Structural requirements for interactions between leucine-sorting signals and clathrin-associated adaptor protein complex AP3

Cytoplasmic tails of LIMPII and the invariant chain contain similar leucine-based sorting signals, but the invariant chain interacts only with AP1 and AP2, whereas LIMPII interacts strongly with AP3. In a series of in vitro experiments, we investigated the effect of residues upstream of the leucine pairs and demonstrated that these residues determine adapter binding, and certain residues favor interactions with AP3. Furthermore, constructs that interacted stronger with AP3 interacted weakly with AP1 and vice versa. Exchanging residues upstream of the leucine-based signal in LIMPII with those of the invariant chain reduced LIMPII binding to AP3 in vitro, and in vivo the corresponding LIMPII mutant was rerouted via the plasma membrane like the invariant chain. These preferential interactions of different leucine signals with different AP complexes may thus be the determining step sorting proteins from the trans-Golgi network to their final destinations. Proteins that interact with AP3 are sorted directly to endosomes/lysosomes, whereas proteins that interact with AP1 are sorted via a different route. At the same time, constructs that exhibited specificity for either AP1 or AP3 might still interact with AP2, suggesting that AP2 may recognize a wider variety of leucine signals. This is consistent with the suggested role of AP2 in internalization of proteins containing general leucine-based signals, including proteins that have been missorted to the plasma membrane.     

Antibody: the flexible adaptor molecule

Antibodies are flexible adaptor molecules linking target and potential killer i.e. antigen and effector. With the discovery of effector binding sites on antibodies we can begin to visualize at the molecular level how this adaptor role is fulfilled.     

Structural requirements for mitochondrial mutagenesis

Ethidium bromide (3, 8-diamino-5-ethyl-6-phenylphenanthridinium bromide) and euflavine (3, 6-diamino-10-methylacridinium chloride) are superficially similar in structure and ability to intercalate into DNA. However, they exhibit qualitative differences in their ability to bring about a mitochondrial mutation (ρ⁺ → ρ^?) in Saccharomyces cerevisiae. This investigation tried to establish and compare the essential structural prerequisites in three series of planar, heterocyclic dyes: the phenanthridines (P series), the acridines (A series), and molecules with different heteroatoms related to acridines (X series). Compounds capable of bringing about the mutation in the complete absence of growth and energy sources are restricted to di-primary amines in the P series: quaternization of the ring nitrogen, and an aromatic side chain at C-6 also appear essential. Compounds in the A series are mutagenic only with growing cells; quaternization (C₁ through C₄) is essential. The 10-allyl derivative is unusual; it is highly effective even in buffer supplemented only with an energy source. The results are interpreted in terms of a model that requires interaction of the mutagen with the mitochondrial inner membrane as well as with its DNA.     

Evidence for evolving Toll-IL-1 receptor-containing adaptor molecule function in vertebrates

In mammals, Toll-IL-1R-containing adaptor molecule 1 (TICAM1)-dependent TLR pathways induce NF-kappaB and IFN-beta responses. TICAM1 activates NF-kappaB through two different pathways involving its interactions with TNFR-associated factor 6 and receptor-interacting protein 1. It also activates IFN regulatory factor 3/7 through its interaction with TANK-binding kinase-1, leading to the robust up-regulation of IFN-beta. In this study, we describe the role of zebrafish (Danio rerio) TICAM1 in activating NF-kappaB and zebrafish type I IFN. Zebrafish IFN is unique in that it cannot be categorized as being alpha- or beta-like. Through comprehensive sequence, phylogenetic, and syntenic analyses, we fully describe the identification of a zebrafish TICAM1 ortholog. Zebrafish TICAM1 exhibits sequence divergence from its mammalian orthologs and our data demonstrate that these sequence differences have functional consequences. Zebrafish TICAM1 activates zebrafish IFN; however, it does so in an apparently IFN regulatory factor 3/7-independent manner. Furthermore, zebrafish TICAM1 does not interact with zebrafish TNFR-associated factor 6, thus NF-kappaB activation is dependent upon its interaction with receptor-interacting protein 1. Comparative genome analysis suggests that TICAM1 and TICAM2 evolved from a common vertebrate TICAM ancestor following a gene duplication event and that TICAM2 was lost in teleosts following the divergence of the rayfin and lobefin fishes 450 million years ago. These studies provide evidence, for the first time, of the evolving function of a vertebrate TLR pathway.     

