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ANG-(1-7) improves the function of the remodeling heart. Although this peptide is generated directly within the myocardium, the effects of ANG-(1-7) on cardiac fibroblasts that play a critical role in cardiac remodeling are largely unknown. We tested the hypothesis that specific binding of ANG-(1-7) to cardiac fibroblasts regulates cellular functions that are involved in cardiac remodeling. [sup.125]I-labeled ANG-(1-7) binding assays identified specific binding sites of ANG-(1-7) on adult rat cardiac fibroblasts (ARCFs) with an affinity of 11.3 nM and a density of 131 fmol/mg protein. At nanomolar concentrations, ANG-(1-7) interacted with specific sites that were distinct from ANG II type 1 and type 2 receptors without increasing cytosolic [Ca.sup.2+] concentration. At these concentrations, ANG-(I-7) had inhibitory effects on collagen synthesis as assessed by [[sup.3]H]proline incorporation and decreased mRNA expression of growth factors in ARCFs. These effects of ANG-(1-7) contrasted with effects of ANG II. Pretreatment of ARCFs with ANG-(1-7) inhibited ANG II-induced increases in collagen synthesis and in mRNA expression of growth factors, including endothelin-1 and leukemia inhibitory factor. ANG-(1-7) pretreatment also inhibited the stimulatory effects of conditioned medium from ANG II-treated ARCFs on [[sup.3]H]leucine incorporation and atrial natriuretic factor mRNA expression, markers of hypertrophy, in cardiomyocytes. Thus ANG-(1-7) interacted with specific receptors on ARCFs to exert potential antifibrotic and antitrophic effects that could reverse ANG II effects. These results suggest that ANG-(1-7) may play an important role in the heart in regulating cardiac remodeling. angiotensin receptors; cardiac remodeling; renin-angiotensin system

The P1par family of active plasmid partition systems consists of at least six members, broadly distributed in a variety of plasmid types and bacterial genera. Each encodes two Par proteins and contains a cis-acting parS site. Individual par systems can show distinct species specificities; the proteins from one type cannot function with the parS site of another. P1par-versus-P7par specificity resides within two hexamer BoxB repeats encoded by parS that contact the ParB protein near the carboxy terminus. Here, we examine the species specificity differences between Yersinia pestis pMT1parS and Escherichia coli P1 and P7parS. pMT1parS site specificity could be altered to that of either P1 or P7 by point mutation changes in the BoxB repeats. Just one base change in a single BoxB repeat sometimes sufficed. The BoxB sequence appears to be able to adopt a number of forms that define exclusive interactions with different ParB species. The looped parS structure may facilitate this repertoire of interaction specificities. Different P1par family members have different partition-mediated incompatibility specificities. This property defines whether two related plasmids can coexist in the same cell and is important in promoting the evolution of new plasmid species. BoxB sequence changes that switch species specificity between P1, P7, and pMT1 species switched partition-mediated plasmid incompatibility in concert. Thus, there is a direct mechanistic link between species specificity and partition-mediated incompatibility, and the BoxB-ParB interaction can be regarded as a special mechanism for facilitating plasmid evolution.

The geometrical and electronic structures of the copper-superoxo, -hydroperoxo, and -oxo species and their potential reactivity as an oxidant in the protein environment of dopamine beta-monooxygenase (DBM) are addressed. The findings reveal the energy profiles for dopamine hydroxylation by the copper-superoxo and -oxo species using small models of these oxidant extracted from the quantum mechanical (QM) region of the QM/molecular mechanical (MM) model.

The equilibrium and thermodynamic binding characteristics of wild-type eIF4E (eukaryotic multisubunit initiation factor) and a high-affinity mutant, eIF4K(sub K119A), are compared with those of cap analogues and eIF4G peptides. The results show that the binding affinity for both the mRNA cap and eIF4G peptides can be simultaneously altered by point mutations distant from either binding site.

Interaction between the complement degradation product C3bi and leukocyte integrin alphaMbeta2, which are critical for host defense against foreign pathogens and in tumor cell surveillance is analyzed by a detailed mapping of the C3bi binding site. The results support a mosaic model, in which different ligands engage different amino acids to bind alphaMI-domain, accounting for the broad recognition capacity of integrin alphaMbeta2.

The in-gel fluorescence anisotropy of phospholamban (PLB) labeled with the biarsenical fluorophore FlAsH is measured at three different sites on the cytoplasmic domain. The relative dispositions of PLB cytoplasmic domains help to discriminate among models of pentamer quaternary conformation.

