results of this study indicate that DNA-binding antibodies of high specificity can be developed by using the modular nature of both immunoglobulins and transcription factors.

The Ran/TC4 GTPase-binding domain: identification by expression cloning and characterization of a conserved sequence motif.

Ran/TC4 is an essential, nuclear GTPase implicated in the initiation of DNA replication, entry into and exit from mitosis, and in nuclear RNA and protein transport through the nuclear pore complex. This diversity of functions suggests that Ran interacts with a large number of down-stream targets. Using an overlay assay, we detected a family of putative target proteins that associate with GTP-bound Ran. The sequence of only one such protein, HTF9a/RanBP1, is known. We have now cloned two additional Ran-binding proteins, allowing identification of a distinctive, highly conserved sequence motif of approximately 150 residues. This motif represents a minimal Ran-binding domain that stabilizes the GTP-bound state of Ran. The isolated domain also functions as a coactivator of Ran-GTPase-activating protein. Mutation of a conserved residue within the Ran-binding domain of HTF9a protein drastically reduced Ran binding. Ran-binding proteins coimmunoprecipitated with epitope-tagged Ran from cell lysates, suggesting that these proteins may associate in vivo. A previously uncharacterized Caenorhabditis elegans gene could encode a protein (96 kDa) possessing two Ran-binding domains. This open reading frame also contains similarities to nucleoporins, suggesting a functional link between Ran and nuclear pore complexes.