Cooperative effects in hydrogen-bonding of protein secondary structure elements: a systematic analysis of crystal data using Secbase

A systematic analysis of the hydrogen-bonding geometry in helices and beta sheets has been performed. The distances and angles between the backbone carbonyl O and amide N atoms were correlated considering more than 1500 protein chains in crystal structures determined to a resolution better than 1.5 A. They reveal statistically significant trends in the H-bond geometry across the different secondary structural elements. The analysis has been performed using Secbase, a modular extension of Relibase (Receptor Ligand Database) which integrates information about secondary structural elements assigned to individual protein structures with the various search facilities implemented into Relibase. A comparison of the mean hydrogen-bond distances in alpha helices and 3(10) helices of increasing length shows opposing trends. Whereas in alpha helices the mean H-bond distance shrinks with increasing helix length and turn number, the corresponding mean dimension in 3(10) helices expands in a comparable series. Comparing similarly the hydrogen-bond lengths in beta sheets there is no difference to be found between the mean H-bond length in antiparallel and parallel beta sheets along the strand direction. In contrast, an interesting systematic trend appears to be given for the hydrogen bonds perpendicular to the strands bridging across an extended sheet. With increasing number of accumulated strands, which results in a growing number of back-to-back piling hydrogen bonds across the strands, a slight decrease of the mean H-bond distance is apparent in parallel beta sheets whereas such trends are obviously not given in antiparallel beta sheets. This observation suggests that cooperative effects mutually polarizing spatially well-aligned hydrogen bonds are present either in alpha helices and parallel beta sheets whereas such influences seem to be lacking in 3(10) helices and antiparallel beta sheets.     

Crystal structure of a purine/pyrimidine phosphoribosyltransferase-related protein from Thermus thermophilus HB8

Adenine phosphoribosyltransferase (APRTase) is a widely distributed enzyme involved in the salvage of adenine to form an adenine nucleotide. We crystallized and determined the X-ray crystallographic structure of a purine/pyrimidine phosphoribosyltransferase-related protein from the thermophilic bacterium, Thermus thermophilus HB8. The crystal space group was C2 with unit cell dimensions of a = 167.42 A, b = 61.41 A, c = 102.39 A, beta = 94.0 degrees . Initial phases were determined to 2.6 A using the multiple wavelength anomalous dispersion method and selenomethionine substituted protein (Se-MAD), and refined using a 1.9 A "native" data set. The asymmetric unit contains two pairs of identical dimers, each related by noncrystallographic two-fold symmetry. The fifth monomer forms a similar dimer across a crystallographic two-fold axis. These dimers appear to be the biological unit with both monomers contributing to an unusual highly charged arginine-rich bridge region separating the two active sites. Comparison with distantly related APRTases reveal similarities and differences of the active site.     

Candidate nsSNPs that can affect the functions and interactions of cell cycle proteins

Nonsynonymous single nucleotide polymorphisms (nsSNPs) alter the encoded amino acid sequence, and are thus likely to affect the function of the proteins, and represent potential disease-modifiers. There is an enormous number of nsSNPs in the human population, and the major challenge lies in distinguishing the functionally significant and potentially disease-related ones from the rest. In this study, we analyzed the genetic variations that can alter the functions and the interactions of a group of cell cycle proteins (n = 60) and the proteins interacting with them (n = 26) using computational tools. As a result, we extracted 249 nsSNPs from 77 cell cycle proteins and their interaction partners from public SNP databases. Only 31 (12.4%) of the nsSNPs were validated. The majority (64.5%) of the validated SNPs were rare (minor allele frequencies < 5%). Evolutionary conservation analysis using the SIFT tool suggested that 16.1% of the validated nsSNPs may disrupt the protein function. In addition, 58% of the validated nsSNPs were located in functional protein domains/motifs, which together with the evolutionary conservation analysis enabled us to infer possible biological consequences of the nsSNPs in our set. Our study strongly suggests the presence of naturally occurring genetic variations in the cell cycle proteins that may affect their interactions and functions with possible roles in complex human diseases, such as cancer.     