NKG2D splice variants: a reexamination of adaptor molecule associations

NKG2D is a homodimeric C-type lectin-related receptor expressed on natural killer (NK) cells and T cells. In mice, alternative deoxyribonucleic acid (DNA) splicing generates two isoforms of NKG2D that differ in the length of their cytoplasmic domains. Their ability to induce cellular activation is mediated via association with two membrane-bound, signaling adaptor molecules, DAP10 and DAP12. It has been reported that the long form of NKG2D associates exclusively with DAP10, whereas the short variant can interact with either adaptor. The short isoform was reported to be almost undetectable in naïve NK cells. Using two distinct cell types, we demonstrate that like the short isoform, the long variant of NKG2D also associates not only with DAP10 but also with DAP12. Using reporter cells (70Z/3), we demonstrate that DAP12 can compete equally with DAP10 for association with both variants of NKG2D when DAP10 and DAP12 are coexpressed. Cross-linking either isoform of NKG2D induces a calcium flux when associated exclusively with DAP10 or DAP12. Moreover, using quantitative polymerase chain reaction (PCR), we also show that the short isoform of NKG2D is expressed in naïve NK cells. Our data suggest that signaling via mouse NKG2D isoforms is more complex than originally presented.     

Structural requirements for heparan sulphate self-association

To investigate heparan sulphate self-association, various sub-fractions of beef-lung heparan sulphate have been subjected to affinity chromatography on heparan sulphate-agarose. A particular variant of heparan sulphate was chiefly bound to matrices substituted with the same or cognate heparan sulphates. N-desulphation and N-acetylation abolished the chain-chain interaction. Also, dermatan sulphates and chondroitin sulphates showed affinity for heparan sulphate-agarose. [3H]Heparan sulphates that were bound to a heparan sulphate-agarose were desorbed by elution with the corresponding heparan sulphate chains and also with unrelated heparan sulphates, heparin, and the galactosaminoglycans to various degrees. However, the corresponding heparan sulphate species was the most efficient at low concentrations. Dextran sulphate was unable to desorb bound heparan sulphate. When the corresponding heparan sulphate was N-desulphated/N-acetylated, carboxyl-reduced, or periodate-oxidised (D-glucuronate), the modified polymer was unable to displace [3H]heparan sulphate from heparan sulphate-agarose. The displacing ability of heparin was also destroyed by periodate oxidation. It is concluded that self-interaction between heparan sulphate chains is strongly dependent on the overall molecular conformation. The N-sulphate and carboxylate groups as well as the integrity of the D-glucuronate residue are all essential for maintaining the proper secondary structure.     

Structural requirements for formyl homooligopeptide chemoattractants

Using solution peptide synthesis, we have made three series of N alpha-formylated homooligopeptides, from the dipeptide to the heptapeptide, derived from L-methionine, L-norleucine, and S-methyl-L-cysteine and related to the chemotactic peptide N alpha-formylmethionylleucylphenylalanine. Compounds were prepared to determine the combined effects of the main-chain length and the presence of a sulfur atom in side-chain gamma- and delta-positions. Each peptide was tested for its ability to induce rabbit peritoneal polymorphonuclear leukocytes in the presence of cytochalasin B to secrete granule enzymes. In parallel, a conformational analysis was carried out in the solid state and in solution, using infrared absorption and circular dichroism. We examined these peptides in solvents of widely different polarities, i.e., chloroform, 2,2,2-trifluoroethanol, 1,1,1,3,3,3-hexafluoropropan-2-ol, and mixed organic-aqueous media. The tendencies to form antiparallel-chain beta-associated and folded structures were determined. The biological and conformational data are described in terms of a model of the chemotactic peptide receptor of rabbit neutrophils recently proposed by Freer et al. (1982) [Freer, R.J., Day, A.R., Muthukumarswamy, N., Pinon, D., Wu, A., Showell, H.J., & Becker, E.L. (1982) Biochemistry 21, 257-263]. In the three N alpha-formylated C-methoxy homooligopeptide series tested, the highest level of activity attained is at the tetrapeptide or pentapeptide stage, confirming the suggestion that the formylpeptide receptor is large enough to accommodate a peptide with at least four amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural requirements for CD43 function