The 3-D structure of the peptidyl-tRNA hydrolase from the archaea Sulfolobus solfataricus (suPth2) is described at 1.8 Angstrom resolution. The results clearly distinguish the active site of the archael/eucaryal hydrolases from that of the bacterial/eucaryal ones, where a histidine serves as the catalytic base.

Triplex structures were generated by targeting a DNA duplex with photoreactive psoralen molecule (psoTFO), where TFO is triplex-formation oligonucleotides, of different lengths in order to investigate the mechanism of photoadduct-formation by psoralen photo-cross-linking. Results demonstrated that UV radiation of triplexes formed between a psoTFO and a DNA duplex generated two distinct groups of psoralen photoadducts, monoadducts and psoralen interstand cross-link products.

An attempt is made to demonstrate that TenA is a thiaminase II and to determine the structure of TenI. The TenA structure suggests that the degradation of thiamen by TenA likely proceeds via the same addition-elimination mechanism described for thiaminase I and the structure suggests that TenI is unable to bind thiamin phosphate.

Inactivation of the double mutant containing empty catalytic sites or MgADP bound to one catalytic site with CuCl(sub 2) cross-linked when at least two catalytic sites contained MgADP. The catalytic site at the alpha(sub TP)-beta(sub TP) interface with bound MgADP in crystal structures represents a catalytic site containing inhibitory MgADP.

Two homologous, single-point core mutants of a 57-residue, hyperthermophilic variant of the B1 domain of protein G (HTB1) are characterized. The larger of the two species was found to possess structural features that are reminiscent of an emerging class of protein assemblies prone to beta-sheet-mediated aggregation.

The reaction mechanisms of the Beckmann rearrangement (BR) of cyclohexanone oxime to epsilon-caprolactam in the gas phase and catalyzed by modernite are investigated. It was concluded that the actual turnover of a reaction catalyzed by Bronsted acid (BA) sites might be slowed by the relatively high desorption energy of the product and frequent readsorption and desorption at an increased concentration of BA sites.

It is shown that acid-base catalysis is essential for extradiol dioxygenase catalysis and that two suitably positioned histidine residues are utilized for acid base catalysis in the active sites of class III extradiol dioxygenases. The subtle acid-base catalysis appears to be required only for extradiol cleavage, since the active sites of intradiol catechol dioxygenases do not contain additional acid-base residues.

Structural analyses of the pseudomonas ACC deaminase in complex with various substrates/inhibitors reveal detailed interactions between these substrates/inhibitors and protein residues at the active site of the enzyme. Structural data implicates Tyr294 as the key catalytic residue in ACC deaminase.

The high-affinity metal-binding site of isolated F(sub 1)-ATPase from beef heart mitochondria is studied by high-field (HF) continuous wave electron paramagnetic resonance (CW-EPR) and pulsed EPR spectroscopy, using Mn(super II) as a paramagnetic probe.

The issue regarding whether the normal binding of AA is 'methyl-end first' or 'carboxylate-end first' is investigated by mutating the sLO-1 active site residues, Trp500 and Arg707, and determining their activation and catalytic kinetics. The results demonstrate that both Trp500 and Arg707 are critical to product and substrate binding in the active site, which indicates that the substrate binds 'carboxyl-end first'.

Intracellular receptor DAF -12 regulates dauer formation and development age and affects Caenorhabditis elegans life span. A procedure for multiple rounds of selection and amplification that yielded fragments bearing DAF-12-binding sites is developed beginning with C. elegans genomic DNA.

Myeloperoxidase (MPO), a hemoprotein that uses H2O2 as the electron acceptor in the catalysis of oxidative reactions, is implicated as a participant in inflammatory injury and cardiovascular diseases. It is demonstrated that molecular oxygen (O(sub 2)) binds to ferrous MPO (MPO-Fe(II) in a distinct and novel mechanism by using spectral and rapid kinetic measurements.

Amperometric O2 and H2O2 sensors are used to kinetically examine catalase activity (conversion of H2O2 to (1/2)O2 and H2O) and peroxygenase activity (H2O(sub 2-) dependent oxygenation of substrates) of mushroom tyrosinase. It is found that the catalase activity of mushroom tyrosinase is three-order of magnitude greater than that of mollusk hemocyanin.

