Conformational characteristics of peptides and unanticipated results from crystal structure analyses

Preferred conformation and types of molecular folding are some of the topics that can be addressed by structure analysis using x-ray diffraction of single crystals. The conformations of small linear peptide molecules with 2-6 residues are affected by polarity of solvent, presence of water molecules, hydrogen bonding with neighboring molecules, and other packing forces. Larger peptides, both cyclic and linear, have many intramolecular hydrogen bonds, the effect of which outweighs any intermolecular attractions. Numerous polymorphs of decapeptides grown from a variety of solvents, with different cocrystallized solvents, show a constant conformation for each peptide. Large conformational changes occur, however, upon complexation with metal ions. A new form of free valinomycin grown from DMSO exhibits near three-fold symmetry with only three intramolecular hydrogen bonds. The peptide is in the form of a shallow bowl with a hydrophobic exterior. Near the bottom of the interior of the bowl are three carbonyl oxygens, spaced and directed so that they are in position to form three ligands to a K+, e.g., complexation can be completed by the three lobes containing the beta-bends closing over and encapsulating the K+ ion. In another example, free antamanide and the biologically inactive perhydro analogue, in which four phenyl groups become cyclic hexyl groups, have essentially the same folding of backbone and side chains. The conformation changes drastically upon complexation with Li+ or Na+. However, the metal ion complex of natural antamanide has a hydrophobic globlar form whereas the metal ion complex of the inactive perhydro analogue has a polar band around the middle. The structure results indicate that the antamanide molecule is in a complexed form during its biological activity. Single crystal x-ray diffraction structure analyses have identified the manner in which water molecules are essential to creating minipolar areas on apolar helices. Completely apolar peptides, such as membrane-active peptides, can acquire amphiphilic character by insertion of a water molecule into the helical backbone of Boc-Aib-Ala-Leu-Aib-Ala-Leu-Aib-Ala-Leu-Aib-OMe, for example. The C-terminal half assumes an alpha-helix conformation, whereas the N-terminal half is distorted by an insertion of a water molecule W(1) between N(Ala5) and O(Ala2), forming hydrogen bonds N(5)H...W(1) and W(1)...O(2). The distortion of the helix exposes C = O(Aib1) and C = O(Aib4) to the outside environment with the consequence of attracting additional water molecules. The leucyl side chains are on the other side of the molecule. Thus a helix with an apolar sequence can mimic an amphiphilic helix.     

Mechanism of calcite crystal growth inhibition by the N-terminal undecapeptide of lithostathine

Pancreatic juice is supersaturated with calcium carbonate. Calcite crystals therefore may occur, obstruct pancreatic ducts, and finally cause a lithiasis. Human lithostathine, a protein synthesized by the pancreas, inhibits the growth of calcite crystals by inducing a habit modification: the rhombohedral (10 14) usual habit is transformed into a needle-like habit through the (11 0) crystal form. A similar observation was made with the N-terminal undecapeptide (pE(1)R(11)) of lithostathine. We therefore aimed at discovering how peptides inhibit calcium salt crystal growth. We solved the complete x-ray structure of lithostathine, including the flexible N-terminal domain, at 1.3 A. Docking studies of pE(1)R(11) with the (10 14) and (11 0) faces through molecular dynamics simulation resulted in three successive steps. First, the undecapeptide progressively unfolded as it approached the calcite surface. Second, mobile lateral chains of amino acids made hydrogen bonds with the calcite surface. Last, electrostatic bonds between calcium ions and peptide bonds stabilized and anchored pE(1)R(11) on the crystal surface. pE(1)R(11)-calcite interaction was stronger with the (11 0) face than with the (10 14) face, confirming earlier experimental observations. Energy contributions showed that the peptide backbone governed the binding more than did the lateral chains. The ability of peptides to inhibit crystal growth is therefore essentially based on backbone flexibility.     

