Structural differences between hypoxanthine phosphoribosyltransferase family members highlight opportunities for antiparasitic drug design in neglected diseases.

Approaches to identify novel druggable targets for treating neglected diseases include computational studies that predict possible interactions of drugs and their molecular targets. Hypoxanthine phosphoribosyltransferase (HPRT) plays a central role in the purine salvage pathway. This enzyme is essential for the survival of the protozoan parasite T. cruzi, the causal agent of Chagas disease, and other parasites related to neglected diseases. Here we found dissimilar functional behaviours between TcHPRT and the human homologue, HsHPRT, in the presence of substrate analogues that can lie in differences in their oligomeric assemblies and structural features. To shed light on this issue, we carried out a comparative structural analysis between both enzymes. Our results show that HsHPRT is considerably more resistant to controlled proteolysis than TcHPRT. Moreover, we observed a variation in the length of two key loops depending on the structural arrangement of each protein (groups D1T1 and D1T1'). Such variations might be involved in inter-subunit communication or influencing the oligomeric state. Besides, to understand the molecular basis that govern D1T1 and D1T1' folding groups, we explored the distribution of charges on the interaction surfaces of TcHPRT and HsHPRT, respectively. To know whether the rigidity degree bears effect on the active site, we studied the flexibility of both proteins. The analysis performed here illuminates the underlying reasons and significance behind each protein's preference for one or the other quaternary arrangement that can be exploited for therapeutic approaches.

Structural glance into a novel anti-staphylococcal peptide.

Novel antimicrobial peptides are valuable molecules for developing anti-infective drugs to counteract the contemporary spread of microbial drug-resistance. Here we focus on a novel peptide (RKWVWWRNR-NH2) derived from the fragment 107-115 of the human lysozyme that displays a 20-fold increase in anti-staphylococcal activity. The conformational analysis of this peptide and its interaction with model lipidic phases-as assayed by circular dichroism and fluorescence spectroscopy-show a noteworthy spectral change, which might be related to its anti-staphylococcal activity. The secondary structure of peptide [K(108)W(111)] 107-115 hLz was dramatically affected through a single substitution at position 111 (Ala by Trp). Therefore, this conformational change might improve the interaction of the novel peptide with the bacterial plasma membrane. These results highlight the role of peptide secondary structure and the distribution of polar/nonpolar residues for the effective interaction of this peptide with the bacterial plasma membrane, a key step for triggering its lethal effect. This knowledge may contribute to the rational design of a new generation of antimicrobial peptides with increased efficacy developed from natural sources by simple screening tools.

Zoledronate repositioning as a potential trypanocidal drug. Trypanosoma cruzi HPRT an alternative target to be considered.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and affects 7 million people worldwide. Considering the side effects and drug resistance shown by current treatments, the development of new anti-Chagas therapies is an urgent need. T. cruzi hypoxanthine phosphoribosyltransferase (TcHPRT), the key enzyme of the purine salvage pathway, is essential for the survival of trypanosomatids. Previously, we assessed the inhibitory effect of different bisphosphonates (BPs), HPRT substrate analogues, on the activity of the isolated enzyme. BPs are used as a treatment for bone diseases and growth inhibition studies on T. cruzi have associated BPs action with the farnesyl diphosphate synthase inhibition. Here, we demonstrated significant growth inhibition of epimastigotes in the presence of BPs and a strong correlation with our previous results on the isolated TcHPRT, suggesting this enzyme as a possible and important target for these drugs. We also found that the parasites exhibited a delay at S phase in the presence of zoledronate pointing out enzymes involved in the cell cycle, such as TcHPRT, as intracellular targets. Moreover, we validated that micromolar concentrations of zoledronate are capable to interfere with the progression of cell infection by this parasite. Altogether, our findings allow us to propose the repositioning of zoledronate as a promising candidate against Chagas disease and TcHPRT as a new target for future rational design of antiparasitic drugs.

