Characterization of peptides that bind the tumor-associated Thomsen-Friedenreich antigen selected from bacteriophage display libraries

Peptides with high affinities and specificities for numerous proteins and nucleic acids have been previously identified from random peptide bacteriophage display libraries. Here, random peptide bacteriophage display libraries were used to identify sequences that bound the cancer-associated Thomsen-Friedenreich glycoantigen (T antigen). The T antigen, present on most malignant cells, contains an immunodominant Gal beta1 --> 3GalNAc alpha disaccharide unmasked on the surfaces of most carcinomas. This antigen has been postulated to be involved in tumor cell aggregation and metastasis. Two 15 amino acid random peptide bacteriophage display libraries were affinity selected with glycoproteins displaying T antigen on their surfaces. Sequence analysis revealed that many of the peptides shared homology with sugar recognition sites in several carbohydrate-binding proteins. A comparison of affinity selected sequences from both libraries yielded a common motif (W-Y-A-W/F-S-P) rich in aromatic amino acids. Four peptides, corresponding to the affinity selected sequences, were chemically synthesized and characterized for their carbohydrate recognition properties. The synthetic peptides exhibited high specificities and affinities to T antigen displayed on asialofetuin or conjugated to bovine serum albumin (Kd = 5 nM for MAP-P30 binding to asialofetuin) as well as free T-antigen disaccharide in solution (Kd = 10 microM for MAP-P30, 20 microM for P10). Two peptides, P30 and P10, demonstrated high affinities and specificities for both asialofetuin and T antigen in solution. Iodination of a lone tyrosine residue in each sequence dramatically reduced their abilities to bind T antigen, suggesting that the tyrosine residue plays an important role in carbohydrate recognition. That these peptides are of functional significance is evidenced by the ability of both P30 and P10 to inhibit asialofetuin-mediated melanoma cell aggregation in vitro and to compete with peanut lectin for binding to T antigen displayed on the surface of MDA-MB-435 breast carcinoma cells in situ.     

Probing the basis of antibody reactivity with a panel of constrained peptide libraries displayed by filamentous phage

The structural requirements for peptide binding to an antibody may be elucidated by probing it with a variety of peptides having different constraints. To this end, we have constructed and screened a panel of peptide libraries displayed by filamentous bacteriophage. The peptides in most of the libraries have the potential for constraint by fixed Cys residues, which have been placed at different sites within a randomized amino acid sequence of varying length. When taken together, the binding data obtained from screening the panel with a given antibody allow one to determine the types of constraints that promote binding, as well as the residues that are critical for binding. We describe the construction of 11, pVIII-displayed, peptide libraries, whose sizes range from 150 million to 10 billion clones. The libraries were screened with a number of polyclonal and monoclonal antibodies against peptides, proteins and carbohydrates. Cross-reactivity with peptides was always found for antibodies produced against peptides, linear epitopes on folded proteins and, surprisingly, carbohydrates, whereas antibodies against discontinuous epitopes on proteins were found less frequently. The implications of these results are discussed in terms of the structural basis for cross-reactivity with peptides.     

Specific detection of sialyl Lewis X determinant carried on the mucin GlcNAcbeta1-->6GalNAcalpha core structure as a tumor-associated antigen

