Cluster Of Basic Amino Acids Research Articles

PAP-1, a novel target protein of phosphorylation by pim-1 kinase.

Protooncogene, pim-1, has been reported to be a predisposition for lymphomagenesis along with myc, and its protein product, Pim-1, has been shown to be a serine/threonine protein kinase, whose activity is involved in proliferation and differentiation of blood cells. The signal transduction pathways neither to nor from Pim-1, however, have been clarified. We have cloned a cDNA encoding a novel Pim-1 binding protein, PAP-1, comprising 213 amino acids with a basic amino-acid cluster near the C-terminus. PAP-1 was colocalized with Pim-1 in human HeLa cell nuclei. The in vitro binding assays using GST fusion proteins of the wild-type and various deletion mutants revealed that the whole molecule of Pim-1 is required for the binding activity to PAP-1 and that Pim-1 binds to the region from amino-acid numbers 1-147 of PAP-1, or to two segments in the region. The association of PAP-1 with Pim-1 was also shown in vivo in transfected cells. Furthermore, PAP-1 was phosphorylated in vitro by Pim-1, but not a kinase-negative Pim-1 mutant. The two serine residues of PAP-1 at amino acids 204 and 206 near the C-terminus were phosphorylated by Pim-1. PAP-1 is thus thought to be a target protein for Pim-1 kinase.

Chicken Y-box proteins chk-YB-1b and chk-YB-2 repress translation by sequence-specific interaction with single-stranded RNA

Y-Box proteins comprise a large family of multifunctional proteins with a wide spectrum of activities in both transcription and translational regulation of gene expression. Earlier, we have reported on the involvement of chk-YB-2 in transcriptional regulation of Rous sarcoma virus long terminal repeats and the involvement of chk-YB-1b in transcriptional regulation of alpha1(I) collagen genes. Here, we have investigated the potential role of chk-YB-2 and chk-YB-1b in RNA metabolism. We report that chk-YB-2 and chk-YB-1b are localized predominantly in the cytoplasm and that they both can bind single-stranded RNA in a sequence-specific and reversible manner. Well-conserved cold-shock domain, N-terminal proline-rich domain and the alternating clusters of acidic and basic amino acids located in the C-terminal ends of these two proteins were all found to be necessary for their RNA-binding ability. Further, we demonstrate that these two proteins inhibit translation in vitro and that binding to RNA is required for this inhibition. The significance of these results is discussed.

Histone deacetylase-independent transcriptional repression by methyl-CpG-binding protein 2.

Methyl-CpG-binding protein 2 (MeCP2) contains a transcriptional repression domain (TRD), which can act by recruitment of a large transcriptional co-repressor complex containing histone deacetylases HDAC1 and 2. We demonstrate here that transient transcription from the SV40 enhancer/promoter or the SV40 promoter is strongly repressed in a histone deacetylase-independent manner, since repression is not alleviated by Trichostatin A (TSA). In a mutational analysis, repression depends on a conserved 30 residue sequence containing two clusters of basic amino acids. Mutation of the first of these clusters inhibits in vitro interaction between TRD and mSin3A. Furthermore, a subdomain of the TRD containing the conserved 30-residue sequence and 16 flanking amino acids was sufficient to compromise VP16-activated transcription. In summary, our results indicate an alternative, histone deacetylase-independent pathway of transcriptional repression by MeCP2.

Characterization of the nuclear localization signal in the DNA helicase responsible for Bloom syndrome.

Bloom syndrome (BS) is a rare genetic disorder characterized by small body size, photosensitivity, immunodeficiency and a high predisposition to various types of cancer. BLM was identified as the causative gene for BS. The BLM protein is homologous to DNA helicase and has two basic amino acid clusters in its C-terminal region. Previously, we reported that the distal arm of these basic amino acids clusters in the BLM protein functioned as the nuclear localization signal (NLS) of the protein. In this study, we generated plasmid constructs for expression of enhanced green fluorescent protein (EGFP) fused with various BLM protein variants having a mutation with deletions or substitutions in the basic amino acid and analyzed the subcellular localization of the expressed proteins. The EGFP-fused protein containing the basic amino acid cluster region proximal to the C-terminus of BLM helicase was localized exclusively in the nucleus. However, the EGFP-BLM proteins that lacked either Arg1344 or Lys1346 distributed in both the cytoplasm and the nucleus equally. Deletion of Arg1347 also resulted in localization in both the nucleus and cytoplasm, and substitution of Arg1344, Lys1346, Arg1347 or Arg1348 with non-basic amino acids reduced the nuclear localization of BLM protein. Mouse BLM protein which also migrate to the nucleus has two basic amino acid clusters in the C-terminus and the basic amino acids (Lys1346-Pro1347-Lys1348-Arg1349-Arg1350) proximal to the C-terminus are conserved between mouse and human. These findings suggest that the Arg1344-Ser1345-Lys1346-Arg1347 sequence at the C-terminus of the human BLM protein is essential for nuclear localization of this protein.