Transmembrane channels based on tartaric acid-gramicidin A hybrids.

The gramicidin A transmembrane channel is believed to consist of two head-to-head beta helices. Computer-generated models were used to formulate the structure of new single-chain channel molecules based on the gramicidin motif. The chemical synthesis of two tartaric acid-gramicidin A hybrids and single-channel analyses of their conducting properties are reported. These studies illustrate the rational design and synthesis of long-lived channels with tunable conductance properties and provide support for current molecular models of the natural (dimeric) gramicidin channel.

Intervening in the ß-barrel structure of lipid binding proteins: consequences on folding, ligand-binding and aggregation propensity.

Natural ß-folds manage to fold up successfully. By contrast, attempts to dissect fragments or peptides from well folded ß-sheet proteins have met with insurmountable difficulties. Here we briefly review selected successful cases of intervention on the well-known scaffold of intestinal fatty acid binding protein (IFABP). Lessons from these examples might set guidelines along the design of proteins belonging to this class. Impact of modifications on topology, binding and aggregation is highlighted. With the aid of abridged variants of IFABP we focus on key structural features responsible for the assembly into oligomeric forms or aggregates.

Detection and characterization of an ovine placental lactogen stable intermediate in the urea-induced unfolding process.

The urea-induced equilibrium unfolding of ovine placental lactogen, purified from ovine placenta, was followed by size-exclusion chromatography, far-UV CD, and intrinsic tryptophan fluorescence. The data obtained by each of these methods showed a poor fit to a two-state model involving only a native and an unfolded form. A satisfactory fit required, instead, a model that involved a stable, partially folded form in addition to the native and unfolded ones. The results obtained from the best-fitting theoretical curves for the three-state model indicated that this intermediate state, which is the predominant species in solution at 3.6 M of urea activity, is compact, largely alpha-helical, and changes considerably the native-like tertiary packing around its tryptophan residues. These findings suggest that this stable intermediate exhibits properties similar to those that characterize the molten globule state.

The membrane topology of the amino-terminal domain of the red cell calcium pump.

A systematic study of the membrane-associated regions in the plasma membrane Ca2+ pump of erythrocytes has been performed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine ([125I]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane-bound probe, [3H]bis-phosphatidylethanolamine (trifluoromethyl) phenyldiazirine. A significant light-dependent labeling of an M(r) 135,000-140,000 peptide, corresponding to the full Ca2+ pump, was observed with both probes. After proteolysis of the pump labeled with each probe and isolation of fragments by SDS-PAGE, a common pattern of labeled peptides was observed. Similarly, labeling of the Ca2+ pump with [125I]TID, either in isolated red blood cell membranes or after the enzyme was purified, yields a similar pattern of labeled peptides. Taken together, these results validate the use of either probe to study the lipid interface of the membrane-embedded region of this protein, and sustain the notion that the conformation of the pump is maintained throughout the procedures of solubilization, affinity purification, and reconstitution into proteoliposomes. In this work, we put special emphasis on a detailed analysis of the N-terminal domain of the Ca2+ pump. A labeled peptide of M(r) 40,000 belonging to this region was purified and further digested with V8 protease. The specific incorporation of [125I]TID to proteolytic fragments pertaining to the amino-terminal region indicates the existence of two transmembrane stretches in this domain. A theoretical analysis based on the amino acid sequence 1-322 predicts two segments with high probability of membrane insertion, in agreement with the experimental data. Each segment shows a periodicity pattern of hydrophobicity and variability compatible with alpha-helical structure. These results strongly suggest the existence of a transmembrane helical hairpin motif near the N-terminus of the Ca2+ pump.

A membrane protein associated with the prolactin receptor. Studies with a photoactivatable human growth hormone derivative.