Sialyl Lewis X serves as a ligand for selectins and is proposed to be implicated in hematogenous metastasis of cancers. When a cultured human breast cancer cell line, MCF-7, which does not express sialyl Lewis X, was transfected with human fucosyltransferase VI cDNA, a strong expression of sialyl Lewis X was induced on transfectant cells. The transfectant cells were found to be also reactive to the antibody NCC-ST-439, which was initially raised against human gastric cancer cells and later was shown to recognize a tumor-associated carbohydrate antigen in breast, gastric, and colon cancers. This suggested that the antigen recognized by NCC-ST-439 is closely related to sialyl Lewis X. Subsequent studies indicated that NCC-ST-439 specifically reacts to NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6GalNAcalpha1 -->R, the sialyl Lewis X on the mucin GlcNAcbeta1-->6 GalNAcalpha structure. The antibody was not reactive to the conventional sialyl Lewis X determinants on straight and/or branched polylactosamine structures including NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->3Galbeta1-->4 Glcbeta1-->R and NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6Galbeta1-->4 Glcbeta1-->R. This was in clear contrast to most of the known anti-sialyl Lewis X antibodies, which do not discriminate internal structures carrying the sialyl Lewis X determinant. On the other hand, the newly generated monoclonal antibody GSC154-27 had a specificity completely the reverse of the specificity of NCC-ST-439 in that it was strongly reactive to the conventional sialyl Lewis X determinants in straight and branched polylactosamine structures, while far less reactive to the sialyl Lewis X determinant on the mucin GlcNAcbeta1-->6GalNAcalpha core structure. A set of these two antibodies would be useful in discriminating the molecular species of sialyl Lewis X expressed by malignant cells and in studying their functional significance.     

Human interleukin-2-activated adherent natural killer cells recognize a conserved antigen found on tumor cells and protozoan parasites

Plastic adherent interleukin-2-activated human natural killer (NK) cells (ALAK) lyse many different histological types of tumor target cells. In order to effect their function as cytotoxic mediators of innate immunity, ALAKs may 'recognize' antigen(s) of protozoan parasites, select virus-infected cells and they may release certain cytokines in response to bacterial antigens. In the present study, we demonstrate that CD3-/CD56dim/CD16dim/monoclonal antibody 5C6bright human ALAKs bind to an antigenic determinant on tumor cells independent of target cell H-2 allotype expression. The conserved antigen was originally obtained from the protozoan Tetrahymena pyriformis, however it is also located on the membranes of many ALAK-sensitive tumor cells. The sequence of this protein, i.e. NK target antigen/NKTag, was previously deduced from cDNA. One ALAK cognate determinant of NKTag was identified by inhibition of cytotoxicity using NKTag-derived synthetic peptides. Biotinylated synthetic peptide [amino acids (aa) 58-74] bound to ALAKs, and synthetic peptides corresponding to this sequence inhibited ALAK lysis of U937 target cells. Inhibition effects of peptide binding were nonreversible. To determine the requirements for recognition by ALAKs of this antigenic determinant, the cognate peptide aa 55-74 was truncated to 17-, 14-, 10-, 7- and 6-mer lengths and tested for inhibition of cytotoxicity. All inhibited except the 6-mer. A possible mechanism of peptide inhibition of cytotoxicity following ALAK binding to an antigenic determinant was a requirement for recognition of one anchor peptide (arginine) and receptor occupancy by a minimum of five to six additional amino acids. In antibody-dependent cell-mediated cytotoxicity experiments, synthetic peptide (aa 68-74) inhibited ALAK killing of anti-H-2d-sensitized P815 targets. This same peptide also inhibited conventional lysis of nonsensitized P815 and IM-9 targets. However, the cognate synthetic peptide (aa 58-74) did not inhibit conjugate formation between ALAKs and U937 target cells. These data demonstrate that ALAK binding to a soluble monomeric peptide inhibited cytotoxicity. Peptide binding appeared to negatively regulate cytotoxicity, and the inhibitory effects following peptide binding were nonreversible. Effector:target cell conjugate formation was not affected by peptide binding, however, recognition was required because inhibition was specific for the amino acid sequence of the synthetic peptide.     