Glycosaminoglycan-Binding Properties and Secondary Structure of the C-Terminus of Netrin-1

Netrins are soluble neurite-outgrowth-promoting proteins related to the laminin B2 chain. Since these proteins and their receptor DCC (the “deleted in colorectal carcinoma” gene product) bind heparin, glycosaminoglycans may modulate their biological actions in a similar fashion as described for several other ligand-receptor systems. Here we show that a polypeptide encompassing the C-terminal cluster of basic amino acids of netrin-1 (i) adopts an α-helical conformation in water-trifluoroethanol mixtures according to circular dichroism experiments and (ii) binds electrostatically to heparin with high affinity under physiological ionic conditions (KD = 15 nM for the binding to immobilized heparin according to surface plasmon resonance, KD = 50 nM in solution as determined with isothermal titration calorimetry). These data indicate that the cluster of basic amino acids at the C-terminus of netrin-1 forms an α-helical structural element which can contribute to the glycosaminoglycan-binding activity of this neurotrophic guidance molecule.

Architecture and function of the human endonucleases RNase P and RNase MRP.

In the past decade, important advances have been made in our knowledge of the composition of human RNase MRP and RNase P complexes. Both ribonucleoprotein particles function as endonucleases and contain RNA components that are structurally related. RNase MRP has been suggested to be involved in the processing of precursor rRNA; RNase P, in the maturation of tRNA. Here we give an overview of current data on the structure and function of human RNase MRP and RNase P particles, with emphasis on their molecular composition. At present, seven protein subunits, probably all associated with both ribonucleoprotein particles, have been isolated and their corresponding cDNAs cloned. Although no known structural motifs can be identified in the amino acid sequences of these proteins, the majority is clearly rich in basic residues. For two protein subunits, a cluster of basic amino acids have been shown to be involved in nucleolar accumulation, whereas another protein, which lacks such a region, probably enters the nucleolus by way of a piggyback mechanism. The binding regions for several of the protein subunits on the RNA have been identified, and the data have been used to create a putative structural model for the RNase MRP particle. The rather obscure situation concerning the association of the autoantigenic Th-40 protein and its possible relationship with one of the subunits, Rpp38, is discussed.

Mutational analysis of the signal for a nuclear localization of proteins which accumulate specifically during meiosis in lily microsporocytes.

LIM5 and LIM13 are novel meiosis-associated genes isolated from Lilium longiflorum. The presence of a hydrophobic N-terminal region predicted from the amino acid sequence has suggested that they function as extracellular structural components. However, both proteins also contain clusters of basic amino acids which may function as nuclear localization signals. To investigate the cellular localization of the protein, we tagged the C-termini of LIM5 and LIM13 with a green fluorescent protein. Transient expression of fusion proteins in onion epidermal cells revealed nuclear localization activity of both proteins. Mutational analysis indicated that amino acid sequences that constitute bipartite-type nuclear localization signals are necessary and sufficient for the intracellular localization of both proteins.

Nuclear targeting determinants of the phage P1 cre DNA recombinase.