Poly-(L-alanine) expansions form core beta-sheets that nucleate amyloid assembly

Expansion to a total of 11-17 sequential alanine residues from the normal number of 10 in the polyadenine-binding protein nuclear-1 (PABPN1) results in formation of intranuclear, fibrillar inclusions in skeletal muscle and hypothalamic neurons in adult-onset, dominantly inherited oculopharyngeal muscular dystrophy (OPMD). To understand the role that homopolymeric length may play in the protein misfolding that leads to the inclusions, we analyzed the self-assembly of synthetic poly-(L-alanine) peptides having 3-20 residues. We found that the conformational transition and structure of polyalanine (polyAla) assemblies in solution are not only length-dependent but also are determined by concentration, temperature, and incubation time. No beta-sheet complex was detected for those peptides characterized by n < 8, where n is number of alanine residues. A second group of peptides with 7 < n < 15 showed varying levels of complex formation, while for those peptides having n > 15, the interconversion process from the monomeric to the beta-sheet complex was complete under any of the tested experimental conditions. Unlike the typical tinctorial properties of amyloid fibrils, polyalanine fibrils did not show fluorescence with thioflavin T or apple-green birefringence with Congo red; however, like amyloid, X-ray diffraction showed that the peptide chains in these fibrils were oriented normal to the fibril axis (i.e., in the cross-beta arrangement). Neighboring beta-sheets are quarter-staggered in the hydrogen-bonding direction such that the alanine side-chains were closely packed in the intersheet space. Strong van der Waals contacts between side-chains in this arrangement likely account for the high stability of the macromolecular fibrillar complex in solution over a wide range of temperature (5-85 degrees C), and pH (2-10.5), and its resistance to denaturant (< 8 M urea) and to proteases (protease K, trypsin). We postulate that a similar stabilization of an expanded polyalanine stretch could form a core beta-sheet structure that mediates the intermolecular association of mutant proteins into fibrillar inclusions in human pathologies.     

Common binding site for disialyllactose and tri-peptide in C-fragment of tetanus neurotoxin

Clostridial neurotoxins are comprised of botulinum (BoNT) and tetanus (TeNT), which share significant structural and functional similarity. Crystal structures of the binding domain of TeNT complexed with disialyllactose (DiSia) and a tri-peptide Tyr-Glu-Trp (YEW) have been determined to 2.3 and 2.2 A, respectively. Both DiSia and YEW bind in a shallow cleft region on the surface of the molecule in the beta-trefoil domain, interacting with a set of common residues, Asp1147, Asp1214, Asn1216, and Arg1226. DiSia and YEW binding at the same site in tetanus toxin provides a putative site that could be occupied either by a ganglioside moiety or a peptide. Soaking experiments with a mixture of YEW and DiSia show that YEW competes with DiSia, suggesting that YEW can be used to block ganglioside binding. A comparison with the TeNT binding domain in complex with small molecules, BoNT/A and /B, provides insight into the different modes of ganglioside binding.     

Structure of the tetrameric form of human L-Xylulose reductase: probing the inhibitor-binding site with molecular modeling and site-directed mutagenesis

L-Xylulose reductase (XR) is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. In this study we report the structure of the biological tetramer of human XR in complex with NADP(+) and a competitive inhibitor solved at 2.3 A resolution. A single subunit of human XR is formed by a centrally positioned, seven-stranded, parallel beta-sheet surrounded on either side by two arrays of three alpha-helices. Two helices located away from the main body of the protein form the variable substrate-binding cleft, while the dinucleotide coenzyme-binding motif is formed by a classical Rossmann fold. The tetrameric structure of XR, which is held together via salt bridges formed by the guanidino group of Arg203 from one monomer and the carboxylate group of the C-terminal residue Cys244 from the neighboring monomer, explains the ability of human XR to prevent the cold inactivation seen in the rodent forms of the enzyme. The orientations of Arg203 and Cys244 are maintained by a network of hydrogen bonds and main-chain interactions of Gln137, Glu238, Phe241, and Trp242. These interactions are similar to those defining the quaternary structure of the closely related carbonyl reductase from mouse lung. Molecular modeling and site-directed mutagenesis identified the active site residues His146 and Trp191 as forming essential contacts with inhibitors of XR. These results could provide a structural basis in the design of potent and specific inhibitors for human XR.     

