Structure of Plasmodium falciparum triose-phosphate isomerase-2-phosphoglycerate complex at 1.1-A resolution

Triose-phosphate isomerase, a key enzyme of the glycolytic pathway, catalyzes the isomerization of dihydroxy acetone phosphate and glyceraldehyde 3-phosphate. In this communication we report the crystal structure of Plasmodium falciparum triose-phosphate isomerase complexed to the inhibitor 2-phosphoglycerate at 1.1-A resolution. The crystallographic asymmetric unit contains a dimeric molecule. The inhibitor bound to one of the subunits in which the flexible catalytic loop 6 is in the open conformation has been cleaved into two fragments presumably due to radiation damage. The cleavage products have been tentatively identified as 2-oxoglycerate and meta-phosphate. The intact 2-phosphoglycerate bound to the active site of the other subunit has been observed in two different orientations. The active site loop in this subunit is in both open and "closed" conformations, although the open form is predominant. Concomitant with the loop closure, Phe-96, Leu-167, and residues 208-211 (YGGS) are also observed in dual conformations in the B-subunit. Detailed comparison of the active-site geometry in the present case to the Saccharomyces cerevisiae triose-phosphate isomerase-dihydroxy acetone phosphate and Leishmania mexicana triose-phosphate isomerase-phosphoglycolate complexes, which have also been determined at atomic resolution, shows that certain interactions are common to the three structures, although 2-phosphoglycerate is neither a substrate nor a transition state analogue.     

A C-H triplebond O hydrogen bond stabilized polypeptide chain reversal motif at the C terminus of helices in proteins

The serendipitous observation of a C-H cdots, three dots, centered O hydrogen bond mediated polypeptide chain reversal in synthetic peptide helices has led to a search for the occurrence of a similar motif in protein structures. From a dataset of 634 proteins, 1304 helices terminating in a Schellman motif have been examined. The C-H triplebond O interaction between the T-4 C(alpha)H and T+1 Cz doublebond O group (C triplebond O< or =3.5A) becomes possible only when the T+1 residue adopts an extended beta conformation (T is defined as the helix terminating residue adopting an alpha(L) conformation). In all, 111 examples of this chain reversal motif have been identified and the compositional and conformational preferences at positions T-4, T, and T+1 determined. A marked preference for residues like Ser, Glu and Gln is observed at T-4 position with the motif being further stabilized by the formation of a side-chain-backbone O triplebond H-N hydrogen bond involving the side-chain of residue T-4 and the N-H group of residue T+3. In as many as 57 examples, the segment following the helix was extended with three to four successive residues in beta conformation. In a majority of these cases, the succeeding beta strand lies approximately antiparallel with the helix, suggesting that the backbone C-H triplebond O interactions may provide a means of registering helices and strands in an antiparallel orientation. Two examples were identified in which extended registry was detected with two sets of C-H cdots, three dots, centered O hydrogen bonds between (T-4) C(alpha)H triplebond O (T+1) and (T-8) C(alpha)H triplebondC doublebond O (T+3).     

Structure of the Plasmodium falciparum triosephosphate isomerase-phosphoglycolate complex in two crystal forms: characterization of catalytic loop open and closed conformations in the ligand-bound state

Triosephosphate isomerase (TIM) has been the subject of many structural and mechanistic studies. At position 96, there is a highly conserved Ser residue, which is proximal to the catalytic site. Thus far, no specific role has been ascribed to this residue. Plasmodium falciparum TIM (PfTIM), a fully catalytically active enzyme, is unique in possessing a Phe residue at position 96. The structure of PfTIM complexed to phosphoglycolate (PG), a transition state analogue, has been determined in an effort to probe the effects of the mutation at residue 96 on the nature of inhibitor-enzyme interactions and the orientation of the critical catalytic loop (loop 6, residues 166-176) in TIM. Crystal structures of PfTIM complexed to phosphoglycolate in orthorhombic (P2(1)2(1)2(1)) and monoclinic (C2) forms were determined and refined at resolutions of 2.8 and 1.9 A, respectively. The P2(1)2(1)2(1) form contains two dimers in the asymmetric unit. In the C2 form, the molecular and crystal 2-fold axes are coincident, leading to a monomer in the asymmetric unit. The catalytic loop adopts the open state in the P2(1)2(1)2(1) form and the closed conformation in the C2 crystal. The open conformation of the loop in the P2(1)2(1)2(1) form appears to be a consequence of the Ser to Phe mutation at residue 96. The steric clash between Phe96 and Ile172 probably impedes loop closure in PfTIM-ligand complexes. The PfTIM-PG complex is the first example of a TIM-ligand complex being observed in both loop open and closed forms. In the C2 form (loop closed), Phe96 and Leu167 adopt alternative conformations that are different from the ones observed in the open form, permitting loop closure. These structures provide strong support for the view that loop closure is not essential for ligand binding and that dynamic loop movement may occur in both free and ligand-bound forms of the enzyme.     

Conformational analysis of cyclolinopeptide A, a cyclic nonapeptide: nuclear Overhauser effect and energy minimization studies

The conformation of cyclolinopeptide A [cyclo(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val)], a naturally occurring cyclic nonapeptide has been investigated in dimethylsulfoxide solution by 270 MHz 1H-nmr. A complete assignment of all C alpha H and NH resonances has been accomplished using two-dimensional correlated spectroscopy and nuclear Overhauser effects (NOEs). Analysis of interresidue NOEs and JHNC alpha H values permit construction of a molecular model for the cyclic peptide backbone. The crude model derived from nmr has been used as a starting point for energy minimization, which yields a refined structure largely compatible with nmr observations. The major features of the conformation of cyclolinopeptide A are a Type VI beta-turn centered at Pro(1)-Pro(2), with a cis peptide bond between these residues and a gamma-turn (C7 structure) centered at Ile(6). Two intramolecular hydrogen bonds Val(9) CO--Phe(3)NH (4----1) and Leu(5) CO--Ile(7)NH (3----1) are observed in the low-energy conformation. The limited solvent accessibility observed for the Val(9) and Leu(5) NH groups in the nmr studies are rationalized in terms of steric shielding.     