The enterohepatic circulation of bile salts is facilitated by the apical sodium-dependent bile acid transporter (ASBT, SLC10A2) and plays a key role in cholesterol metabolism. The localization of potential substrate-binding sites may aid future development of more specific therapeutic strategies against hypercholesterolemia and related cardiovascular diseases.

Tear lipocalin (TL) is a major component of human tears, which shows pH-dependent endogenous ligand binding. Details of findings suggests that apposition of the loops at low pH excludes the ligand from the intracavitary binding site

Electrogenic events associated with the activity of the melibiose permease (MelB), a transporter from Escherichia coli were studied by means of the solid supported membrane (SSM) technique. Results demonstrate that melibiose binding to MelB results in an electrogenic conformational change.

The crystal structures of HbA2 and HbE at 2.20 and 1.74 angstorm resolution, respectively, in the R2 states, which are used to provide the probable explanations of the thermal stability and instability of HbA2 and HbE is presented. A high resolution crystal structure of ferrocyanide-bound HbA2 is also presented, which throws light on the location and the mode of binding of ferrocyanide anion with hemoglobin, predominantly using the residues involved in diphosphoglycerate (DPG) binding.

Design, self-assembly and properties of interlocked supramolecular complexes that show stimulus-responsive assembly and disassembly and therefore function like a molecular machine is presented. The reversible switching between two states is based on the transcis isomerism of diazo functionality induced by light and thermal energy which leads to blocking and opening of the binding site in the rod component.

Nonamidated gastrins such as progastrin and glycine-extended gastrin 17 (Ggly) induce cell proliferation, migration in vitro, and colonicmucosal proliferation in vivo. The aim of the study was to define the minimum biologically active fragment of Ggly and to determine whether ferric ions are required for the activity.

The role of key residues in the active site of LysRS2 (lysS encoded) from Escherichia coli is investigated. Based on the structure of L-lysine complexed with E. coli LysRS2 (lysS), residues implicated in amino acid recognition and discrimination was systematically replaced.

A specific agonist of the endothelin-B receptor subtype (ET(sub B)), a few photosensitive analogues is developed to investigate the binding domain of the receptor. Using the photoaffinity-labeling technique, a key segment of the ET(sub B) receptor participating in the interaction with the ET-1 of IRL-1620 ligands is identified.

A study is conducted towards the synthesis and characterization of Mn-oxo complexes to understand the structural and physicochemical properties of the WO site in PSII. A major emphasis is put towards complexes that had direct or indirect relevance to the PSII Mn (sub 4) site.

The effects of the acidic C-tail of HMGB1, a nonhistone chromosomal protein in higher eukaryotic nuclei, on the proteinEs function and behaviour on chromatin are investigated. The terminal sequence of the HMGB1 acidic C-tail, DDDDE, interacts with core histone N-tails, which is a key factor for the linker DNA-dependent binding of HMGB1 to the nucleosome, cause modulation of DNA and nucleosome and cause transcription stimulation in cultured cells.

Design and synthesis of de novo cytochrome c is carried out by using consensus sequence for the heme-binding site of a designed four-helix bundle and the apoproteins consist of either a histidine or a methionine residue positioned at the sixth coordination site. The structural and functional properties of artificially synthesized cytochrome c is investigated and compared with those of natural cytochromes, their variants and designed cytochrome b.

Structural changes occurring during the activation of transglutaminase Factor XIII (FXIII) is explored by protein modification experiments. Experiment that involved the labeling of cysteine and lysine side chains of the serine protease thrombin, FXIII activating enzyme, before and after activation reaction depicted differences in the labeling patterns of activated and nonactivated FXIII, occurred either due to physiological action with thrombin or nonpreteolytic exposure to calcium.

Surface plasmon resonance (SPR) is used to study the kinetic rate constant of the binding reaction of botulinum A (BoNT/A) with ganglioside GT1b. The neurotoxin botulinum bind to polysialylated gangliosides by undergoing conformational change that cause increase in alpha-helix content and a decrease in beta-sheet contents, and binding ability is affected by the ionic strength of the buffer.

Based on the crystal structure of glutamate racemase (MurI) from the hyperthermophilic bacterium Aquifex pyrophilus, molecular dynamics (MD) simulations of six different systems are carried out to analyse stereochemistry, substrate ligation, and active site protonation state of individual system. The study provides an insight into the mechanism of glutamate racemase in the catalysis during the racemization reaction of glutamate.