The occurrence of a glycine cleavage system in mammalian brain

A major catabolic route for glycine in liver is its conversion to methylene tetra-hydrofolate (methylene THF), CO₂ and NH₃, catalysed by the glycine cleavage system described by Yoshida and Kikuchi (1970). In view of the role of glycine as a putative neuro-transmitter, the occurrence of this system in mammalian brain was investigated. Our studies demonstrated: (a) that the production of ¹⁴CO₂ from [1-¹⁴C]glycine required the presence of tetrahydrofolate, NAD, dithiothreitol and pyridoxal phosphate in the reaction mixture; (b) that besides CO₂l -serine was the other major product of the reaction; (c) that glyoxylate did not function as an intermediate in this reaction; and (d) that enzymatic activity appeared to be associated with membranes. All of these properties resembled those previously described for the hepatic system.     

The occurrence of glycine in bacterial lipopolysaccharides

Abstract The aminoacyl analysis of endotoxic lipopolysaccharides (LPS) isolated from several bacteria revealed essential amounts of glycine, among the inherent LPS components. Significant amounts of the glycine was detected in lipopolysaccharides isolated from over 30 strains of Escherichia, Salmonella, Hafnia, Citrobacter and Shigella species. Glycine as a single amino acid was found only in a core part of LPS. Molar ratio of glycine in core oligosaccharide fraction ranged from 0.2 to 0.6 per 3 heptoses. The oligosaccharide enriched in glycine was isolated using the HPLC. The amino acid appeared to be terminally located in a core oligosaccharide. The labelling of the lipopolysaccharide cores was achieved when the bacteria were cultivated in the presence of radioactive [¹⁴C]glycine. The labelled core oligosaccharide released the radioactivity during treatment with mild alkali or acid (0.1 M NaOH or HCl, 100°C, 4 h). The radioactivity in SDS-polyacrylamide gel electrophoresis migrated exclusively with LPS. The results indicate that amino acid is an integral constituent of core oligosaccharide in lipopolysaccharide.     

The 1.8 A crystal structure of catechol 1,2-dioxygenase reveals a novel hydrophobic helical zipper as a subunit linker

BACKGROUND: Intradiol dioxygenases catalyze the critical ring-cleavage step in the conversion of catecholate derivatives to citric acid cycle intermediates. Catechol 1,2-dioxygenases (1, 2-CTDs) have a rudimentary design structure - a homodimer with one catalytic non-heme ferric ion per monomer, that is (alphaFe(3+))(2). This is in contrast to the archetypical intradiol dioxygenase protocatechuate 3,4-dioxygenase (3,4-PCD), which forms more diverse oligomers, such as (alphabetaFe(3+))(2-12). RESULTS: The crystal structure of 1,2-CTD from Acinetobacter sp. ADP1 (Ac 1,2-CTD) was solved by single isomorphous replacement and refined to 2.0 A resolution. The structures of the enzyme complexed with catechol and 4-methylcatechol were also determined at resolutions of 1.9 A and 1.8 A, respectively. While the characteristics of the iron ligands are similar, Ac 1,2-CTD differs from 3,4-PCDs in that only one subunit is used to fashion each active-site cavity. In addition, a novel 'helical zipper', consisting of five N-terminal helices from each subunit, forms the molecular dimer axis. Two phospholipids were unexpectedly found to bind within an 8 x 35 A hydrophobic tunnel along this axis. CONCLUSIONS: The helical zipper domain of Ac 1, 2-CTD has no equivalent in other proteins of known structure. Sequence analysis suggests the domain is a common motif in all members of the 1,2-CTD family. Complexes with catechol and 4-methylcatechol are the highest resolution complex structures to date of an intradiol dioxygenase. Furthermore, they confirm several observations seen in 3,4-PCDs, including ligand displacement upon binding exogenous ligands. The structures presented here are the first of a new family of intradiol dioxygenases.     

The crystal structure of the ligand binding module of axonin-1/TAG-1 suggests a zipper mechanism for neural cell adhesion

We have determined the crystal structure of the ligand binding fragment of the neural cell adhesion molecule axonin-1/TAG-1 comprising the first four immunoglobulin (Ig) domains. The overall structure of axonin-1(Ig1-4) is U-shaped due to contacts between domains 1 and 4 and domains 2 and 3. In the crystals, these molecules are aligned in a string with adjacent molecules oriented in an anti-parallel fashion and their C termini perpendicular to the string. This arrangement suggests that cell adhesion by homophilic axonin-1 interaction occurs by the formation of a linear zipper-like array in which the axonin-1 molecules are alternately provided by the two apposed membranes. In accordance with this model, mutations in a loop critical for the formation of the zipper resulted in the loss of the homophilic binding capacity of axonin-1.     