Peptide design: influence of a guest Aib-Pro segment on the stereochemistry of an oligo-val sequence--solution conformations and crystal structure of Boc-(Val)2-Aib-Pro-(Val)3-OMe

The peptide Boc-Val-Val-Aib-Pro-Val-Val-Val-OMe has been synthesized to investigate the effect of introduction of a strong beta-turn promoting guest segment into an oligopeptide with a tendency to form extended structures. 1H-nmr studies in solution using analysis of NH group solvent accessibility and nuclear Overhauser effects suggest an appreciable solvent dependence of conformations. In chloroform a 3(10)-helical structure is favored, while in dimethylsulfoxide an Aib-Pro beta-turn with extended arms on either side is suggested. In the crystal, the backbone forms a somewhat distorted 3(10)-helix despite the presence of a Pro residue in the middle. Among the four possible intrahelical hydrogen bonds three are of the 4----1 type and one 5----1. Head-to-tail NH...O = C hydrogen bonds link the helical molecules into continuous columns. The space group is P2(1)2(1)2(1) a = 11.320(2), b = 19.889(3), and c = 21.247(3) A.     

Ionophore-mediated transmembrane movement of divalent cations in small unilamellar liposomes: An evaluation of the chlortetracycline fluorescence technique and correlations with black lipid membrane studies

Summary Conceptual advances in the field of membrane transport have, in the main, utilized artificial membranes, both planar and vesicular. Systems of biological interest,viz., cells and organelles, resemble vesicles in size and geometry. Methods are, therefore, required to extend the results obtained with planar membranes to liposome systems. In this report we present an analysis of a fluorescence technique, using the divalent cation probe chlortetracycline, in small, unilamellar vesicles, for the study of divalent cation fluxes. An ion carrier (X537 A) and a pore former (alamethicin) have been studied. The rate of rise of fluorescence signal and the transmembrane ion gradient have been related to transmembrane current and potential, respectively. A second power dependence of ion conduction-including the electrically silent portion thereof — on X537 A concentration, has been observed. An exponential dependence of current on transmembrane potential in the case of alamethicin is also confirmed. Possible errors in the technique are discussed.     

X-Pro peptides. A theoretical study of the hydrogen bonded conformations of (α-aminoisobutyryl-l-prolyl)n sequences

Intramolecularly hydrogen bonded conformations of (Aib-Pro)_n sequences have been analysed theoretically. Both 4→1 (C₁₀ and 3→1 (C₇ hydrogen bonded regular structures are shown to be stereochemically feasible. Conformational energies for the helical structures have been estimated using classical potential energy methods. Both C₁₀ and C₇ conformations have very similar energies. Pyrrolidine ring puckering has a pronounced effect on the energies, and only Cγ-endo puckered Pro residues can be accommodated. The theoretical calculations using spectroscopic data suggest that the recently proposed novel 3₁₀ helical conformation for benzyloxycarbonyl(Aib-Pro)₄-methyl ester is in solution, is indeed energetically and stereochemically favourable.     

Employing a Crude Toxin Preparation from Sarocladium oryzae as a Molecular Sieve to Select Sheath Rot-resistant Somaclones of Rice

This study was undertaken to explore the possibilities of using in vitro techniques for selection of sheath rot-resistant soma clones of rice cv Co43. Oryza nivara and O. longistaminata possessing sheath rot-resistance were included in the study, with a view to comparing the reaction of the susceptible somaclones of O. Sativa cv Co43 to a crude toxin preparation from culture filtrate of the pathogenic fungus, Sarocladium oryzae. A few somaclones of Co43, selecfed on the crude toxin preparation, regenerated into plantlets and their reaction to the toxin was reconfirmed up to R₁ generation.     

Plasmodium falciparum hypoxanthine guanine phosphoribosyltransferase. Stability studies on the product-activated enzyme

Hypoxanthine guanine phosphoribosyltransferases (HGPRTs) catalyze the conversion of 6-oxopurine bases to their respective nucleotides, the phosphoribosyl group being derived from phosphoribosyl pyrophosphate. Recombinant Plasmodium falciparum HGPRT, on purification, has negligible activity, and previous reports have shown that high activities can be achieved upon incubation of recombinant enzyme with the substrates hypoxanthine and phosphoribosyl pyrophosphate [Keough DT, Ng AL, Winzor DJ, Emmerson BT & de Jersey J (1999) Mol Biochem Parasitol98, 29-41; Sujay Subbayya IN & Balaram H (2000) Biochem Biophys Res Commun279, 433-437]. In this report, we show that activation is effected by the product, Inosine monophosphate (IMP), and not by the substrates. Studies carried out on Plasmodium falciparum HGPRT and on a temperature-sensitive mutant, L44F, show that the enzymes are destabilized in the presence of the substrates and the product, IMP. These stability studies suggest that the active, product-bound form of the enzyme is less stable than the ligand-free, unactivated enzyme. Equilibrium isothermal-unfolding studies indicate that the active form is destabilized by 2-3 kcal x mol(-1) compared with the unactivated state. This presents a unique example of an enzyme that attains its active conformation of lower stability by product binding. This property of ligand-mediated activation is not seen with recombinant human HGPRT, which is highly active in the unliganded state. The reversibility between highly active and weakly active states suggests a novel mechanism for the regulation of enzyme activity in P. falciparum.