The catalytic mechanism of Thermobifida fusca Ce19A-90 is determined by constructing, cloning and expressing 12 mutant genes with changes in five conserved residues of Ce19A-68 in Escherichia coli. The results revealed the function of E424 as the catalytic acid, requirement of D55 and D58 as catalytic base activity, and important role of Y206 in binding catalysis and processivity, while Y318 is important for binding of crystalline cellulose substrates and is required for processivity.

The independent generation of 5-(2'deoxycytidinyl)methyl radical and that this radical can give rise to the predicted novel intrastrand cross-link lesion in dinucleoside monophosphates of d(mCG) and D(GmC) is reported. The 5-(2'-deoxycytidinyl)methyl radical can be specifically generated by 254-nm irradiation of 5-(phenyl-thiomethyl)-2'-deoxycytidine in dinucleoside monophosphates d(mCSPLG) and D(GmCSPL).

Three series of polymers are carefully formulated with increasing amounts of template while keeping the polymer components constant. The trends N and K(sub n) for each series of polymers, which were graphed versus percenttemplate, suggest multiple functional monomers in the binding sites of noncovalent molecularly imprinted polymers (MIPs).

Polar residue hot spots have been observed at protein-protein binding sites. Here we show that hot spots occur predominantly at the interfaces of macromolecular complexes, distinguishing binding sites from the remainder of the surface. Consequently, hot spots can be used to define binding epitopes. We further show a correspondence between energy hot spots and structurally conserved residues. The number of structurally conserved residues, particularly of high ranking energy hot spots, increases with the binding site contact size. This finding may suggest that effectively dispersing hot spots within a large contact area, rather than compactly clustering them, may be a strategy to sustain essential key interactions while still allowing certain protein flexibility at the interface. Thus, most conserved polar residues at the binding interfaces confer rigidity to minimize the entropic cost on binding, whereas surrounding residues form a flexible cushion. Furthermore, our finding that similar residue hot spots occur across different protein families suggests that affinity and specificity are not necessarily coupled: higher affinity does not directly imply greater specificity. Conservation of Trp on the protein surface indicates a highly likely binding site. To a lesser extent, conservation of Phe and Met also imply a binding site. For all three residues, there is a significant conservation in binding sites, whereas there is no conservation on the exposed surface. A hybrid strategy, mapping sequence alignment onto a single structure illustrates the possibility of binding site identification around these three residues. protein-protein interfaces | hot spots | molecular recognition | binding site prediction | residue conservation

The [S.sub.N]2 displacement of [Cl.sup.-] from 1,2-dichloroethane by acetate (C[H.sub.3]C[O.sup.-.sub.2]) in water and by the carboxylate of the active site aspartate in the haloalkane dehalogenase of Xanthobacter autothropicus have been compared by using molecular dynamics simulations. In aqueous solution, six families of contact-pair structures (I-VI) were identified, and their relative concentrations and dissociation rate constants were determined. The near attack conformers (NACs) required for the [S.sub.N]2 displacement reaction are members of the IV (C[H.sub.3]CO[O.sup.-]***C[H.sub.2](Cl)C[H.sub.2]Cl) family and are formed in the sequence II [right arrow] III [right arrow] IV [right arrow] NAC. The NAC subclass is defined by the--CO[O.sup.-]***C--Cl contact distance of [less than or equal to] 3.41 [Angstrom] and the--CO[O.sup.-]***C--Cl angle of 157-180[degrees]. The mole percentage of NACs is 0.16%, based on the 1 M standard state. This result may be compared with 13.4 mole percentage of NACs in the Michaelis complex in the enzyme. It follows that NAC formation in the enzyme is favored by 2.6 kcal/mol. Because reaction coordinates from S to TS, both in water and in the enzyme, pass via NAC (i.e., S [right arrow] NAC [right arrow] TS), the reduction in the S [right arrow] NAC barrier by 2.6 kcal/mol accounts for [approximately equal to] 25% of the reduction of total barrier in the S [right arrow] TS (10.7 kcal/mol). The remaining 75% of the advantage of the enzymatic reaction revolves around the efficiency of NAC [right arrow] TS step. This process, based on previous studies, is discussed briefly.