Multiplicity of neuropeptide Y receptors: cloning of a third distinct subtype in the zebrafish

Five different receptor subtypes for neuropeptide Y (NPY) have recently been cloned in mammals. We have discovered three distinct subtypes by PCR in the zebrafish, Danio rerio, and describe here one of these called zYc. The protein sequence identity is 46-51% to mammalian subtypes Y1, Y4 and Y6 and to zebrafish Ya, i.e., the same degree of identity as these subtypes display to one another. The identity to zYb is higher, 75%, indicating that zYb and zYc share a more recent ancestor. The zYc receptor binds NPY and PYY (peptide YY) from mammals as well as zebrafish with high affinities and has a Kd of 16 pM for 125I-pPYY. The pharmacological profile is similar to, but distinct-from, mammalian Y1. zYc inhibits cAMP synthesis. This work suggests that NPY has more receptor subtypes than any other peptide that binds to G protein-coupled receptors. Work is in progress to see if the zebrafish receptors are present in mammals.     

Antigenic structure of the central conserved region of protein G of bovine respiratory syncytial virus

Epitopes were resolved at the amino acid level for nine monoclonal antibodies (MAbs) directed against the central conserved region of protein G of bovine respiratory syncytial virus (BRSV-G). Peptide binding studies showed which amino acids in the epitope contributed to antibody binding. The details of the epitopes were compared with the high-resolution structure of a synthetic peptide corresponding to the central conserved region of BRSV-G, and this indicated which face of the central conserved region is the antigenic structure. The major linear epitope of the central conserved region of BRSV-G is located at the tip of the loop, overlapping a relatively flat surface formed by a double disulfide-bonded cystine noose. At least one, but possibly two sulfur atoms of a disulfide bridge that line the conserved pocket at the center of the flat surface, is a major contributor to antibody binding. Some of the residue positions in the epitope have mutated during the evolution of RSV-G, which suggests that the virus escaped antibody recognition with these mutations. Mutations that occur at positions 177 and 180 may have only a local effect on the antigenic surface, without influencing the structure of the backbone, whereas mutations at positions 183 and 184 will probably have major structural consequences. The study explains the antigenic, structural, and functional importance of each residue in the cystine noose which provides information for peptide vaccine design. Additionally, analysis of the epitopes demonstrated that two point mutations at positions 180 and 205 define the preliminary classification of BRSV subgroups.     

Selection of phage-displayed peptides mimicking an extracellular epitope of human MDR1-P-glycoprotein

To study the structural conformation of the MM4.17 monoclonal antibody (mAb) epitope, twenty-six mAb MM4.17-specific phage clones were affinity-isolated and their inserts characterized for amino acid composition and homology with MDR1 gene product (MDR1-P-glycoprotein). The resulting sequence alignment shows that a unique consensus sequence, which corresponds to the previously mapped TRIDDPET linear peptide identified through synthetic peptide scanning, could not be identified. However, similarities between the inserts of positive phage clones and P-glycoprotein primary structure, consisting in two or three amino acid-long sequences, were observed. An analysis of the over-represented amino acid residues in the inserts of positive clones, and their comparison with the sequence of the antigen was also performed. The two different procedures led to the identification of four regions in which these similarities are clustered, indicating that four different antigen regions, one of which includes the TRIDDPET linear amino acid sequence, might participate in forming the structure of monoclonal antibody MM4.17 epitope.     

Nonspecific binding of Clostridium difficile toxin A to murine immunoglobulins occurs via the fab component

Clostridium difficile toxin A binds nonspecifically to a mouse monoclonal antibody (MAb) immunoglobulin G3 lambda chain [IgG3(lambda)], through the Fab component. This binding, which is retained even after boiling the MAb, is temperature dependent, with more toxin bound at 4 than 37 degrees C (P = 0.0024). The nonspecific binding was decreased by incubation of the IgG3 lambda MAb with alpha- or beta-galactosidase (P = 0.0001 and 0.029, respectively), indicating that toxin A binds to a carbohydrate moiety on the Fab. However, binding was not blocked by the Bandeiraea simplicifolia lectin BS-1, indicating that a terminal alpha-galactose may not be involved. Binding was also not affected by competitive assays with Lewis X antigen. The dependence on carbohydrate moieties in nonspecific binding was also shown for two other MAbs, IgA(kappa) and IgM(lambda), with demonstration of a significant reduction in binding with alpha-galactosidase (P = 0.0001 and 0.0002, respectively) but not beta-galactosidase (P = 0.27 and 0.25, respectively).     