Prolactin receptor from rat liver (PRL-R, 42 kDa) was cross-linked to a radiolabeled azidophenacyl derivative of human growth hormone ([125I]AP-hGH) to yield a 63 kDa adduct. In addition, a protein of Mr 50-52 K was detected as a 73 kDa complex. Microsomes incubated with either (a) increasing amounts of [125I]AP-hGH, or (b) a fixed amount of photoprobe and increasing concentrations of unlabeled hGH, showed that the 73/63 kDa band intensity ratio remains constant (0.71-0.77). Once transferred onto nitrocellulose membranes, only the 42 kDa protein is able to bind [125I]AP-hGH or [125I]hGH. Two anti-PRL-R monoclonal antibodies fail to cross-react with proteins of Mr 50-52 K. In membranes solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), a significantly lower amount of the 73 kDa complex is detected. Thus, the 50-52 kDa protein appears to be structurally unrelated to, but is presumably associated with the PRL-R. The 73 kDa complex is also detected under low membrane fluidity conditions (1 degree C), indicating that PRL-R associates to this 50-52 kDa protein prior to hormone binding. Perfusion of rat liver with [125I]AP-hGH shows that this associated protein accompanies the receptor along its intracellular pathway.

Performance of alginate films for retention of L-(+)-ascorbic acid.

In view of acting as controlled delivery systems for nutritional supplementation, therapy or antioxidant activity at interfaces, alginate films of different copolymer composition and glycerol plasticizer levels were developed in the presence of Ca(2+) for achieving higher stability of L-(+)-ascorbic acid (AA). The ability of the alginate network to preserve AA from hydrolysis, tested by storage under vacuum at 25 °C, only decreased with the relative humidity (RH) increase when alginates were mainly constituted by guluronic-guluronic acid blocks (GG), whereas also decreased with the glycerol level increase when mannuronic-mannuronic acid (MM) and/or alternating guluronic-mannuronic (GM+MG) flexible blocks were present in higher proportions. This result could be probably related to the lower capability of the latter alginate block compositions to immobilize water in the network as they are not able to constitute Ca(2+) mediated junction zones where water molecules are highly retained. Films also studied under air storage showed that even at less favorable conditions of RH and glycerol levels, both GG and GM+MG enriched alginate networks in general preserved AA from oxidation. It also demonstrated that hydrolysis is the principal way by which AA is lost when supported in films.

Thermal regulation of membrane lipid fluidity by a two-component system in Bacillus subtilis.

This article describes a simple and robust laboratory exercise on the regulation of membrane unsaturated fatty acid composition in bacteria by a decrease in growth temperature. We take advantage of the well characterized Des pathway of Bacillus subtilis, composed of a Δ5-desaturase (encoded by the des gene) and the canonical two-component system DesK-DesR, to study the transcriptional regulation of des during cold shock. Students analyze the expression of a reporter transcriptional fusion between the des promoter and the bacterial lacZ gene in a wild-type B. subtilis strain and in des or desK-desR mutants grown under different culture conditions. Measurements of ß-galactosidase activity allow them to investigate how the Des pathway works and to assess the role of each component of this regulatory system.

Covalent cross-linking of the bovine somatotropin dimer. Effects on growth-promoting, receptor-binding and immunological activities and preliminary characterization of the self-association.

Bovine somatotropin, at pH 8.5 in 0.02 M-Bicine [NN-bis-(2-hydroxyethyl)glycine]/0.09M-NaCl, showed by frontal analysis the characteristics of a rapid monomer-dimer equilibrium whose dissociation constant was estimated to be 6.6 X 10(-6)M. Reaction of the hormone with dimethyl suberimidate lead to covalent cross-linking of the dimeric species. Under the conditions chosen (0.4 mg of bifunctional imidate and 1 mg of protein/ml at room temperature for 1 h) the cross-linked dimers accounted for 26% of the total protein, and these were isolated by molecular sieving in 0.29M-NH3/0.12M-NaCl. Covalent stabilization greatly diminished the growth-promoting activity and the ability to interact with somatogenic sites in both rat liver in vivo and rabbit liver microsomal fractions. Evidence indicating a non-critical role for amino groups involved in the covalent cross-linking was provided by a nearly equivalent derivative obtained after reaction with 3,3'-dithiobispropionimidate, which had substantial hormonal activity upon cleavage of the disulphide links. Conversely, immunological reactivity as demonstrated by radioimmunoassay was not affected by cross-linking. Details of the least-squares procedure employed to evaluate the self-association equilibrium constant has been deposited as Supplement SUP 50115 (7 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem.J. (1981) 193,5.