Observation of an arsenic adduct in an acetyl esterase crystal structure

The crystal structures of an acetyl esterase, HerE, and its complex with an inhibitor dimethylarsinic acid have been determined at 1.30- and 1.45-A resolution, respectively. Although the natural substrate for the enzyme is unknown, HerE hydrolyzes the acetyl groups from heroin to yield morphine and from phenyl acetate to yield phenol. Recently, the activity of the enzyme toward heroin has been exploited to develop a heroin biosensor, which affords higher sensitivity than other currently available detection methods. The crystal structure reveals a single domain with the canonical alpha/beta hydrolase fold with an acyl binding pocket that snugly accommodates the acetyl substituent of the substrate and three backbone amides that form a tripartite oxyanion hole. In addition, a covalent adduct was observed between the active site serine and dimethylarsinic acid, which inhibits the enzyme. This crystal structure provides the first example of an As-containing compound in a serine esterase active site and the first example of covalent modification of serine by arsenic. Thus, the HerE complex reveals the structural basis for the broad scope inhibition of serine hydrolases by As(V)-containing organic compounds.     

2.6 A resolution crystal structure of helices of the motile major sperm protein (MSP) of Caenorhabditis elegans

The amoeboid locomotion of nematode sperm is mediated by the assembly dynamics of the major sperm protein (MSP). MSP forms fibrous networks based on a hierarchy of macromolecular assemblies: helical subfilaments are built from MSP dimers; filaments are formed from two subfilaments coiling round one another; and filaments themselves supercoil to produce bundles. To provide a structural context for understanding the role of these macromolecular assemblies in cell locomotion, we have determined the 2.6 A resolution structure of crystals of Caenorhabditis elegans MSP that are constructed from helices of MSP chains that are analogous to the subfilaments from which filaments are constructed. Comparison with the crystal structures of dimers and helical assemblies of Ascaris suum MSP has identified five conserved interaction interfaces that suggest how subfilaments interact in filaments and how filaments can form bundles. The interfaces frequently involve the loop containing residues 78-85, which is divergent between MSP homologues, and the loop containing residues 98-103, which is highly conserved.     

Platinum-assisted preparation of PS ligands containing chiral centres; X-ray crystal structure of a dithiophosphinite platinum complex

The design and synthesis of some ligands containing P(III) bonded to sulfur (thiophosphinites) and chiral centres is described in this paper.Their complexes with platinum (II), [PtCl₂L], (L = bidentate dithiophosphinite) have been prepared and characterised and it has been shown that in many cases, the coordination to platinum protects these ligands from decomposition processes operated by moisture and oxygen. The first example of X-ray crystal structure of a platinum coordinated dithiophosphinite is described for complex cis-[PtCl₂L], [L = meso-2,3-bis(diphenylthiophosphinito)-dimethyl-succinate], 4a.     

The crystal structure of the cis-proline to glycine variant (P114G) of ribonuclease A

Replacement of a cis-proline by glycine at position 114 in ribonuclease A leads to a large decrease in thermal stability and simplifies the refolding kinetics. A crystallographic approach was used to determine whether the decrease in thermal stability results from the presence of a cis glycine peptide bond, or from a localized structural rearrangement caused by the isomerization of the mutated cis 114 peptide bond. The structure was solved at 2.0 A resolution and refined to an R-factor of 19.5% and an R(free) of 21.9%. The overall conformation of the protein was similar to that of wild-type ribonuclease A; however, there was a large localized rearrangement of the mutated loop (residues 110-117-a 9.3 A shift of the Calpha atom of residue 114). The peptide bond before Gly114 is in the trans configuration. Interestingly, a large anomalous difference density was found near residue 114, and was attributed to a bound cesium ion present in the crystallization experiment. The trans isomeric configuration of the peptide bond in the folded state of this mutant is consistent with the refolding kinetics previously reported, and the associated protein conformational change provides an explanation for the decreased thermal stability.     

