Structure-based inhibitors of tau aggregation.

Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.

Automatic detection of lung nodules in CT datasets based on stable 3D mass-spring models.

We propose a computer-aided detection (CAD) system which can detect small-sized (from 3mm) pulmonary nodules in spiral CT scans. A pulmonary nodule is a small lesion in the lungs, round-shaped (parenchymal nodule) or worm-shaped (juxtapleural nodule). Both kinds of lesions have a radio-density greater than lung parenchyma, thus appearing white on the images. Lung nodules might indicate a lung cancer and their early stage detection arguably improves the patient survival rate. CT is considered to be the most accurate imaging modality for nodule detection. However, the large amount of data per examination makes the full analysis difficult, leading to omission of nodules by the radiologist. We developed an advanced computerized method for the automatic detection of internal and juxtapleural nodules on low-dose and thin-slice lung CT scan. This method consists of an initial selection of nodule candidates list, the segmentation of each candidate nodule and the classification of the features computed for each segmented nodule candidate.The presented CAD system is aimed to reduce the number of omissions and to decrease the radiologist scan examination time. Our system locates with the same scheme both internal and juxtapleural nodules. For a correct volume segmentation of the lung parenchyma, the system uses a Region Growing (RG) algorithm and an opening process for including the juxtapleural nodules. The segmentation and the extraction of the suspected nodular lesions from CT images by a lung CAD system constitutes a hard task. In order to solve this key problem, we use a new Stable 3D Mass-Spring Model (MSM) combined with a spline curves reconstruction process. Our model represents concurrently the characteristic gray value range, the directed contour information as well as shape knowledge, which leads to a much more robust and efficient segmentation process. For distinguishing the real nodules among nodule candidates, an additional classification step is applied; furthermore, a neural network is applied to reduce the false positives (FPs) after a double-threshold cut. The system performance was tested on a set of 84 scans made available by the Lung Image Database Consortium (LIDC) annotated by four expert radiologists. The detection rate of the system is 97% with 6.1 FPs/CT. A reduction to 2.5 FPs/CT is achieved at 88% sensitivity. We presented a new 3D segmentation technique for lung nodules in CT datasets, using deformable MSMs. The result is a efficient segmentation process able to converge, identifying the shape of the generic ROI, after a few iterations. Our suitable results show that the use of the 3D AC model and the feature analysis based FPs reduction process constitutes an accurate approach to the segmentation and the classification of lung nodules.

Computer-assisted diagnosis (CAD) in mammography: comparison of diagnostic accuracy of a new algorithm (Cyclopus, Medicad) with two commercial systems.

PURPOSE: The study compares the diagnostic accuracy (correct identification of cancer) of a new computer-assisted diagnosis (CAD) system (Cyclopus) with two other commercial systems (R2 and CADx). MATERIALS AND METHODS: Cyclopus was tested on a set of 120 mammograms on which the two compared commercial systems had been previously tested. The set consisted of mammograms reported as negative, preceding 31 interval cancers reviewed as screening error or minimal sign, and of 89 verified negative controls randomly selected from the same screening database. RESULTS: Cyclopus sensitivity was 74.1% (R2=54.8%; CADx=41.9%) and was higher for interval cancers reviewed as screening error (90.9%; R2=54.5%; CADx=81.8%) compared with those reviewed as minimal sign (65.0%; R2=55.0%; CADx=20.0%). Specificity was 15.7% (R2=29.2%; CADx=17.9%). Overall accuracy was 30.8% (R2=35.8%; CADx=24.1%). The positive predictive value of a case with CAD marks [regions of interest (ROI)] was 23.4% (23/98; R2=16.0%; CADx=15.1%). Average ROI number per view among negative controls was 1.13 (R2=0.93; CADx=0.99). Cyclopus was more sensitive for masses compared with isolated microcalcifications (208 vs 62 ROI; R2=90 vs 213; CADx=192 vs 130). CONCLUSIONS: Compared with two other commercial systems, Cyclopus was more sensitive (R2 p=0.14; CADx p=0.02) and less specific (R2 p=0.02; CADx p=0.64).