Purification of gramicidin A.

A simple chromatographic purification of the naturally occurring ion channel-forming pentadecapeptide gramicidin A (gA) is presented. This procedure allows gA to be isolated in gram quantities from the commercially available mixture of isomers after chromatography on silica gel. The gramicidin A obtained in this manner is greater than 95% pure as determined by 1HNMR, HPLC, and amino acid analysis.

Carboxylate groups in bovine somatotropin involved in growth promoting activity. Theoretical models based upon individual kinetic constants to interpret the activity decay after chemical modification.

Modification of approximately one fifth of the carboxylate groups in bovine somatotropin with a water soluble carbodiimide caused loss of growth promoting potency pointing to the existence of residues related to the hormonal activity among those belonging to a fast reacting set. A sigmoidal curve was obtained whether the inactivation process was referred to reaction time or degree of modification. Isoelectrofocusing of derivatives released the native hormone from responsibility for the biological potency exerted by preparations with 1.5-2.6 modified carboxylate groups. Examination of the individual reaction kinetics of the 11 fast reacting residues, in turn, excluded the possibility of the sigmoidal character of the inactivation curve being caused by a nonexponential disappearance of essential residues, as a possible consequence of the chemical modification of others. According to synthetic models, the experimental curve may be the consequence of the effect of cumulative modification of 2 or 3 out of a set of 3 to 8 relevant residues.

Temperature-induced conformational transition of intestinal fatty acid binding protein enhancing ligand binding: a functional, spectroscopic, and molecular modeling study.

Intestinal fatty acid binding protein (IFABP) undergoes a reversible thermal transition between 35 and 50 degreesC, as revealed by circular dichroism spectroscopy in the near-UV region. For the apoprotein, the molar ellipticity measured at 254 nm (possibly implicating the environment around F17 and/or F55) decreases significantly in this temperature range, while in the holoprotein (bound to oleic acid), this phenomenon is not observed. Concomitantly, an increase in the activity of binding to [14C]oleic acid occurs. Nevertheless, other spectroscopic evidence indicates that the beta-barrel structure, the major motif of this protein, is highly stable up to 70 degreesC. No changes associated with conformation were detected for both structures by fourth-derivative analysis of the UV absorption spectra, circular dichroism in the far-UV region, and intrinsic fluorescence measurements. Further structural information arises from experiments in which binding to the anionic fluorescent probes 1-anilinonaphthalene-8-sulfonic acid (ANS) and its dimer bisANS was examined. The fluorescence intensity of bound ANS diminishes monotonically, whereas that of bisANS increases slightly in the temperature range of 35-50 degreesC. Given the different size of these probes, model building suggests that ANS would be able to sense regions located deeply inside the cavity, while bisANS could also reach the vicinity of the small helical domain of this protein. In light of these results, we believe that this subtle conformational transition of IFABP, which positively influences the binding activity, would involve fluctuations at the peripheral "entry portal" region for the ligand. This interpretation is compatible with the discrete disorder observed in this place in apo-IFABP, as evidenced by NMR spectroscopy [Hodsdon, M. E., and Cistola, D. P. (1997) Biochemistry 36, 1450-1460].

A new photoactivatable reagent capable of transferring a radiolabel to target proteins. Application to the human growth hormone-rat liver prolactin receptor interaction.