The regulation of T cell activation and adhesion by CD43 (leukosialin, sialophorin) has been thought to be mainly a function of the large size and negative charge of the extracellular domain of the protein. In this work, we demonstrate that the cytoplasmic tail is both necessary and sufficient for the negative regulatory effect of CD43 on cell-cell adhesion. Expression of mutant CD43 proteins in primary T cells from CD43-deficient mice demonstrated that the antiproliferative effect of CD43 is also dependent upon the cytoplasmic tail. In contrast, Ab-mediated costimulation through CD43 does not require the intracellular domain of CD43. These data demonstrate that CD43 primarily serves as a negative regulator of T cell activation and adhesion, and that this is mediated not exclusively by passive effects of the extracellular domain, but requires participation of the cytoplasmic tail, perhaps through interactions with the cytoskeleton, or alternatively, active regulation of intracellular signaling pathways.     

Structural requirements for the function of a yeast chromosomal replicator

We have investigated the role of small nuclear ribonucleoprotein particles (snRNPs) in the in vitro splicing of messenger RNA precursors by a variety of procedures. Removal of the U-type snRNPs from the nuclear extracts of HeLa cells with protein A-Sepharose-coupled human autoimmune antibodies leads to complete loss of splicing activity. The inhibition of splicing can be prevented by saturating the coupled antibodies with purified nucleoplasmic U snRNPs prior to incubation with nuclear extract. We further demonstrate that an intact 5' terminus of U1 snRNA is required for the functioning of U1 snRNP in the splicing reaction. Antibodies directed against the trimethylated cap structure of the U snRNAs inhibit splicing. Upon removal of the first eight nucleotides of the U1 snRNA in the particles by site-directed hydrolysis with ribonuclease H in the presence of a synthetic complementary oligodeoxynucleotide splicing is completely abolished. These results are in strong support of current models suggesting that a base-pairing interaction between the 5' terminus of the U1 snRNA and the 5' splice site of a mRNA precursor is a prerequisite for proper splicing.     

Structural requirements for the binding of prostaglandins

The binding of prostaglandins and analogs to the lipocyte PGE receptor was shown to exhibit a high degree of structural specificity. Small changes, particularly at the 9-keto or 15-hydroxy position, were found to drastically diminish interaction with the receptor. Studies of a rather substantial number of compounds revealed a close relationship between affinity for the lipocyte PGE receptor and the ability to stimulate cyclic AMP synthesis in the isolated mouse ovary. In general, activities in these two parameters follow the biological potencies generally recognized for these compounds.     

Structural requirements for efficient prion protein conversion

To understand why cross species infection of prion disease often results in inefficient transmission and reduced protein conversion, most research has focussed on defining the effect of variations in PrP primary structures, including sequence compatibility of substrate and seed. By contrast, little research has been aimed at investigating structural differences between different variants of PrP^C and secondary structural requirements for efficient conversion. This is despite a clear role for molecular chaperones in formation of prions in non-mammalian systems, indicating the importance of secondary/tertiary structure during the conversion process. Recent data from our laboratory on the cellular location of disease-specific prion cofactors supports the critical role of specific secondary structural motifs and the stability of these motifs in determining the efficiency of disease-specific prion protein conversion. In this paper we summarise our recent results and build on the hypothesis previously suggested by Wuthrich and colleagues, that stability of certain regions of the prion protein is crucial for protein conversion to abnormal isoforms in vivo. It is suggested that one role for molecular co-factors in the conversion process is to stabilise PrP^C structure in a form that is amenable for conversion to PrP^Sc.     