MAGIC-5: an Italian mammographic database of digitised images for research.

The implementation of a database of digitised mammograms is discussed. The digitised images were collected beginning in 1999 by a community of physicists in collaboration with radiologists in several Italian hospitals as a first step in developing and implementing a computer-aided detection (CAD) system. All 3,369 mammograms were collected from 967 patients and classified according to lesion type and morphology, breast tissue and pathology type. A dedicated graphical user interface was developed to visualise and process mammograms to support the medical diagnosis directly on a high-resolution screen. The database has been the starting point for developing other medical imaging applications, such as a breast CAD, currently being upgraded and optimised for use in a distributed environment with grid services, in the framework of the Instituto Nazionale di Fisicia Nucleare (INFN)-funded Medical Applications on a Grid Infrastructure Connection (MAGIC)-5 project.

A CAD system for nodule detection in low-dose lung CTs based on region growing and a new active contour model.

A computer-aided detection (CAD) system for the selection of lung nodules in computer tomography (CT) images is presented. The system is based on region growing (RG) algorithms and a new active contour model (ACM), implementing a local convex hull, able to draw the correct contour of the lung parenchyma and to include the pleural nodules. The CAD consists of three steps: (1) the lung parenchymal volume is segmented by means of a RG algorithm; the pleural nodules are included through the new ACM technique; (2) a RG algorithm is iteratively applied to the previously segmented volume in order to detect the candidate nodules; (3) a double-threshold cut and a neural network are applied to reduce the false positives (FPs). After having set the parameters on a clinical CT, the system works on whole scans, without the need for any manual selection. The CT database was recorded at the Pisa center of the ITALUNG-CT trial, the first Italian randomized controlled trial for the screening of the lung cancer. The detection rate of the system is 88.5% with 6.6 FPs/CT on 15 CT scans (about 4700 sectional images) with 26 nodules: 15 internal and 11 pleural. A reduction to 2.47 FPs/CT is achieved at 80% efficiency.

A completely automated CAD system for mass detection in a large mammographic database.

Mass localization plays a crucial role in computer-aided detection (CAD) systems for the classification of suspicious regions in mammograms. In this article we present a completely automated classification system for the detection of masses in digitized mammographic images. The tool system we discuss consists in three processing levels: (a) Image segmentation for the localization of regions of interest (ROIs). This step relies on an iterative dynamical threshold algorithm able to select iso-intensity closed contours around gray level maxima of the mammogram. (b) ROI characterization by means of textural features computed from the gray tone spatial dependence matrix (GTSDM), containing second-order spatial statistics information on the pixel gray level intensity. As the images under study were recorded in different centers and with different machine settings, eight GTSDM features were selected so as to be invariant under monotonic transformation. In this way, the images do not need to be normalized, as the adopted features depend on the texture only, rather than on the gray tone levels, too. (c) ROI classification by means of a neural network, with supervision provided by the radiologist's diagnosis. The CAD system was evaluated on a large database of 3369 mammographic images [2307 negative, 1062 pathological (or positive), containing at least one confirmed mass, as diagnosed by an expert radiologist]. To assess the performance of the system, receiver operating characteristic (ROC) and free-response ROC analysis were employed. The area under the ROC curve was found to be Az = 0.783 +/- 0.008 for the ROI-based classification. When evaluating the accuracy of the CAD against the radiologist-drawn boundaries, 4.23 false positives per image are found at 80% of mass sensitivity.

An ancestral nuclear protein assembly: crystal structure of the Methanopyrus kandleri histone.