A new photoactivatable cross-linking reagent, 1-(2'-dithiopyridyl)-2-(5'-azidosalicylamido)ethane (ASDPE), was synthesized. This probe can be easily labeled with 125I in the azidosalicylamido ring and contains an activated disulfide bridge. After reaction of [125I]ASDPE with proteins, the radiolabeled moiety of the probe becomes attached to cysteine residues. Upon partial reduction of human growth hormone (hGH) with dithiothreitol, its C-terminal disulfide bond between residues 182 and 189 was cleaved and the nascent thiol groups were modified with [125I]ASDPE to yield [125I]ASET-hGH [1-(thio-hGH)-2-(3'-[125I]iodo-5'-azidosalicylamido)ethane]. After binding of this hormone derivative to rat liver microsomes, followed by photolysis and subsequent reduction of disulfide bridges, the specific transfer of the radiolabeled moiety to prolactin receptor (PRL-R) was achieved. Partial purification of the radiolabeled receptor by size exclusion chromatography was performed. We anticipate that [125I]ASDPE will be generally useful in pursuing structural and functional studies of target proteins which interact specifically with protein ligands.

Specific modification of carboxyl-groups in bovine growth hormone by a soluble carbodiimide and glycinemethylester.

The reactivity of the carboxyl groups in bovine growth hormone was studied by reaction with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide in the presence of an excess of glycinemethylester. Localization in the molecule of the various kinetically distinguishable carboxyl groups was achieved. Highest reactivity was found in carboxyl groups 30, 125, 127 and 128. These residues were followed, as to reactivity, by a set including carboxyl groups 65, 105, 151, 152, 185 and 190. Modification of approximately one fifth of the carboxyl groups in bovine growth hormone led to an important decrease in its growth promoting activity and capacity to compete with 125I-labeled growth hormone for rat liver binding sites. Demethoxylation restored most of the original biological activity.

Binding in vitro to rat liver receptors does not correlate with activities in vivo of bovine somatotropin. Use of chemically modified derivatives as probes.

Bovine somatotropin with an increasing number of its carboxylate groups modified by reaction with glycine methyl ester in the presence of a water-soluble carbodi-imide was tested for its activity in different bioassays. Only those derivatives which were known to be active in the body-weight-increase bioassay were able to compete with 125I-labelled bovine somatotropin for their specific binding sites in vivo. No difference was found in the rate of clearance of a poorly active derivative as compared with that of native somatotropin. In contrast, both active and inactive derivatives were found to be equally effective in displacing the tracer from its binding sites present in isolated cells and membrane preparations from rat liver. These results suggest that the liver somatogenic receptors studied in vitro are less discriminating than those detected in vivo.

Interaction of unsaturated fatty acids with the red blood cell Ca(2+)-ATPase. Studies with a novel photoactivatable probe.

Unsaturated fatty acids, such as oleic acid, increase both the affinity for Ca2+ and the maximum effect of the Ca(2+)-ATPase of red blood cells [Niggli et al. (1981) J. Biol. Chem. 256, 8588-8592]. With the aim of examining the structural and kinetic details of the interaction between unsaturated fatty acids and the enzyme, we designed and synthesized 8-(5'-azido-O-hexanoylsalicylamido)octanoic acid (AS86), a photoactivatable analogue of unsaturated fatty acids. AS86, interacting noncovalently with the enzyme, shares with oleic acid the following properties: (i) it binds reversibly to the plasma membrane Ca(2+)-ATPase; (ii) in the absence of calmodulin, AS86 shows a biphasic behavior; i.e., at low concentrations it increases the affinity for Ca2+ and the maximum velocity of the enzyme, while at higher concentrations it decreases the maximum velocity; (iii) in the presence of calmodulin, AS86 increases slightly the affinity for Ca2+ and decreases the maximum velocity of the Ca2+ pump; and (iv) AS86 inhibits the activity of the enzyme devoid of its calmodulin-binding domain after proteolysis. When the reagent is covalently bound to the native enzyme, and then activated by calmodulin, increasing amounts of AS86 decrease the maximum velocity along a hyperbolic curve without modifying the apparent affinity for Ca2+. These results could be explained by the eventual existence of two different kind of sites recognizing the reagent: one influencing the affinity for Ca2+ and the other inhibitory of the calmodulin effects. When covalently bound, AS86 exerts its inhibitory effects upon the enzyme lacking the calmodulin-binding domain, thus reflecting that this action is promoted by interaction with a site lying outside this region. The purified enzyme is susceptible to be tagged with 125I-AS86. Both the inhibitory effect on the calmodulin-dependent enzymic activity after covalent binding of AS86 and the photoadduct formation between the enzyme and 125I-AS86 are impaired by the presence of oleic acid in a concentration-dependent fashion. Recognition of photoreactive fatty acid analogues by the purified enzyme could be useful to provide further insight on the location of the interacting sites.

The HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion.

The interaction between influenza virus and target membrane lipids during membrane fusion was studied with hydrophobic photoactivatable probes. Two probes, the newly synthesized bisphospholipid diphosphatidylethanolamine trifluoromethyl [3H]phenyl diazirine and the phospholipid analogue 1-palmitoyl-2(11-[4-[3-(trifluoromethyl)diazirinyl]phenyl]-[2-3H]- undecanoyl]-sn-glycero-3-phosphocholine (Harter, C., Bächi, T., Semenza, G., and Brunner , J. (1988) Biochemistry 27, 1856-1864), were used. Both labeled the HA2 subunit of the virus at low pH. By measuring virus-liposome interactions at 0 degrees C, it could be demonstrated that HA2 was inserted into the target membrane prior to fusion. As we have recently demonstrated, at this temperature, exposure of the fusion peptide of HA2 takes place within 15 s after acidification, but fusion does not start for 4 min (Stegmann, T., White, J. M., and Helenius, A. (1990) EMBO J. 9, 4231-4241). HA2 was labeled at least 2 min before fusion. No labeling of the HA1 subunit was seen. These data indicate that fusion is triggered by a direct interaction of the HA2 subunit of a kinetic intermediate form of HA with the lipids of the target membrane. Most likely, it is the fusion peptide of HA2 that is inserted into the target membrane. Just before fusion, HA is thus an integral membrane protein in both membranes. In contrast, the bromelain-derived ectodomain of HA was labeled by 1-palmitoyl-2(11-[4-[3-(trifluoromethyl)diazirinyl]phenyl]- [2-3H]undecanoyl)-sn-glycerol-3-phosphocholine at low pH but not by diphosphatidylethanolamine trifluoromethyl [3H]phenyl diazirine. This indicates that insertion of the fusion peptide of the bromelain-derived ectodomain of HA into a membrane differs from that of viral HA during fusion.

Differential hydrolysis of immobilized phosphatidylcholines by phospholipases A2 and C.

A novel phospholipid, 1-fatty acyl-2-(12-aminododecyl) phosphatidylcholine (APC), was synthesized and reacted with two different activated agarose matrices, differing in the spacer arm length: N-hydroxysuccinimidylester agarose (1-atom spacer arm) and N-hydroxysuccinimidylester-6-aminohexanoic acid agarose (8-atom spacer arm). Both immobilized phosphatidylcholines were readily degraded by Bacillus cereus phospholipase C at similar rates. By contrast, Crotalus adamanteus phospholipase A2 hydrolyzed long-spacer arm phosphatidylcholine, but had less than one tenth of the activity towards the short-spacer arm one. These results are interpreted in terms of a chain length-related steric hindrance caused by the matrix, affecting phospholipase A2 but not phospholipase C activity, supporting the view that the first involves a deeper burrowing of the substrate into the enzyme molecule.