The integrin [alpha]X[beta]2 (CD11c/CD18, p150,95) binds ligands through the I domain of the [alpha]X subunit. Ligands include the complement factor fragment iC3b, a key component in the innate immune defense, which, together with the expression of [alpha]X[beta]2 on dendritic cells and on other leukocytes, suggests a role in the immune response. We now report the structure of the [alpha]X I domain resolved at 1.65 [Angstrom] by x-ray crystallography. To analyze structural requirements for ligand binding we made a mutation in the [alpha]X I domain C-terminal helix, which increased the affinity for iC3b [approximately equal to] 200-fold to 2.4 [micro]M compared with the wild-type domain affinity of [approximately equal to] 400 [micro]M. Gel permeation chromatography supported a conformational change between the wild-type and mutated domains. Conservation of allosteric regulation in the [alpha]X I domain points to the functional importance of this phenomenon.

Signaling in the human growth hormone (hGH)-human GH receptor system is initiated by a controlled sequential two-step hormone-induced dimerization of two hGH receptors via their extracellular domains (ECDs). Little is currently known about the energetics governing the important regulatory step in receptor signaling (step 2) because of previously existing experimental barriers in characterizing the binding of the second receptor (ECD2). A further complication is that ECD2 binds through contacts from two spatially distinct sites: through its N-terminal domain to hGH, and to ECD1 through its C-terminal domain, which forms a pseudo-2-fold symmetrical interaction between the stems of the two receptors. We report here a detailed evaluation of the energetics of step 2 binding using a modified surface plasmon resonance method that is able to measure accurately the kinetics of the trimolecular binding process and separate the effects of the two binding sites. The binding kinetics of 23 single and 126 ECD1-ECD2 pair-wise alanine mutations was measured. Although both of the ECD2 binding interfaces were found to be important, the ECD1-ECD2 stem-stem contact is the stronger of the two. It was determined that most residues in the binding interfaces act in additive fashion, and that the six residues common in both ECDs contribute very differently to homodimerization depending on which ECD they reside in. This interface is characterized by a binding 'hot-spot' consisting of a core of three residues in ECD1 and two in ECD2. There is no similar hot-spot in the N-terminal domain of ECD2 binding to Site2 of hGH. This study suggests ways to engineer ECD molecules that will bind specifically to either Site1 or Site2 of hGH, providing novel reagents for biophysical and biological studies.

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhP[G.sub.1-80]) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1-1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CC[K.sub.1] and CC[K.sub.2] receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CC[K.sub.1]-R and CC[K.sub.2]-R on IEC cells. High-affinity ([K.sub.d] = 0.5-1.0 nM) binding sites for [sup.I]-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrinlike peptides in the order PG [greater than or equal to] COOH-terminally extended G17 [greater than or equal to] G-Gly > G17 > *CCK-8 (* significant difference; P < 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells. progastrin-preferring receptors; confocal microscopy; in vitro growth effects; Gly-gastrin

We examined the effects of a fragment of the substrate binding domain of DnaK on protein refolding from chemically denatured states. The fragment DnaK384-638, containing a full-length substrate binding domain, tightly binds to the unfolded protein in solution. The effects of DnaK384-638 on the reactivation of [beta]-galactosidase and luciferase were examined at low substrate concentration and low temperature, conditions in which the folding is significantly slow (several days) but the reactivation yield is higher than those in ordinary refolding conditions. In the presence of DnaK384-638, the maximum yield of active [beta]-galactosidase was improved from 45% to 65% after a 48-h refolding reaction. Spectroscopic experiments showed that DnaK384-638 bound to partially structured monomers of [beta]-galactosidase and consequently suppressed aggregation. DnaK384-638 accelerated the refolding of luciferase to attain equilibrium in 8 h. On the other hand, DnaK386-561, which has no affinity for the substrate, had no chaperone activity for the reactivation of these proteins. These results indicate that the substrate binding of DnaK384-638 facilitates slow protein refolding.

Orbital interactions in the adsorption of methanethiolate in various binding sites of a three layer slab model and gold (sub 42) cluster model of gold (111) surface are investigated. Fragment molecular orbital result explains the vertical S-C bonds in the hollow models and the tilted S-C bonds in the on-top and bridge models.