Eukaryotic histone proteins condense DNA into compact structures called nucleosomes. Nucleosomes were viewed as a distinguishing feature of eukaryotes prior to identification of histone orthologs in methanogens. Although evolutionarily distinct from methanogens, the methane-producing hyperthermophile Methanopyrus kandleri produces a novel, 154-residue histone (HMk). Amino acid sequence comparisons show that HMk differs from both methanogenic and eukaryotic histones, in that it contains two histone-fold ms within a single chain. The two HMk histone-fold ms, N and C terminal, are 28% identical in amino acid sequence to each other and approximately 21% identical in amino acid sequence to other histone proteins. Here we present the 1.37-A-resolution crystal structure of HMk and report that the HMk monomer structure is homologous to the eukaryotic histone heterodimers. In the crystal, HMk forms a dimer homologous to [H3-H4](2) in the eukaryotic nucleosome. Based on the spatial similarities to structural ms found in the eukaryotic nucleosome that are important for DNA-binding, we infer that the Methanopyrus histone binds DNA in a manner similar to the eukaryotic histone tetramer [H3-H4](2).

The crystal structure of a heptameric archaeal Sm protein: Implications for the eukaryotic snRNP core.

Sm proteins form the core of small nuclear ribonucleoprotein particles (snRNPs), making them key components of several mRNA-processing assemblies, including the spliceosome. We report the 1.75-A crystal structure of SmAP, an Sm-like archaeal protein that forms a heptameric ring perforated by a cationic pore. In addition to providing direct evidence for such an assembly in eukaryotic snRNPs, this structure (i) shows that SmAP homodimers are structurally similar to human Sm heterodimers, (ii) supports a gene duplication model of Sm protein evolution, and (iii) offers a model of SmAP bound to single-stranded RNA (ssRNA) that explains Sm binding-site specificity. The pronounced electrostatic asymmetry of the SmAP surface imparts directionality to putative SmAP-RNA interactions.

Crystal structures of spin labeled T4 lysozyme mutants: implications for the interpretation of EPR spectra in terms of structure.

High resolution (1.43-1.8 A) crystal structures and the corresponding electron paramagnetic resonance (EPR) spectra were determined for T4 lysozyme derivatives with a disulfide-linked nitroxide side chain [-CH(2)-S-S-CH(2)-(3-[2,2,5,5-tetramethyl pyrroline-1-oxyl]) identical with R1] substituted at solvent-exposed helix surface sites (Lys65, Arg80, Arg119) or a tertiary contact site (Val75). In each case, electron density is clearly resolved for the disulfide group, revealing distinct rotamers of the side chain, defined by the dihedral angles X(1) and X(2). The electron density associated with the nitroxide ring in the different mutants is inversely correlated with its mobility determined from the EPR spectrum. Residue 80R1 assumes a single g(+)()g(+)() conformation (Chi(1) = 286, X(2) = 294). Residue 119R1 has two EPR spectral components, apparently corresponding to two rotamers, one similar to that for 80R1 and the other in a tg(-)() conformation (Chi(1) = 175, X(2) = 54). The latter state is apparently stabilized by interaction of the disulfide with a Gln at i + 4, a situation also observed at 65R1. R1 residues at helix surface site 65 and tertiary contact site 75 make intra- as well as intermolecular contacts in the crystal and serve to identify the kind of molecular interactions possible for the R1 side chain. A single conformation of the entire 75R1 side chain is stabilized by a variety of interactions with the nitroxide ring, including hydrophobic contacts and two unconventional C-H.O hydrogen bonds, one in which the nitroxide acts as a donor (with tyrosine) and the other in which it acts as an acceptor (with phenylalanine). The interactions revealed in these structures provide an important link between the dynamics of the R1 side chain, reflected in the EPR spectrum, and local protein structure. A library of such interactions will provide a basis for the quantitative interpretation of EPR spectra in terms of protein structure and dynamics.

Monomeric structure of the human EphB2 sterile alpha motif domain.