Crystal structure and structural stability of acylphosphatase from hyperthermophilic archaeon Pyrococcus horikoshii OT3

To elucidate the structural basis for the high stability of acylphosphatase (AcP) from Pyrococcus horikoshii OT3, we determined its crystal structure at 1.72 A resolution. P. horikoshii AcP possesses high stability despite its approximately 30% sequence identity with eukaryotic enzymes that have moderate thermostability. The overall fold of P. horikoshii AcP was very similar to the structures of eukaryotic counterparts. The crystal structure of P. horikoshii AcP shows the same fold betaalphabetabetaalphabeta topology and the conserved putative catalytic residues as observed in eukaryotic enzymes. Comparison with the crystal structure of bovine common-type AcP and that of D. melanogaster AcP (AcPDro2) as representative of eukaryotic AcP revealed some significant characteristics in P. horikoshii AcP that likely play important roles in structural stability: (1) shortening of the flexible N-terminal region and long loop; (2) an increased number of ion pairs on the protein surface; (3) stabilization of the loop structure by hydrogen bonds. In P. horikoshii AcP, two ion pair networks were observed one located in the loop structure positioned near the C-terminus, and other on the beta-sheet. The importance of ion pairs for structural stability was confirmed by site-directed mutation and denaturation induced by guanidium chloride.     

Cobalt complexes with cinchonidine and quinidine: effect of C8/C9 stereochemistry and 6'-substitution on intermolecular interactions

The title compounds, (cinchonidinium)trichlorocobalt(II) [(C(19)H(23)ON(2))CoCl(3)] (CoCdn) and (quinidinium)trichlorocobalt(II) [(C(20)H(25)O(2)N(2))CoCl(3)] (CoQd), are zwitterions that differ in absolute configuration and conformation. In both complexes, the sp(3) nitrogen of quinuclidine is protonated, whereas the sp(2) nitrogen of quinoline is linked to the Co(II) atom, which coordinates three chlorine atoms in distorted tetrahedral geometry. The mutual orientations of the quinoline and quinuclidine moieties in CoCdn and CoQd differ significantly because of different hydrogen bonding involving the hydroxyl group. In both complexes, the quinuclidine NH groups and hydroxyl groups are hydrogen-bond donors to the chlorine atoms of Co(II) tetrahedra. In CoQd the hydrogen bonding leads to formation of a nine-membered ring consisting of Co, two chlorines, and a fragment of the quinidine molecule. A comparison of the crystal structures of four Cinchona alkaloid complexes with trichlorocobalt(II) shows that their space groups are determined by the absolute configuration of the alkaloid, whereas the hydrogen-bonding pattern is mainly affected by the substituent in the quinoline ring, i.e., by hydrogen or methoxyl group.     

Preparative enantiomeric separation of new aromatase inhibitors by HPLC on polysaccharide-based chiral stationary phases: Determination of enantiomeric purity and assignment of absolute stereochemistry by X-ray structure analysis

The development of high-performance liquid chromatography methods on polysaccharide-based stationary phases (cellulose or amylose derivatives) has permitted preparative enantioseparations of various 6-[1(imidazol-1-yl)-1-phenylmethyl]-3-methyl-1,3-benzoxazol-2(3H)-one and 6-[1(imidazol-1-yl)-1-phenylmethyl]-3-methyl-1,3-benzothiazol-2(3H)-one, aromatase inhibitors, with satisfactory yields. Analytical enantioseparation methods using both UV and evaporative light-scattering detection (ELSD) were validated to determine the enantiomeric purity of these compounds. Using UV detection, linear calibration curves in the range from 4 x 10(-6) to 4.8 x 10(-4) M range were obtained; repeatability, limits of detection (LD), and quantification (LQ) were determined: LD varied, for the various solutes, from 1 to 80 microg/l and from 2.05 to 10.05 mg/l with UV detection and ELSD, respectively. Single-crystal X-ray analysis was successful in determining the absolute configuration of the individual enantiomers. The relationship between retention order and absolute configuration of the enantiomers was established.