Symmetry and structure of RNA and DNA triple helices

Despite wide interest in nucleic acid triple helices, there has beenno stereochemically satisfactory structure of an RNA triple helixin atomic detail. An RNA triplex structure has previously been proposed based on fiber diffraction and molecular modeling [S. Arnott and P. J. Bond (1973) Nature New Biology, Vol. 244. pp. 99–101; S. Arnott. P. J. Bond. E. Seising, and P. J. C. Smith (1976) Nucleic Acids Research, Vol. 3. pp.2459–2470], but it has nonallowed close contacts at every triplet and is therefore not stereochemically acceptable. We propose here a new modelfor an RNA triple helix in which the three chains have identical backbone conformations and are symmetry related. There are no short contacts. The modeling employs a novel geometrical approach using the linked atom least squares [P. J. C. Smith and S. Arnott (1978) Acta Crystallographica, Vol. A34, pp. 3–11] program and is not based on energy minimization. In general, the method leads to a range of possible structures rather than a unique structure. In the present case, however, the constraints resulting from theintroduction of a third strand limit the possible structures to a very small range of conformation space. This method was used previously to obtain a model for DNA triple helices [G. Raghunathan, H. T. Miles, and V. Sasisekharan (1993) Biochemistry, Vol. 32, pp. 455–462], subsequently confirmed by fiber-type x-ray diffraction of oligomeric crystals [K. Liu. H. T. Miles. K. D. Parris, and V. Sasisekharan (1994) Nature Structural Biology, Vol. 1. pp. 11–12]. The above triple helices have Watson–Crick–Hoogsteen [K. Hoogsteen (1963) Acta Crystallographica, Vol. 16. pp. 907–916] pairing of the three bases. The same modeling method was used to investigate the feasibility of three-dimensional structures based on the three possible alternative hydrogen-bonding schemes: Watson–Crick–reverse Hoogsteen, Donogue [J. Donohue (1953) Proceeding of the national Academy of Science USA, Vol. 39, pp. 470–475] (reverse Watson–Crick)–Hoogsteen, and Donohue–reverse Hoogsteen. We found that none of these can occur in either RNA or DNA helices because they give rise only to structures with prohibitively short contacts between backbone and base atoms in the same chain. © 1995 John Wiley & Sons, Inc.     

Parallel double stranded helices and the tertiary structure of nucleic acids

Thermal denaturation of four oligonucleotides, viz. 3'-d(AT)5pO(CH2)6Opd(AT)5-3'(par(AT], 3'-d(AT)5pO(CH2)6Opd(AT)5-5'(anti(AT],3'-d(A)10pO(CH2) 6Op(T)10-3'(par(A-T], and 3'-d(A)10pO(CH2)6Opd(T)10-5' (anti(A-T], was studied in 0.01 M phosphate buffer, pH 7, in the presence of 0.1, 0.25, 0.5 and 1.0 M NaCl. All the oligomers were found to exist at a lower temperature (0 to 20 degrees C) as complexes composed either of two oligomer molecules (a canonical duplex) or of more oligomer molecules whereas, at a higher temperature (30 to 70 degrees C), they formed hairpins with a parallel (par(AT) and par(A-T] or antiparallel (anti(AT) and anti(A-T) orientation of the chains. Melting curves (A260(T] were used to calculate thermodynamic parameters for the formation of hairpins and "low-temperature" duplexes. Experiments on ethidium bromide binding to the oligonucleotides have shown that the oligomer anti(A-T) exists, at a low ionic strength, as a four stranded complex ("quadruplex") contains two antiparallel helices, d(A).d(T), which have a parallel orientation and are bound to one another owing to the formation of additional hydrogen bonds between nucleic acid bases. The possible biological function of quadruplexes is discussed.     