The sterile alpha motif (SAM) domain is a protein module found in many diverse signaling proteins. SAM domains in some systems have been shown to self-associate. Previous crystal structures of an EphA4-SAM domain dimer (Stapleton, D., Balan, I., Pawson, T., and Sicheri, F. (1999) Nat. Struct. Biol. 6, 44-49) and a possible EphB2-SAM oligomer (Thanos, C. D., Goodwill, K. E., and Bowie, J. U. (1999) Science 283, 833-836) both revealed large interfaces comprising an exchange of N-terminal peptide arms. Within the arm, a conserved hydrophobic residue (Tyr-8 in the EphB2-SAM structure or Phe-910 in the EphA4-SAM structure) is anchored into a hydrophobic cleft on a neighboring molecule. Here we have solved a new crystal form of the human EphB2-SAM domain that has the same overall SAM domain fold yet has no substantial intermolecular contacts. In the new structure, the N-terminal peptide arm of the EphB2-SAM domain protrudes out from the core of the molecule, leaving both the arm (including Tyr-8) and the hydrophobic cleft solvent-exposed. To verify that Tyr-8 is solvent-exposed in solution, we made a Tyr-8 to Ala-8 mutation and found that the EphB2-SAM domain structure and stability were only slightly altered. These results suggest that Tyr-8 is not part of the hydrophobic core of the EphB2-SAM domain and is conserved for functional reasons. Cystallographic evidence suggests a possible role for the N-terminal arm in oligomerization. In the absence of a direct demonstration of biological relevance, however, the functional role of the N-terminal arm remains an open question.

Packed protein bilayers in the 0.90 A resolution structure of a designed alpha helical bundle.

A 12-residue peptide designed to form an alpha-helix and self-associate into an antiparallel 4-alpha-helical bundle yields a 0.9 A crystal structure revealing unanticipated features. The structure was determined by direct phasing with the "Shake-and-Bake" program, and contains four crystallographically distinct 12-mer peptide molecules plus solvent for a total of 479 atoms. The crystal is formed from nearly ideal alpha-helices hydrogen bonded head-to-tail into columns, which in turn pack side-by-side into sheets spanning the width of the crystal. Within each sheet, the alpha-helices run antiparallel and are closely spaced (9-10 A center-to-center). The sheets are more loosely packed against each other (13-14 A between helix centers). Each sheet is amphiphilic: apolar leucine side chains project from one face, charged lysine and glutamate side chains from the other face. The sheets are stacked with two polar faces opposing and two apolar faces opposing. The result is a periodic biomaterial composed of packed protein bilayers, with alternating polar and apolar interfaces. All of the 30 water molecules in the unit cell lie in the polar interface or between the stacked termini of helices. A section through the sheet reveals that the helices packed at the apolar interface resemble the four-alpha-helical bundle of the design, but the helices overhang parts of the adjacent bundles, and the helix crossing angles are less steep than intended (7-11 degrees rather than 18 degrees).

Centrosymmetric bilayers in the 0.75 A resolution structure of a designed alpha-helical peptide, D,L-Alpha-1.

We report the 0.75 A crystal structure of a racemic mixture of the 12-residue designed peptide "Alpha-1" (Acetyl-ELLKKLLEELKG), the L-enantiomer of which is described in the accompanying paper. Equivalent solutions of the centrosymmetric bilayers were determined by two direct phasing programs in space groups P1 and P1bar. The unit cell contains two L-alpha-helices and two D-alpha-helices. The columnar-sheet bilayer motif seen in L-Alpha-1 is maintained in the D,L-Alpha-1 structure except that each sheet of head-to-tail helices is composed of one enantiomer and is related to its neighboring sheets by inversion symmetry. Comparison to the L-Alpha-1 structure provides further insight into peptide design. The high resolution and small asymmetric unit allowed building an intricate model (R = 13.1%, Rfree = 14.5%) that incorporates much of the discrete disorder of peptide and solvent. Ethanolamine and 2-methyl-2,4-pentanediol (MPD) molecules bind near helix termini. Rigid body analysis identifies sites of restricted displacements and torsions. Side-chain discrete disorder propagates into the backbone of one helix but not the other. Although no side chain in Alpha-1 is rigid, the environments in the crystal restrict some of them to no or only one active torsion.

The 1.8 A crystal structure of the ycaC gene product from Escherichia coli reveals an octameric hydrolase of unknown specificity.