The Cre DNA recombinase of bacteriophage P1 has become a useful tool for genomic manipulation in mice and other eukaryotes. Because Cre is of prokaryotic origin, the 38 kDa protein has been presumed to gain access to the eukaryotic nucleus simply because it is sufficiently small to pass through the nuclear pore by passive diffusion. Instead, we show here that Cre carries nuclear targeting determinants that efficiently direct Cre entry into the nucleus of mammalian cells. Fusions of Cre with green fluorescent protein (GFP) identified two regions that are necessary for nuclear localization. Region I contains a cluster of basic amino acids that is essential for nuclear localization and which resembles a bipartite-like nuclear localization signal. Region II exhibits a beta-sheet structure with which the bipartite motif may interact. However, neither region is by itself sufficient for nuclear localization. Nuclear transport in vitro with a 98 kDa GFP-Cre fusion protein shows that Cre does not gain access to the nucleus by passive diffusion, but instead enters the nucleus by means of an energy-dependent process. Thus, Cre is one of the few prokaryotic proteins that have been shown to carry determinants that allow it to target the eukaryotic nucleus.

The Nuclear Localization Signal of the Human Ku70 Is a Variant Bipartite Type Recognized by the Two Components of Nuclear Pore-Targeting Complex

Ku protein is a complex of two subunits, Ku70 and Ku80. Ku is suspected to participate in both DNA double-strand break repair and transcription. Since both of these processes take place in the cell nucleus, we have been investigating the subcellular localization and nuclear transport of Ku proteins. In the present study, we analyzed the subcellular localization and nuclear localization signal (NLS) of Ku70. Fusion proteins of Ku70 and green fluorescent protein (GFP) transiently expressed in cells were clearly localized in the nuclei of interphase cells. Ku70 staining was distributed throughout both the nucleus and the cytoplasm in late telophase to early G1 phase cells. The NLS of Ku70 was located at the region composed of 18 amino acid residues (positions 539 to 556). This region overlapped with the Ku80-independent DNA-binding domain reported previously. The Ku70 NLS consisted of two basic subregions and a nonbasic intervening region. All the subregions were necessary for complete NLS activity. The amino acids in the nonbasic intervening region of Ku70 might be important for full NLS activity not only to provide sufficient length between the two separated clusters of basic amino acids but also to have an adequate amino acid sequence. All of the basic amino acid residues in the basic subregions were conserved among mammalian and avian homologues, confirming their importance in the nuclear translocation of Ku70. The structure of the Ku70 NLS resembled the consensus of a bipartite-type NLS. The Ku70 NLS was mediated to target to the nuclear rim by two components of the nuclear pore-targeting complex, PTAC58 and PTAC97.

Sea Urchin TgBMP2/4 Gene Encoding a Bone Morphogenetic Protein Closely Related to Vertebrate BMP2 and BMP4 with Maximal Expression at the Later Stages of Embryonic Development

We have cloned a gene fragment (namedTgBMP2/4) that encodes a protein homologous to vertebrate bone morphogenetic protein (BMP) 2 and BMP4 in the sea urchinTripneustes gratilla.This peptide sequence contains 204 amino acids with 7 conserved cysteine residues at the C-terminus of the coding region and a cluster of basic amino acids that may serve as a signal for proteolytic cleavage. Sequence comparison and phylogenetic analyses reveal that TgBMP2/4 is closely related to vertebrate BMP2 and BMP4 as well as to amphioxus BMP2/4, with similarity levels ranging from 90% to 94% at the mature C-terminal domain. Northern blot analyses show that a 6.3-kbTgBMP2/4mRNA appears first at the mesenchyme blastula stage and increases to a maximal level at the gastrula and pluteus stages. This expression pattern is different from that of a BMP2/4-related gene previously found in sea urchin.

The role of Pr55(gag) in the annealing of tRNA3Lys to human immunodeficiency virus type 1 genomic RNA.

During human immunodeficiency virus type 1 (HIV-1) assembly, the primer tRNA for the reverse transcriptase-catalyzed synthesis of minus-strand strong-stop cDNA, tRNA3Lys, is selectively packaged into the virus and annealed onto the primer binding site on the RNA genome. Annealing of tRNA3Lys in HIV-1 is independent of polyprotein processing and is facilitated in vitro by p7 nucleocapsid (NCp7). We have previously shown that mutations in clusters of basic amino acids flanking the first Cys-His box in NC sequence inhibit annealing of tRNA3Lys in vivo by 70 to 80%. In this report, we have investigated whether these NC mutations act through Pr55(gag) or Pr160(gag-pol). In vivo placement of tRNA3Lys is measured with total viral RNA as the source of primer tRNA-template in an in vitro reverse transcription assay. Cotransfection of COS cells with a plasmid coding for either mutant Pr55(gag) or mutant Pr160(gag-pol), and with a plasmid containing HIV-1 proviral DNA, shows that only the NC mutations in Pr55(gag) inhibit tRNA3Lys placement. The NC mutations in Pr55(gag) reduce viral infectivity by 95% and are trans-dominant-negative, i.e., they inhibit genomic placement of tRNA3Lys even in the presence of wild-type Pr55(gag). This dominant phenotype may indicate that the mutant Pr55(gag) is disrupting an ordered Pr55(gag) structure responsible for the annealing of tRNA3Lys to genomic RNA.