The occurrence of a multilayered structure in the sperm of a pteridophyte

Summary A multilayered structure, previously recorded only in bryophytes, is reported in spermatids of three species of Equisetum. It is interpreted as comprising four layers, recalling the Vierergruppe of Marchantia spermatids. Unlike the multilayered structure of bryophyte spermatids, a rather compact organelle, that of Equisetum forms a thin strip extending almost the whole diameter of the cell. As in bryophytes the upper layer of the Equisetum multilayered structure is composed of parallel microtubules, extending far beyond the underlying layers. The microtubular band is considered equivalent to the similar structure seen in a variety of plant spermatozoids, but in Equisetum it is much longer and composed of many more microtubules than reported from any other plant spermatozoid. The morphology of the multilayered structure is related to the large size and short, broad form of Equisetum spermatozoids.     

Self‐assembly and chiral recognition of quartz crystal microbalance chiral sensor

A novel chiral sensor based on the self‐assembled monolayer of (6^A‐ω‐mercaptoethylureado‐6^A‐deoxy)heptakis(2,3‐di‐o‐phenylcarbamoyl)‐6^B, 6^C, 6^D, 6^E, 6^F, 6^G‐ hexa‐o‐phenylcarbamoyl‐β‐cyclodextrin (Ph‐β‐CD‐SH) on a quartz crystal transducer for chiral recognition was set up. (R,S)‐(±)‐(3‐Methoxyphenyl)ethylamine were recognized by this QCM chiral sensor with a QCM chiral discrimination factor of 1.33. Furthermore, UV spectroscopy was used to investigate the mechanism of host‐guest interactions between (6^A‐azido‐6^A‐deoxy)heptakis(2,3‐di‐o‐phenylcarbamoyl)‐6^B, 6^C, 6^D, 6^E, 6^F, 6^G‐hexa‐o‐phenylcarbamoyl‐β‐cyclodextrin (Ph‐β‐CD) and (R,S)‐(±)‐(3‐methoxyphenyl) ethylamine. The UV discrimination factor was determined to be 0.066. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Effects of glycine substitutions on the structure and function of gramicidin a channels

Tryptophan residues often are found at the lipid-aqueous interface region of membrane-spanning proteins, including ion channels, where they are thought to be important determinants of protein structure and function. To better understand how Trp residues modulate the function of membrane-spanning channels, we have examined the effects of Trp replacements on the structure and function of gramicidin A channels. Analogues of gramicidin A in which the Trp residues at positions 9, 11, 13, and 15 were sequentially replaced with Gly were synthesized, and the three-dimensional structure of each analogue was determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. Though Trp --> Gly substitutions destabilize the beta6.3-helical gA channel structure, it is possible to determine the structure of analogues with Trp --> Gly substitutions at positions 11, 13, and 15, but not for the analogue with the Trp --> Gly substitution at position 9. The Gly11-, Gly13-, and Gly15-gA analogues form channels that adopt a backbone fold identical to that of native gramicidin A, with only small changes in the side chain conformations of the unsubstituted residues. Single-channel current measurements show that the channel function and lifetime of the analogues are significantly affected by the Trp --> Gly replacements. The conductance variations appear to be caused by sequential removal of the Trp dipoles, which alter the ion-dipole interactions that modulate ion movement. The lifetime variations did not appear to follow a clear pattern.     

Packing of transmembrane helices in bacteriorhodopsin folding: structure and thermodynamics

We propose a coarse-grained (CG) model to study the native structure and physical properties of helical membrane proteins (HMPs) using off-lattice computer simulations. Instead of considering sequence heterogeneity explicitly, we model its effect on the packing of helices by employing a mean packing parameter r(0), which is calculated from an all-atom (AA) model. Specifically, this CG model is applied to investigate the packing of helices in bacteriorhodopsin (BR), and predicts the seven helix bundle structure of BR with a root mean square deviation (RMSD) in coordinates of helix backbone atoms (N, C, C(alpha)) of 3.99 A from its crystal structure. This predicted structure is further refined in an AA model by Amber and the refined structure has a RMSD (in coordinates of helix backbone atoms) of 2.64 A. The predicted packing position, tilting angle, and orientation angle of each helix in the refined structure are consistent with experimental data and their physical origins can be well understood in our model. Our results show that a reasonably good structure of BR can be predicted by using such a dual-scale approach, provided that its secondary structure is known. Starting from a random initial configuration, the folded structure can be obtained in days using a regular desktop computer. Various thermodynamic properties of helix packing of BR are also investigated in this CG model.