Identification of a carbohydrate recognition domain in filamentous hemagglutinin from Bordetella pertussis

The adherence of Bordetella pertussis to ciliated cells and macrophages is critical to colonization and infection of the respiratory tract. Adherence to both types of cells involves the recognition of eukaryotic carbohydrates by the bacterial adhesin filamentous hemagglutinin (Fha). The carbohydrate recognition domain (CRD) of Fha is considered an important antigen for subcomponent vaccines to maximize the generation of antiadherence antibodies capable of protecting against colonization. For identification of the CRD of Fha, a bank of eight monoclonal antibodies (MAbs) that mapped to four contiguous regions were tested for their ability to block Fha binding to lactosylceramide or to block bacterial binding to ciliated cells. Only MAb 12.5A9, which maps to amino acid residues 1141 to 1279, blocked both Fha binding to lactosylceramide and bacterial binding to ciliated cells. An 18-kDa polypeptide corresponding to this region was expressed in Escherichia coli. Cell lysates containing this protein bound to lactosylceramide in a manner identical to that of native Fha. Mutant strains of B. pertussis that contained an in-frame deletion of the coding sequence for this region produced a truncated Fha that showed negligible cross-reactivity with MAb 12.5A9. In an adherence assay, these mutant strains failed to bind efficiently to either ciliated cells or macrophages. The numbers of adherent bacteria for these strains were reduced to the number obtained with a nonadherent strain. We conclude that the region defined by residues 1141 to 1279 of Fha constitutes a CRD critical for bacterial adherence and represents a potential candidate for a subcomponent vaccine.     

A potent nonpeptide neuropeptide Y Y1 receptor antagonist, a benzodiazepine derivative

We describe here a nonpeptide neuropeptide Y Y1 receptor antagonist, 2,4-dioxo-1,5-bis(2-oxo-2-orthotolyl-ethyl)-3-[3-[3-([3-[3-(3-p iperidin-1-ylmethyl-phenoxy)-propylcarbamoyl]-propyl]-car bamoyloxymethyl)-phenyl]-ureido]-2,3,4,5-tetrahydro-1H-benzo[b][1,4]diaz epine (Compound 1), which was previously synthesized as a linked type of dual cholecystokinin (CCK)-B and histamine H2 receptor antagonist. Compound 1 competitively inhibited [125I]peptide YY (PYY) binding to Y1 receptors in human neuroblastoma SK-N-MC cells with Ki of 6.4 +/- 1.0 nM, while it had no effect on [125I]PYY binding to Y2 or Y5 receptors even at 1 microM. Functionally, Compound 1 inhibited the Y1 receptor-mediated increase in cytosolic free Ca2+ concentration and Y1 receptor-mediated attenuation of cAMP accumulation in a dose-dependent manner with IC50 values of 95 +/- 5 and 320 +/- 10 nM in SK-N-MC cells, respectively. Neither its CCK-B receptor antagonistic moiety of Compound 1 (Compound 2) nor its histamine H2 receptor antagonistic moiety of Compound 1 (Compound 3) had any effect on [125I]PYY binding, suggesting that the entire structure of Compound 1 is essential for Y1 receptor blocking activity. It showed no significant activity (IC50 > 1 microM) in 30 receptor binding assays and 5 enzyme assays, with the exception of CCK-B and histamine H2 receptors. We conclude that Compound 1 is a useful molecule not only for studying the physiological role of neuropeptide Y but also for exploring more specific Y1 receptor antagonists.     