BINDING OF INTERFERON γ BY GLYCOSAMINOGLYCANS: A STRATEGY FOR LOCALIZATION AND/OR INHIBITION OF ITS ACTIVITY

Glycosaminoglycans (GAGs) are a group of negatively charged molecules present in many tissues as components of the extracellular matrix, basement and cellular membranes. This work analysed the ability of this group of substances to interact with human interferon γ and the effect of those interactions on its biologic activity. A variety of GAGs (heparin, heparan sulfate, chondroitin sulfate and hyaluronic acid), and a related sulfated polysaccharide (dextran sulfate), were found to interact with IFN-γ as determined by inhibition of the binding of [125I]IFN-γ to COLO-205 cells and binding to wells coated with GAGs. These interactions were inhibited by synthetic peptides mimicking the sequences of the basic amino acid cluster located at the C-terminal end of mouse and human IFN-γ, or by poly-L-lysine, suggesting that ionic interactions between the positively-charged C-terminus and negatively charged groups in GAGs were involved. IFN-γ molecules bound to plate-immobilized or endothelial cell surface GAGs retained biological activity, since they could induce major histocompatibility complex (MHC) class II expression on COLO-205 cells, suggesting that cell surface GAGs might be able to present IFN-γ to its receptors. These results suggest important regulatory roles for GAGs on the activity of IFN-γ in vivo.

A Receptor-like Protein-tyrosine Phosphatase PTPζ/RPTPβ Binds a Heparin-binding Growth Factor Midkine: INVOLVEMENT OF ARGININE 78 OF MIDKINE IN THE HIGH AFFINITY BINDING TO PTPζ

Midkine is a 13-kDa heparin-binding growth factor with 45% sequence identity to pleiotrophin. Pleiotrophin has been demonstrated to bind to protein-tyrosine phosphatase zeta (PTPzeta) with high affinity. In this study, we examined the binding of midkine to PTPzeta by solid-phase binding assay. Midkine and pleiotrophin binding to PTPzeta were equally inhibited by soluble pleiotrophin and also by some specific glycosaminoglycans. For both bindings, Scatchard analysis revealed low (3.0 nM) and high (0.58 nM) affinity binding sites. These results suggested that PTPzeta is a common receptor for midkine and pleiotrophin. Midkine is structurally divided into the N- and C-terminal halves, and the latter exhibited full activity for PTPzeta binding and neuronal migration induction. The C-terminal half contains two heparin-binding sites consisting of clusters of basic amino acids, Clusters I and II. A mutation at Arg78 in Cluster I resulted in loss of the high affinity binding and reduced neuronal migration-inducing activity, while mutations at Lys83 and Lys84 in Cluster II showed almost no effect on either activity. Chondroitinase ABC-treated PTPzeta exhibited similar low affinity binding both to the native midkine and midkine mutants at Arg78. These results suggested that Arg78 in midkine plays an essential role in high affinity binding to PTPzeta by interacting with the chondroitin sulfate portion of this receptor.

Mutational analysis of apolipoprotein B mRNA editing enzyme (APOBEC1): structure–function relationships of RNA editing and dimerization