BACKGROUND: The ycaC gene comprises a 621 base pair open reading frame in Escherichia coli. The ycaC gene product (ycaCgp) is uncharacterized and has no assigned function. The closest sequence homologs with an assigned function belong to a family of bacterial hydrolases that catalyze isochorismatase-like reactions, but these have only low sequence similarity to ycaCgp (approximately 20% amino acid identity). The ycaCgp was obtained and identified during crystallization trials of an unrelated E. coli protein with which it co-purified. RESULTS: The 1.8 A crystal structure of ycaCgp reveals an octameric complex comprised of two tetrameric rings. A large three-layer (alphabetaalpha) sandwich domain and a small helical domain form the folded structure of the monomeric unit. Comparisons with sequence and structure databases suggest that ycaCgp belongs to a diverse family of bacterial hydrolases. The most closely related three-dimensional structure is that of the D2 tetrameric N-carbamoylsarcosine amidohydrolase (CSHase) from an Arthrobacter species. A conspicuous cleft between two ycaCgp subunits contains several conserved residues including Cys118, which we propose to be catalytic. In the active site, a nonprolyl cis peptide bond precedes Val114 and coincides with a cis peptide bond in CSHase in a region of dissimilar sequence. The crystal structure reveals a probable error or mutation relative to the reported genomic sequence. CONCLUSIONS: Although the specific function of ycaCgp is not yet known, structural studies solidify the relationship of this protein to other hydrolases and illuminate its active site and key elements of the catalytic mechanism.

NarL dimerization? Suggestive evidence from a new crystal form.

The structure of the Escherichia coli response regulator NarL has been solved in a new, monoclinic space group, and compared with the earlier orthorhombic crystal structure. Because the monoclinic crystal has two independent NarL molecules per asymmetric unit, we now have three completely independent snapshots of the NarL molecule: two from the monoclinic form and one from the orthorhombic. Comparison of these three structures shows the following: (a) The pairing of N and C domains of the NarL molecule proposed from the earlier analysis is in fact correct, although the polypeptide chain connecting domains was, and remains, disordered and not completely visible. The new structure exhibits identical relative orientation of N and C domains, and supplies some of the missing residues, leaving a gap of only seven amino acids. (b) Examination of corresponding features in the three independent NarL molecules shows that deformations in structure produced by crystal packing are negligible. (c) The "telephone receiver" model of NarL activation is confirmed. The N domain of NarL blocks the binding of DNA to the C domain that would be expected from the helix-turn-helix structure of the C domain. Hence, binding can only occur after significant displacement of N and C domains. (d) NarL monomers have a strong tendency toward dimerization involving contacts between helixes alpha 1 in the two monomers, and this may have mechanistic significance in DNA binding. Analogous involvement of helix alpha 1 in intermolecular contacts is also found in UhpA and in the CheY/CheZ complex.

Structure of a DNA analog of the primer for HIV-1 RT second strand synthesis.

The non-self-complementary DNA decamer C-A-A-A-G-A-A-A-A-G/C-T-T-T-T-C-T-T-T-G is a DNA/DNA analogue of a portion of the polypurine tract or PPT, which is a RNA/DNA hybrid that serves as a primer for synthesis of the (+) DNA strand by HIV reverse transcriptase (RT), and which is not digested by the RNase H domain of reverse transcriptase following (-) strand synthesis. The same unusual conformation that eludes RNase H, thought to be a change in width of minor groove, may also be responsible for the inhibition of HIV RT by minor groove binding drugs such as distamycin and their bis-linked derivatives. The present X-ray crystal structure of this DNA decamer exhibits the usual properties of A-tract B-DNA under biologically relevant conditions: large propeller twist of base-pairs, narrowed minor groove, and a straight helix axis. Groove narrowing is fully developed in the A-A-A-A region, but not in the A-A-A region, which previous investigators have proposed as being too short to exhibit typical A-tract properties. The RNA/DNA hybrid produced by HIV reverse transcriptase during (-) strand synthesis presumably forms a "heteromerous" or H-helix with narrower minor groove than an A-helical RNA/RNA duplex. If the narrowing of minor groove in A-tract H-helices is comparable to that seen in A-tract B-helices, then the narrowed minor groove of the polypurine tract could make the second primer site both (1) impervious to RNase H digestion, and (2) susceptible to inhibition by minor groove binding drugs.