Three-dimensional structure of a human Fab with high affinity for tetanus toxoid

BACKGROUND: The wide range of antibody specificity and affinity results from the differing shapes and chemical compositions of their binding sites. These shapes range from discrete grooves in antibodies elicited by linear oligomers of nucleotides and carbohydrates to shallow depressions or flat surfaces for accommodation of proteins, peptides and large organic compounds. OBJECTIVES: To determine the Fab structure of a high-affinity human antitoxin antibody. To explore structural features which enable the antibody to bind to intact tetanus toxoid, peptides derived from the sequence of the natural immunogen and antigenic mimics identified by combinatorial chemistry. To explain why this Fab shows a remarkable tendency to produce crystals consistently diffracting to d spacings of 1.7-1.8 A. To use this information to engineer a strong tendency to crystallize into the design of other Fabs. STUDY DESIGN: The protein was crystallized in hanging or sitting drops by a microseeding technique in polyethylene glycol (PEG) 8000. Crystals were subjected to X-ray analysis and the three-dimensional structure of the Fab was determined by the molecular replacement method. Interactive computer graphics were employed to fit models to electron density maps, survey the structure in multiple views and discover the crystal packing motif of the protein. RESULTS: Exceptionally large single crystals of this protein have been obtained, one measuring 5 x 3 x 2 mm (l x w x d). The latter was cut into six irregular pieces, each retaining the features of the original in diffracting to high resolution (1.8 A) with little decay in the X-ray beam. In an individual Fab, the active site is relatively flat and it seems likely that the protein antigen and derivative peptides are tightly held on the outer surface without significant penetration into the interior. There is no free space to accommodate even a dipeptide between VH and VL. One of the unique features of the B7-15A2 Fab is a large aliphatic ridge dominating the center of the active site. The CDR3 of the H chain contributes significantly to this ridge, as well as to adjoining regions projected to be important for the docking of the antigen. Both the ease of crystallization and the favorable diffraction properties are mainly attributable to the tight packing of the protein molecules in the crystal lattice. DISCUSSION: The B7-15A2 active site provides a stable and well defined platform for high affinity docking of proteins, peptides and their mimotopes. The advantages for future developments are suggested by the analysis of the crystal properties. It should be possible to incorporate the features promoting crystallization, close packing and resistance to radiation damage into engineered human antibodies without altering the desired specificities and affinities of their active sites.     

Preliminary studies for interaction of a cyclobutane thymine dimer region with its cognate antibody

A monoclonal antibody (TDM-2) specific to a UV-induced cyclobutane pyrimidine dimer has previously been established. We investigated here the structural requirements of antigen recognition by the antibody using chemically synthesized antigen analogs. TDM-2 bound to the cis-syn, but not the trans-syn thymine dimer, and recognized not only the cyclobutane ring but also the 5'- or 3'-side phosphate groups flanking the cyclobutane dimer site. The antibody showed strong binding to photodimer-containing single stranded DNA but indicated little binding to double stranded DNA.     

NMR solution structure of the receptor binding domain of Pseudomonas aeruginosa pilin strain P1. Identification of a beta-turn

The solution structure of the peptide antigen from the receptor binding domain of Pseudomonas aeruginosa strain P1 has been determined using two-dimensional 1H NMR techniques. Ensembles of solution conformations for the trans form of this 23-residue disulfide bridged peptide have been generated using a simulated annealing procedure in conjunction with distance and torsion angle restraints derived from NMR data. Comparison of the NMR-derived solution structures of the P1 peptide with those previously determined for the 17-residue PAK, PAO and KB7 strain peptides [McInnes, C., et al. (1993) Biochemistry 32, 13432-13440; Campbell, A.P., et al. (1995) Biochemistry 34, 16255-16268] reveals the common structural motif of a beta-turn, which may be the necessary structural requirement for recognition of a common cell surface receptor and a common cross-reactive antibody to which all four strains bind. The importance of this conserved beta-turn in the PAK, PAO, KB7 and P1 peptides is discussed with regard to the design of a synthetic peptide vaccine effective against multiple strains of Pseudomonas aeruginosa infections.     