APOBEC1 is the catalytic subunit of an enzyme complex that mediates apolipoprotein (apo) B mRNA editing. It dimerizes in vitro and requires complementation factor(s) for its editing activity. We have performed a systematic analysis of the structure–functional relationship of APOBEC1 by targeted mutagenesis of various sequence motifs within the protein. Using in vitro RNA editing assay, we found that basic amino acid clusters at the amino-terminal region R15R16R17 and R33K34, are essential for apoB mRNA editing. Mutation of R15R16R17 to K15K16K17 and mutation of R33K34 simultaneously to A33A34 almost completely abolished in vitro editing activity. The carboxy-terminal region of APOBEC1 contains a leucine-rich motif. Deletion analysis of this region indicates that residues 181 to 210 are important for in vitro apoB mRNA editing. Single amino acid substitutions demonstrate that L182, I185, and L189 are important residues required for normal editing function. Furthermore, the double mutant P190A/P191A also lost >90% of editing activity which suggests that a β turn in this region of the molecule may be essential for proper functioning of APOBEC1. It was suggested that dimerization of APOBEC1 creates an active structure for deamination of apoB mRNA. When we examined the dimerization potential of truncated APOBEC1s using both amino and carboxy termini deletion mutants, we found that amino-terminal deletions up to residue A117 did not impair dimerization activity whereas carboxy-terminal deletions showed diminished dimerization. The systematic and extensive mutagenesis experiments in this study provide information on the role of various sequence motifs identified in APOBEC1 in enzyme catalysis and dimerization.—Teng, B-B., S. Ochsner, Q. Zhang, K. V. Soman, P. P. Lau, and L. Chan. Mutational analysis of apolipoprotein B mRNA editing enzyme (APOBEC1): structure–function relationships of RNA editing and dimerization. J. Lipid Res. 1999. 40: 623–635.

Activation of Ryanodine Receptors by Imperatoxin A and a Peptide Segment of the II-III Loop of the Dihydropyridine Receptor

Excitation-contraction coupling in skeletal muscle is believed to be triggered by direct protein-protein interactions between the sarcolemmal dihydropyridine-sensitive Ca2+ channel and the Ca2+ release channel/ryanodine receptor (RyR) of sarcoplasmic reticulum. A 138-amino acid cytoplasmic loop between repeats II and III of the alpha1 subunit of the skeletal dihydropyridine receptor (the II-III loop) interacts with a region of the RyR to elicit Ca2+ release. In addition, small segments (10-20 amino acid residues) of the II-III loop retain the capacity to activate Ca2+ release. Imperatoxin A, a 33-amino acid peptide from the scorpion Pandinus imperator, binds directly to the RyR and displays structural and functional homology with an activating segment of the II-III loop (Glu666-Leu690). Mutations in a structural motif composed of a cluster of basic amino acids followed by Ser or Thr dramatically reduce or completely abolish the capacity of the peptides to activate RyRs. Thus, the Imperatoxin A-RyR interaction mimics critical molecular characteristics of the II-III loop-RyR interaction and may be a useful tool to elucidate the molecular mechanism that couples membrane depolarization to sarcoplasmic reticulum Ca2+ release in vivo.

Nucleocytoplasmic Protein Transport and Recycling of Ran.

The active transport of proteins into and out of the nucleus is mediated by specific signals, the nuclear localization signal (NLS) and nuclear export signal (NES), respectively. The best characterized NLS is that of the SV40 large T antigen, which contains a cluster of basic amino acids. The NESs were first identified in the protein kinase inhibitor (PKI) and HIV Rev protein, which are rich in leucine residues. The SV40 T-NLS containing transport substrates are carried into the nucleus by an importin alpha/beta heterodimer. Importin alpha recognizes the NLS and acts as an adapter between the NLS and importin beta, whereas importin beta interacts with importin alpha bound to the NLS, and acts as a carrier of the NLS/importin alpha/beta trimer. It is generally thought that importin alpha and beta are part of a large protein family. The leucine rich NES-containing proteins are exported from the nucleus by one of the importin beta family molecules, CRM1/exportin 1. A Ras-like small GTPase Ran plays a crucial role in both import/export pathways and determines the directionality of nuclear transport. It has recently been demonstrated in living cells that Ran actually shuttles between the nucleus and the cytoplasm and that the recycling of Ran is essential for the nuclear transport. Furthermore, it has been shown that nuclear transport factor 2 (NTF2) mediates the nuclear import of RanGDP. This review largely focuses on the issue concerning the functional divergence of importin alpha family molecules and the role of Ran in nucleocytoplasmic protein transport.

Mutational analysis of Raf-1 cysteine rich domain: requirement for a cluster of basic aminoacids for interaction with phosphatidylserine.