Crystal structure of a covalent DNA-drug adduct: anthramycin bound to C-C-A-A-C-G-T-T-G-G and a molecular explanation of specificity.

A 2.3-A X-ray crystal structure analysis has been carried out on the antitumor drug anthramycin, covalently bound to a ten base pair DNA double helix of sequence C-C-A-A-C-G-T-T-G-G. One drug molecule sits within the minor groove at each end of the helix, covalently bound through its C11 position to the N2 amine of the penultimate guanine of the chain. The stereochemical conformation is C11S, C11aS. The natural twist of the anthramycin molecule in the C11aS conformation matches the twist of the minor groove as it winds along the helix; a C11aR drug would only fit into a left-handed helix. The C11S attachment is roughly equatorial to the overall plane of the molecule, whereas a C11R attachment would be axial and would obstruct the fitting of the drug into the groove. The six-membered ring of anthramycin points toward the 3' end of the chain to which it is covalently attached or toward the end of the helix. The acrylamide tail attached to the five-membered ring extends back along the minor groove toward the center of the helix, binding in a manner reminiscent of netropsin or distamycin. The drug-DNA complex is stabilized by hydrogen bonds from C9-OH, N10, and the end of the acrylamide tail to base pair edges on the floor of the minor groove. The origin of anthramycin specificity for three successive purines arises not from specific hydrogen bonds but from the low twist angles adopted by purine-purine steps in a B-DNA helix. Binding of anthramycin induces a low twist at T-G in the T-G-G sequence of this DNA-drug complex, by comparison with the structure of the free DNA. The origin of anthramycin's preference for adenines flanking the alkylated guanine arises from a netropsin-like fitting of the acrylamide tail into the minor groove.

Crystallization studies of the human immunodeficiency virus (HIV-1) Tat protein and its trans-activation response element (TAR) RNA.

Small single crystals are reported of a complex between a small peptide fragment of the HIV-1 Tat protein and a fragment of the RNA to which it binds. Tat is responsible for enhancing the level of expression of the human immunodeficiency virus type 1 (HIV-1) and is a logical target for AIDS therapy. Tat may function to increase the level of transcription initiation or to prevent premature termination of transcripts. In vitro, Tat binds through its basic domain (two Lys and six Arg in nine residues) to a three-nucleotide bulge of a stem-loop RNA structure called TAR. Complex formation between Tat and TAR is necessary for Tat activity. Peptides which contain the basic region of Tat also bind to TAR RNA. We have carried out crystallization experiments on a 27-nucleotide fragment of TAR RNA and on complexes between two Tat peptides and TAR.

Refined molecular structure of pig pancreatic alpha-amylase at 2.1 A resolution.