Structural requirements for dermorphin opioid receptor binding

Structural features influencing binding activity of dermorphin to opioid receptors have been investigated in the rat brain through the synthesis and evaluation of binding affinity of a series of synthetic dermorphin analogs. Tritiated dermorphin was used as primary ligand. The single population of high affinity dermorphin binding sites present in the rat brain is clearly of an opioid nature since bound radiolabeled dermorphin was fully displaced with high affinity either by morphine or naloxone. Displacement of tritiated dermorphin by all alkaloid opiates or dermorphin related peptides tested was monophasic, consistent with simple competitive inhibition at a single population of binding sites. Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) was the most potent competitor in all experiments. The D-configuration of the amino acid residue in position 2 was found to be of crucial importance for binding. Replacement of D-Ala2 with L-Ala led to a deleterious effect, this analog being 1/5000th as potent as dermorphin in displacing bound tritiated dermorphin from its receptor. Shorter dermorphin homologs, dermorphin-(1-4)-NH2 and dermorphin-(1-3)-NH2, were found to be 20 and 40-fold less potent, respectively, than dermorphin. The C-terminal carboxamide function is of significant importance for manifestation of the full intrinsic binding potency of dermorphin. Deamidated dermorphin had 1/5th the potency of the parent peptide. This suggests that while the whole dermorphin sequence is required for the expression of the full intrinsic binding activity of the molecule, the N-terminal tripeptide is a key structure as it contains the features which allow receptor recognition.     

Identification of a novel death domain-containing adaptor molecule for ectodysplasin-A receptor that is mutated in crinkled mice

Hypohydrotic Ectodermal Dysplasia (HED) is a genetic disease seen in humans and mice. It is characterized by loss of hair, sweat glands, and teeth. The predominant X-linked form results from mutations in ectodysplasin-A (EDA), a TNF-like ligand. A phenotypically indistinguishable autosomal form of the disease results from mutations in the receptor for EDA (EDAR). EDAR is a NF-kappaB-activating, death domain-containing member of the TNF receptor family. crinkled, a distinct autosomal form of HED, was discovered in a mouse strain in which both the ligand (EDA) and receptor (EDAR) were wild-type, suggestive of a disruption further downstream in the signaling pathway. Employing a forward genetic approach, we have cloned crinkled (CR) and find it to encode a novel death domain-containing adaptor. crinkled binds EDAR through a homotypic death domain interaction and mediates engagement of the NF-kappaB pathway, possibly by recruiting TRAF2 to the receptor-signaling complex. This is an unprecedented example of naturally occurring mutations in ligand, receptor, or adaptor giving rise to the same phenotypic disease characterized by a defect in the proper development of epidermal appendages.     

Structural requirements for conserved arginine of parathyroid hormone

Arg-20 is one of two residues conserved in all peptides known to activate the parathyroid hormone (PTH) receptor. Previous studies have failed to find any naturally encoded analogues of residue 20 that had any adenylyl cyclase (AC) stimulating activity. In this work we have studied substitutions of Arg-20 with nonencoded amino acids and conformationally constrained analogues with side chains mimicking that of Arg. No analogue had more than 20% of the AC-stimulating ability of the natural Arg-20-bearing peptide. In descending order of activity, the most active analogues had (S)-4-piperidyl-(N-amidino)glycine (PipGly), norleucine (Nle), citrulline (Cit), or ornithine (Orn) at residue 20. Analogues with Arg-20 substituted with L-4-piperidyl-(N-amidino)alanine, Lys, Glu, Ala, Gln, (S)-2-amino-4-[(2-amino)pyrimidinyl]butanoic acid, or L-(4-guanidino)phenylalanine had very low or negligible activity. Low or negligible activities of Lys or Orn analogues suggested ionic interactions play a minor role in the Arg interaction with the receptor. The conformational constraints imposed by the PipGly ring had a negative effect on its ability to substitute for Arg. The side-chain H-bonding potential of the Cit ureimido group was likely an important factor in its mimicry of Arg. The increase in amphiphilicity, as demonstrated by its greater high-performance liquid chromatographic retention, and increased alpha-helix, as shown by circular dichroic spectroscopy, likely contributed to the activity of the Nle-20 analogue. The data demonstrated that specific H-bonding, hydrophobicity of the side chain, stabilization of alpha-helix, and possibly specific cation positioning were all important in the interaction of Arg-20 with receptor groups.     