Activation of Raf-1 kinase is preceded by a translocation of Raf-1 to the plasma membrane in response to external stimuli. The membrane localization of Raf-1 is facilitated through its interaction with activated Ras and with membrane phospholipids. Previous evidence suggests that the interaction of Raf-1 with Ras is mediated by two distinct domains within the N-terminal region of Raf-1 comprising amino acid residues 51-131 and residues 139-184, the latter of which codes for a zinc containing cysteine-rich domain. The cysteine-rich domain of Raf-1 is also reported to associate with other proteins, such as 14-3-3, and for selectively binding acidic phospholipids, particularly phosphatidylserine (PS). In the present study, we have investigated the consequences of progressive deletions and point mutations within the cysteine-rich domain of Raf-1 on its ability to bind PS. A reduced interaction with PS was observed in vitro for all deletion mutants of Raf-1 expressed either as full-length proteins or as fragments containing the isolated cysteine-rich domain. In particular, the cluster of basic amino acids R143, K144, and K148 appeared to be critical for interaction with PS, since substitution of all three residues to alanine resulted in a protein that failed to interact with liposomes enriched for PS. Expression of Raf-1 in vivo, containing point mutations in the cysteine-rich domain resulted in a truncated polypeptide that lacked both the Ras and PS binding sites and could no longer translocate to the plasma membrane upon serum stimulation. These results indicate that the basic residues 143, 144 and 148 in the anterior half of Raf-1 cysteine-rich domain play a role in the association with the lipid bilayer and possibly in protein stability, therefore they might contribute to Raf-1 localization and subsequent activation.

Regulation of RasGRP via a phorbol ester-responsive C1 domain.

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.

High-resolution solution NMR structure of the minimal active domain of the human immunodeficiency virus type-2 nucleocapsid protein.

The retroviral nucleocapsid (NC) protein is a multifunctional protein essential for RNA genome packaging and viral infectivity. The NC protein, NCp8, of the human immunodeficiency virus type-II (HIV-2) is a 49 amino acid peptide containing two zinc fingers, of the type C-X2-C-X4-H-X4-C, connected by seven amino acid residues, called the "basic amino acid cluster." It has been shown that the N-terminal zinc finger flanked by the basic amino acid cluster is the minimal active domain for the specific binding to viral RNA and other functions. However, the structure-activity relationships of NCp8 have not been investigated in detail. In the present study, the three-dimensional structure of a 29 amino acid peptide, including the minimal active domain (NCp8-fl), was determined by two-dimensional 1H NMR spectroscopy with simulated annealing calculations. A total of 15 converged structures of NCp8-fl were obtained on the basis of 355 experimental constraints, including 343 distance constraints obtained from nuclear Overhauser effect connectivities, 12 torsion angle (phi, chi1) constraints, and four constraints for zinc binding. The root-mean-square deviation of the 15 converged structures was 0.29 +/- 0.04 A for the backbone atoms (N, C(alpha), C) and 1.27 +/- 0.13 A for all heavy atoms. Interestingly, the basic amino acid cluster itself was defined well, with a loop-like conformation in which three arginine residues in the cluster and one arginine residue in the zinc finger are located approximately in the same plane of the molecule and are exposed to the solvent. The structure-activity relationships are discussed on the basis of the comparison of this well-defined structure with those of other NC proteins.

The HHQK Domain of β-Amyloid Provides a Structural Basis for the Immunopathology of Alzheimer's Disease

The beta-amyloid peptide 1-42 (Abeta1-42), a major component of neuritic and core plaques found in Alzheimer's disease, activates microglia to kill neurons. Selective modifications of amino acids near the N terminus of Abeta showed that residues 13-16, the HHQK domain, bind to microglial cells. This same cluster of basic amino acids is also known as a domain with high binding affinity for heparan sulfate. Both Abeta binding to microglia and Abeta induction of microglial killing of neurons were sensitive to heparitinase cleavage and to competition with heparan sulfate, suggesting membrane-associated heparan sulfate mediated plaque-microglia interactions through the HHQK domain. Importantly, small peptides containing HHQK inhibited Abeta1-42 cell binding as well as plaque induction of neurotoxicity in human microglia. In vivo experiments confirmed that the HHQK peptide reduces rat brain inflammation elicited after infusion of Abeta peptides or implantation of native plaque fragments. Strategies which exploit HHQK-like agents may offer a specific therapy to block plaque-induced microgliosis and, in this way, slow the neuronal loss and dementia of Alzheimer's disease.