Modelling the P2Y purinoceptor using rhodopsin as template

The P2Y1 purinoceptor cloned from chick brain (Webb, T. et al (1993) FEBS Lett., 324, 219-225) is a 362 amino acid, 41 kDa protein. To locate residues tentatively involved in ligand recognition a molecular model of the P2Y purinoceptor has been constructed. The model was based on the primary sequence and structural homology with the G-protein coupled photoreceptor rhodopsin, in analogy to the method proposed by Ballesteros and Weinstein ((1995) Meth. Neurosci. 25, 366-428). Transmembrane helices were constructed from the amino acid sequence, minimized individually, and positioned in a helical bundle. The helical bundle was then minimized using the Amber forcefield in Discover (BIOSYM Technologies) to obtain the final model. Several residues that have been shown to be critical in ligand binding in other GPCRs are conserved in the P2Y1 purinoceptor. According to our model the side chains of these conserved residues are facing the internal cleft in which ligand binding likely occurs. The model also suggests four basic residues (H121 in TM3, H266 and K269 in TM6 and R299 in TM7) near the extracellular surface that might be involved in ligand binding. These basic residues might be essential in coordinating the triphosphate chain of the endogenous ligand adenosine 5'-triphosphate (ATP). Potential binding sites for agonists have been explored by docking several derivatives (including newly synthesized N6-derivatives) into the model. The N6-phenylethyl substituent is tolerated pharmacologically, and in our model this substituent occupies a region predominantly defined by aromatic residues such as F51 (TM1), Y100 (TM2) and F120 (TM3). The dimethylated analogue of ATP, N6,N6-dimethyl-adenosine 5'-triphosphate, is less well tolerated pharmacologically, and our model predicts that the attenuated activity is due to interference with hydrogen bonding capacity to Q296 (TM7).     

NCCRP-1: a novel receptor protein sequenced from teleost nonspecific cytotoxic cells

The recognition structure responsible for binding to conventional antigen on target cells has not previously been described for nonspecific cytotoxic cells (NCC) or for mammalian natural killer (NK) cells. Although several biochemical pathways may be available for initiation of the lytic cycle in NCC, evidence presented indicates that initial contact with a tumor cell or protozoan parasite is facilitated by recognition of a target antigen by a membrane protein of Mr 34,000 on NCC (NCC receptor protein, NCCRP-1). Binding to NCCRP-1 by monoclonal antibody 5C6, by target cell antigen or by cognate synthetic peptide initiates a signalling response leading to increased cytotoxicity. In the present study, three 20-mer microsequences were obtained from tryptic digests of purified NCCRP-1. Degenerate primers were synthesized (based on each peptide sequence) and were used for RT-PCR with mRNA purified from homogeneous NCC populations. An NCCRP-1 specific cDNA sequence was used to synthesize nondegenerate primers. These primers were used in a 5'/3' RACE PCR to obtain the entire NCCRP-1 specific cDNA. A deduced aa sequence consisted of 235 aa with a derived molecular weight of 30,628 Da. NCCRP-1 is proline rich (9%), has two glycosylation sites and 18% of all amino acids are potential phosphorylation sites (serine, threonine, tyrosine). The identity of the protein was confirmed by finding the previously microsequenced peptides in the derived sequence. Homology searches revealed that NCCRP-1 is a novel protein. Northern blot analysis of mRNA content from teleost NCC, B-cells and T-cells revealed only one band in NCC preparations. Functional studies demonstrated a decrease in membrane NCCRP-1 expression and inhibition of NCC cytotoxicity following treatment with NCCRP-1 anti-sense oligonucleotides. Treatment of NCC with sense oligonucleotides had no inhibitory effects on cytotoxicity. An algorithm predicting the membrane conformation of NCCRP-1 suggests one extracellular proline-rich domain, a transmembrane portion of 15 18 aa and a cytoplasmic tail composed of a high frequency of phosphorylation sites. Current studies suggest that NCC and NCCRP-1 may participate in innate resistance functions in teleost fish.     