Structural requirements for nucleophilic substrates of carboxypeptidase Y

The composition and structural aspects of the amino and carboxylic acid groups required for incorporation into peptides by transpeptidation and inhibition of hydrolysis in carboxypeptidase Y-catalyzed reactions were studied. Separation of these two groups by even one carbon prevents incorporation by transpeptidation and does not inhibit incorporation of other amino acids into model peptides. Substitution of phosphonic or sulfonic acids for the carboxylic acid group also results in loss of incorporation by transpeptidation. Only the sulfonic acid analog of glycine causes inhibition of hydrolysis and this inhibition is lost when serine is included in the reaction. d-Serine is not incorporated by carboxypeptidase Y, and its presence in the reaction mixture does not inhibit the incorporation of the L-isomer.     

Structural requirements for the antitubercular quaternized triflupromazine pharmacophore

Quaternized triflupromazine derivatives (QTDs) must possess benzyl groups attached to the quaternary nitrogen in order to have significant antitubercular potency. Replacing the quaternary amine with a triazole abolishes antitubercular activity. A modest halogen substitution effect exists, with the 4-bromophenyl QTD 3 having the best selectivity index (>21). All N-benzyl QTDs 1-4 similarly inhibit non-replicating, persistent Mycobacterium tuberculosis with MIC<8μM, and compounds 1-3 were all nontoxic to mammalian cells in vitro (IC(50)>128μM).     

Structural requirements for expression of factor Va activity

Thrombin activated factor Va (factor VIIa, residues 1-709 and 1546-2196) has an apparent dissociation constant (Kd,app) for factor Xa within prothrombinase of approximately 0.5 nM. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp697, Asp1509, and Asp1514 to produce a molecule (factor VNN) that is composed of a Mr 100,000 heavy chain (amino acid residues 1-696) and a Mr 80,000 light chain (amino acid residues 1509/1514-2196). Factor VNN, has a Kd,app for factor Xa of 4 nm and reduced clotting activity. Cleavage of factor VIIa by NN at Asp697 results in a cofactor that loses approximately 60-80% of its clotting activity. An enzyme from Russell's viper venom (RVV) cleaves human factor V at Arg1018 and Arg1545 to produce a Mr 150,000 heavy chain and Mr 74,000 light chain (factor VRVV, residues 1-1018 and 1546-2196). The RVV species has affinity for factor Xa and clotting activity similar to the thrombin-activated factor Va. Cleavage of factor VNN at Arg1545 by alpha-thrombin (factor VNN/IIa) or RVV (factor VNN/RVV) leads to enhanced affinity of the cofactor for factor Xa (Kd,app approximately 0.5 nM). A synthetic peptide containing the last 13 residues from the heavy chain of factor Va (amino acid sequence 697-709, D13R) was found to be a competitive inhibitor of prothrombinase with respect to prothrombin. The peptide was also found to specifically interact with thrombin-agarose. These data demonstrate that 1) cleavage at Arg1545 and formation of the light chain of factor VIIa is essential for high affinity binding and function of factor Xa within prothrombinase and 2) a binding site for prothrombin is contributed by amino acid residues 697-709 of the heavy chain of the cofactor.     

Structural requirements for key residues and auxiliary portions of a BLUF domain

BlrB in Rhodobacter sphaeroides is a single domain, flavin-based blue light sensor protein in the BLUF family of photoreceptors. Consistent with other members of this family, blue light excitation induces a putative signaling state characterized by a 10 nm red shift in the UV-visible absorbance spectrum. Structural and spectroscopic characterization of truncated BlrB constructs establishes that the C-terminal 50 amino acids of this protein are essential to its structural integrity despite not being part of the canonical BLUF domain architecture. Mutagenesis studies support the critical roles of Tyr9, Asn33, and Gln51 for flavin binding and the integrity of the BLUF domain fold. Comparison of solution NMR spectra of BlrB acquired under dark and light conditions indicates very limited light-dependent conformational changes except for a few interesting residues: Trp92, Met94, and Ile127. Notably, the Ile127 side chain experiences significant chemical shift changes despite the fact that it is far ( approximately 15 A) from the flavin chromophore in the C-terminal extension. These data suggest that the light-induced signal is propagated from the flavin through the beta sheet to the last two alpha helices in the C-terminal extension, potentially providing a mechanism to transmit this change to initiate a cellular response to blue light.