A subset of microtubule-associated proteins, including cytoplasmic linker protein (CLIP)-170, dynactin, EB1, adenomatous polyposis coli, cytoplasmic dynein, CLASPs, and LIS-1, has been shown recently to target to the plus ends of microtubules. The mechanisms and functions of this binding specificity are not understood, although a role in encouraging microtubule elongation has been proposed. To extend previous work on the role of dynactin in organelle transport, we analyzed [p150.sup.Glued] by live-cell imaging. Time-lapse analysis of [p150.sup.Glued] revealed targeting to the plus ends of growing microtubules, requiring the N[H.sub.2]-terminal cytoskeleton-associated protein-glycine rich domain, but not EB1 or CLIP-170. Effectors of protein kinase A modulated microtubule binding and suggested [p150.sup.Glued] phosphorylation as a factor in plus-end binding specificity. Using a phosphosensitive monoclonal antibody, we mapped the site of [p150.sup.Glued] phosphorylation to Ser-19. In vivo and in vitro analysis of phosphorylation site mutants revealed that [p150.sup.Glued] phosphorylation mediates dynamic binding to microtubules. To address the function of dynamic binding, we imaged GFP-[p150.sup.Glued] during the dynein-dependent transport of Golgi membranes. Live-cell analysis revealed a transient interaction between Golgi membranes and GFP-[p150.sup.Glued]-labeled microtubules just prior to transport, implicating microtubules and dynactin in a searchcapture mechanism for minus-end-directed organelles.

Research has been conducted on the binding site of Ag-Gly-Asp (RGD) triad within integrin. The identification of this binding site has been carried out via the superimposition of the contact domains on the X-ray crystal structure of the alpha (sub)v beta (sub)3 ectopic domain and the details are reported.

Bacteriophage T4 tail fibers have a quaternary structure of bent rigid rods, 3 x 160 nm in size. The four proteins which make up these organelles are able to self-assemble in an essentially irreversible manner. To use the self-assembly domains of these proteins as elements in construction of mesoscale structures, we must be able to rearrange these domains without affecting the self-assembly properties and add internal binding sites for other functional elements. Here we present results on several alterations of the P37 component of the T4 tail fiber that change its length and add novel protein sequences into the protein. One of these sequences is an antibody binding site that is used to inactivate phage carrying the modified gene.

We describe a general method based on principal coordinates analysis to predict the effects of single-nucleotide polymorphisms within regulatory sequences on DNA-protein interactions. We use binding data for the transcription factor NF-[kappa]B as a test system. The method incorporates the effects of interactions between base pair positions in the binding site, and we demonstrate that such interactions are present for NF-[kappa]B. Prediction accuracy is higher than with profile models, confirmed by crossvalidation and by the experimental verification of our predictions for additional sequences. The binding affinities of all potential NF-[kappa]B sites on human chromosome 22, together with the effects of known single-nucleotide polymorphisms, are calculated to determine likely functional variants. We propose that this approach may be valuable, either on its own or in combination with other methods, when standard profile models are disadvantaged by complex internucleotide interactions.

The GGAs (Golgi-localizing, [gamma]-adaptin ear homology domain, ARF-binding proteins) are a family of proteins implicated in protein trafficking from the Golgi to endosomes/lysosomes. These proteins have modular structures with an N-terminal VHS (VPS-27, Hfs, and STAM) domain followed by a GAT (GGA and TOM1) domain, a connecting hinge segment, and a C-terminal GAE ([gamma]-adaptin ear) domain. Isolated VHS domains have been shown to bind specifically to acidic cluster (AC)-dileucine motifs present in the cytoplasmic tails of the mannose 6-phosphate receptors. Here we report that full-length cytoplasmic GGA1 and GGA3 but not GGA2 bind the cation-independent mannose 6-phosphate receptor very poorly because of autoinhibition. This inhibition is caused by the binding of an AC-LL sequence present in the hinge segment to the ligand-binding site in the VHS domain. The inhibition depends on the phosphorylation of a serine located three residues upstream of the AC-LL motif. The serine is phosphorylated by casein kinase 2 in in vitro assays. Substitution of the GGA1 inhibitory sequence into the analogous location in GGA2, which lacks the AC-LL motif, results in autoinhibition of the latter protein. These data indicate that the activity of GGA1 and GGA3 is regulated by cycles of phosphorylation/dephosphorylation.

Many anticancer drugs interact directly with DNA to exert their biological functions. To date, all noncovalent, intercalating drugs interact with DNA exclusively by inserting their chromophores into base steps to form elongated and unwound duplex structures without disrupting the flanking base pairs. By using actinomycin D (ActD)-5'-GXC/CYG-5' complexes as examples, we have found a rather unusual interaction mode for the intercalated drug; the central Watson-Crick X/Y base pairs are looped out and displaced by the ActD chromophore. The looped-out bases are not disordered but interact perpendicularly with the base/chromophore and form specific H bonds with DNA. Such a complex structure provides intriguing insights into how ligand interacts with DNA and enlarges the repertoires for sequence-specific DNA recognition.