The structure of pig pancreatic alpha-amylase has been determined by X-ray diffraction analysis using multiple isomorphous replacement in a crystal of space group P2(1)2(1)2(1) (a = 70.6 A, b = 114.8 A, c = 118.8 A) containing nearly 75% solvent. The structure was refined by simulated annealing and Powell minimization, as monitored by 2Fo-Fc difference Fourier syntheses, to a conventional R of 0.168 at 2.1 A resolution. The final model consists of all 496 amino acid residues, a chloride and a calcium ion, 145 water molecules and an endogenous disaccharide molecule that contiguously links protein molecules related by the 2(1) crystallographic operator along x. The protein is composed of a large domain (amino acid residues 1 to 403) featuring a central alpha ta-barrel of eight parallel strands and connecting helices with a prominent excursion between strand beta 3 and helix alpha 3 (amino acid residues 100 to 168). The final 93 amino acid residues at the carboxyl terminus form a second small domain consisting of a compact Greek key beta-barrel. The domains are tightly associated through hydrophobic interfaces. The beta 3/alpha 3 excursion and portions of the central alpha/beta-barrel provide four protein ligands to the tightly bound Ca ion; three water molecules complete the coordination. The Cl- ion is bound within one end of the alpha/beta-barrel by two arginine residues in a manner suggesting a plausible mechanism for its allosteric activation of the enzyme. A crystalline complex of the pancreatic alpha-amylase with alpha-cyclodextrin, a cyclic substrate analog of six glucose residues, reveals, in difference Fourier maps, three unique binding sites. One of the alpha-cyclodextrin sites is near the center of the long polysaccharide binding cleft that traverses one end of the alpha/beta-barrel, another is at the extreme of this cleft. By symmetry this can also be considered as two half sites located at the extremes of the active site cleft. This latter alpha-cyclodextrin displaces the endogenous disaccharide when it binds and, along with the first sugar ring, delineates the extended starch binding site. The third alpha-cyclodextrin binds at an "accessory site" near the edge of the protein and is quite distant from the polysaccharide binding cleft. Its presence explains the multivalency of alpha-amylase binding to dextrins in solution. The extended active site cleft is formed by large, sweeping, connecting loops at one end of the alpha/beta-barrel. These include three sequence segments that are highly conserved among alpha-amylases.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystal structure of the unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate.

The crystal structure of unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase from Nicotiana tabacum complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate, was determined to 2.7 A resolution by X-ray crystallography. The transition state analog binds at the active site in an extended conformation. As compared to the binding of the same analog in the activated enzyme, the analog binds in a reverse orientation. The active site Lys 201 is within hydrogen bonding distance of the carboxyl oxygen of the analog. Loop 6 (residues 330-339) remains open and flexible upon binding of the analog in the unactivated enzyme, in contrast to the closed and ordered loop 6 in the activated enzyme complex. The transition state analog is exposed to solvent due to the open conformation of loop 6.

Crystal structure of canine and bovine granulocyte-colony stimulating factor (G-CSF).

The crystal structures of recombinant canine and bovine granulocyte colony stimulating factor (G-CSF) have been determined by X-ray crystallography, using molecular replacement with recombinant human G-CSF as a model. G-CSF is a member of the cytokine family of glycoproteins that stimulate the differentiation and proliferation of blood cells. Human, bovine and canine G-CSF all have a molecular mass of about 19 kDa and share an amino acid sequence identity of about 80%. Two crystal forms of canine G-CSF have been solved. Form I recombinant canine G-CSF (rcG-CSFI; space group C2) contains one molecule in the asymmetric unit while form II canine G-CSF (rcG-CSFII; space group P2(1)) has two molecules in the asymmetric unit and bovine G-CSF (rbG-CSF; space group P2(1)2(1)2(1)) contains one molecule in the asymmetric unit. rcG-CSFI has been refined to an R factor of 20.7% with data to 2.3 A resolution and rcG-CSFII has been refined to an R factor of 19.3% with data to 2.2 A resolution. rbG-CSF has been refined to an R factor of 21.3% with data to 1.7 A resolution. The structure of human, canine and bovine G-CSF is an antiparallel 4-alpha-helical bundle with up-up-down-down connectivity. With the exception of one highly exposed loop (residues 66 to 74), the human, canine and bovine structures are very similar to each other. Using our series of G-CSF crystal structures we developed a function that describes the probability that a particular residue position (i) contributes to a G-CSF receptor binding site based on two principles, (1) high sequence conservation in the primary sequence of human, bovine, canine and murine G-CSF and (2) conservation of high solvent accessibility in the human, bovine and canine crystal structures. On the basis of this probability function as well as a comparison of G-CSF to the crystal structure of human growth hormone (hGH) complexed with the extracellular domain of the human growth hormone receptor (hGHbp), residues that contribute to potential G-CSF receptor binding sites are identified.