Monoclonal antibody against bovine Lactoferricin and its epitopic site

Bovine lactoferricin (LFcin B) is a strong antimicrobial peptide derived from N-lobe of lactoferrin. To study the immunochemical and structural properties of LFcin B, monoclonal antibody (mAb) was prepared and the amino acid sequence concerning with the binding to mAb has been identified. Mice injected with LFcin B showed no production of antibody specific to this peptide, whereas those with LFcin B-KLH conjugate produced anti-LFcin B antibodies. None of the mAb reacted with bovine lactoferrin C-lobe, human lactoferrin or LFcin H. By the reactivity of the mAb against the peptides synthesized on cellulose membranes using SPOTs and against chemically modified derivatives of LFcin B, the antigenic determinant of LFcin B was identified to be the sequence of "QWR".     

Marked decrease of neuropeptide Y Y2 receptor binding sites in the hippocampus in murine prion disease

Using autoradiographic binding methodology with monoiodinated peptide YY together with the agonists neuropeptide Y (NPY) and NPY (13-36), as well as in situ hybridization with oligonucleotide probes complementary to the NPY Y2 receptor (Y2-R) mRNA, we have studied whether or not intracerebral prion inoculation affects Y2-Rs in male CD-1 mice. Monoiodinated peptide YY binding, mainly representing Y2-Rs, was down-regulated by 85% in the CA1 strata oriens and radiatum and by 50-65% in the CA3 stratum oriens 110-140 days postinoculation. In the CA3 stratum radiatum, where the mossy fibers from the dentate granule cells project, there was a significant decrease in PYY binding at 110-120 days. Y2-R mRNA, moderately expressed both in the CA1 and CA3 pyramidal cell layers and the granule cell layer in the dentate gyrus, showed a slight, but not significant, decrease in CA3 neurons 130 days postinoculation. The results indicate that the accumulation of the scrapie prion protein in the CA1-3 region strongly inhibits NPY binding at the Y2-Rs, which, however, is only marginally due to reduced Y2-R mRNA expression. The loss of the ability of NPY to bind to inhibitory Y2-Rs may cause dysfunction of hippocampal circuits and may contribute to the clinical symptoms in mouse scrapie.     

The collagen triple-helix structure

Recent advances, principally through the study of peptide models, have led to an enhanced understanding of the structure and function of the collagen triple helix. In particular, the first crystal structure has clearly shown the highly ordered hydration network critical for stabilizing both the molecular conformation and the interactions between triple helices. The sequence dependent nature of the conformational features is also under active investigation by NMR and other techniques. The triple-helix motif has now been identified in proteins other than collagens, and it has been established as being important in many specific biological interactions as well as being a structural element. The nature of recognition and the degree of specificity for interactions involving triple helices may differ from globular proteins. Triple-helix binding domains consist of linear sequences along the helix, making them amenable to characterization by simple model peptides. The application of structural techniques to such model peptides can serve to clarify the interactions involved in triple-helix recognition and binding and can help explain the varying impact of different structural alterations found in mutant collagens in diseased states.     

Structure of a Numb PTB domain-peptide complex suggests a basis for diverse binding specificity

The phosphotyrosine-binding (PTB) domain of Numb, a protein involved in asymmetric cell division, has recently been shown to bind to the adapter protein Lnx through an LDNPAY sequence, to the Numb-associated kinase (Nak) through a sequence that does not contain an NPXY motif and to GP(p)Y-containing peptides obtained from library screening. We show here that these diverse peptide sequences bind with comparable affinities to the Numb PTB domain at a common binding site on the surface of the protein. The NMR structure of the Numb PTB domain in complex with a GPpY-containing peptide reveals a novel mechanism of binding with the peptide in a helical turn that does not hydrogen bond to the PTB domain beta-sheet. These results suggest that PTB domains can potentially have multiple modes of peptide recognition and provide a structural basis from which the multiple functions of the Numb PTB domain during asymmetric cell division could arise.