Polyalanine Model Research Articles

Protein structure determination by electron diffraction using a single three-dimensional nanocrystal.

Three-dimensional nanometre-sized crystals of macromolecules currently resist structure elucidation by single-crystal X-ray crystallography. Here, a single nanocrystal with a diffracting volume of only 0.14 µm3, i.e. no more than 6 × 105 unit cells, provided sufficient information to determine the structure of a rare dimeric polymorph of hen egg-white lysozyme by electron crystallography. This is at least an order of magnitude smaller than was previously possible. The molecular-replacement solution, based on a monomeric polyalanine model, provided sufficient phasing power to show side-chain density, and automated model building was used to reconstruct the side chains. Diffraction data were acquired using the rotation method with parallel beam diffraction on a Titan Krios transmission electron microscope equipped with a novel in-house-designed 1024 × 1024 pixel Timepix hybrid pixel detector for low-dose diffraction data collection. Favourable detector characteristics include the ability to accurately discriminate single high-energy electrons from X-rays and count them, fast readout to finely sample reciprocal space and a high dynamic range. This work, together with other recent milestones, suggests that electron crystallography can provide an attractive alternative in determining biological structures.

Crystallization and preliminary X-ray analysis of the open form of human ecto-5'-nucleotidase (CD73).

Eukaryotic ecto-5'-nucleotidase (e5NT) catalyses the hydrolysis of extracellular AMP to adenosine and plays a pivotal role in switching on adenosine signalling via the P1 receptors of the purinergic signalling pathway. With such an important regulatory role, e5NT has become an appealing new drug target, with potential applications in the treatment of inflammation, chronic pain, hypoxia and cancer. In order to gain insight into the structure and function of the eukaryotic e5NT enzymes and to assist in structure-based drug design, the crystal structure of human e5NT has been solved. Recombinant human e5NT comprising four asparagine-to-aspartate surface mutations targeting potential glycosylation sites was refolded from bacterial inclusion bodies. Refolded and purified human e5NT crystallized in space group P4(3)32 and a data set to 1.85 Å resolution was obtained. The structure could be solved by molecular replacement using a polyalanine model generated from Thermus thermophilus 5'-nucleotidase (5NT). An anomalous data set revealed the presence of a metal-ion binding site, as well as calcium and chloride ion-binding sites. Structural comparisons with bacterial 5NT homologues showed that the human e5NT crystal structure has an open conformation in which the metal- and substrate-binding sites are distant from each other. Here, the crystallization and preliminary X-ray crystallographic analysis of an open structural conformation of human e5NT are described.

Abstract 4746: Structural studies of human glioma pathogenesis-related protein 1(GLIPR1)

Abstract Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide, to direct its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory protein, antigen 5, pathogenesis related-1) domain, and a transmembrane domain. Phase I/II clinical trial involving direct injection of neoadjuvant GLIPR1 prior to radical prostatectomy are underway (IND #13033). GLIPR1 gene therapy has been shown to be anti-metastatic in an orthotopic mouse model of prostate cancer. GLIPR1 is also currently being investigated for glioblastoma targeted therapy. We present here crystal structures of a truncated soluble domain of the human GLIPR1 protein, sGLIPR1, solved by molecular replacement (MR) using a truncated polyalanine model of the CAP domain of stecrisp, a snake venom CRISP (cysteine-rich secretory protein), in PHASER. The correct MR solution could only be obtained by removing all loops in the search model. While, the native structure was refined to 1.85 Å, that of the Zn2+ complex was refined to 2.2 Å. The latter structure reveals that the putative binding cavity coordinates Zn2+ similarly to snake venom CRISPs which are involved in Zn2+-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop / turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. We also propose a model for the structure of the full-length, membrane-bound GLIPR1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4746. doi:1538-7445.AM2012-4746

Structure of a two-CAP-domain protein from the human hookworm parasiteNecator americanus

Major proteins secreted by the infective larval stage hookworms upon host entry include Ancylostoma secreted proteins (ASPs), which are characterized by one or two CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) domains. The CAP domain has been reported in diverse phylogenetically unrelated proteins, but has no confirmed function. The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite Necator americanus was refined to a resolution limit of 2.2 Å. The structure was solved by molecular replacement (MR) using Na-ASP-2, a one-CAP-domain ASP, as the search model. The correct MR solution could only be obtained by truncating the polyalanine model of Na-ASP-2 and removing several loops. The structure reveals two CAP domains linked by an extended loop. Overall, the carboxyl-terminal CAP domain is more similar to Na-ASP-2 than to the amino-terminal CAP domain. A large central cavity extends from the amino-terminal CAP domain to the carboxyl-terminal CAP domain, encompassing the putative CAP-binding cavity. The putative CAP-binding cavity is a characteristic cavity in the carboxyl-terminal CAP domain that contains a His and Glu pair. These residues are conserved in all single-CAP-domain proteins, but are absent in the amino-terminal CAP domain. The conserved His residues are oriented such that they appear to be capable of directly coordinating a zinc ion as observed for CAP proteins from reptile venoms. This first structure of a two-CAP-domain ASP can serve as a template for homology modeling of other two-CAP-domain proteins.

Biochemical Basis of the Interaction between Cystic Fibrosis Transmembrane Conductance Regulator and Immunoglobulin-like Repeats of Filamin

Mutations in the chloride channel cystic fibrosis transmembrane regulator (CFTR) cause cystic fibrosis, a genetic disorder characterized by defects in CFTR biosynthesis, localization to the cell surface, or activation by regulatory factors. It was discovered recently that surface localization of CFTR is stabilized by an interaction between the CFTR N terminus and the multidomain cytoskeletal protein filamin. The details of the CFTR-filamin interaction, however, are unclear. Using x-ray crystallography, we show how the CFTR N terminus binds to immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). CFTR binds to beta-strands C and D of FlnA-Ig21 using backbone-backbone hydrogen bonds, a linchpin serine residue, and hydrophobic side-chain packing. We use NMR to determine that the CFTR N terminus also binds to several other immunoglobulin-like repeats from filamin A in vitro. Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTR-filamin interaction. We show that FlnA-Ig repeats transfected into cultured Calu-3 cells disrupt CFTR-filamin interaction and reduce surface levels of CFTR. Our findings suggest that filamin A stabilizes surface CFTR by anchoring it to the actin cytoskeleton through interactions with multiple filamin Ig repeats. Such an interaction mode may allow filamins to cluster multiple CFTR molecules and to promote colocalization of CFTR and other filamin-binding proteins in the apical plasma membrane of epithelial cells.

The Structural Basis for the Large Powerstroke of Myosin VI

Due to a unique addition to the lever arm-positioning region (converter), class VI myosins move in the opposite direction (toward the minus-end of actin filaments) compared to other characterized myosin classes. However, the large size of the myosin VI lever arm swing (powerstroke) cannot be explained by our current view of the structural transitions that occur within the myosin motor. We have solved the crystal structure of a fragment of the myosin VI motor in the structural state that represents the starting point for movement on actin; the pre-powerstroke state. Unexpectedly, the converter itself rearranges to achieve a conformation that has not been seen for other myosins. This results in a much larger powerstroke than is achievable without the converter rearrangement. Moreover, it provides a new mechanism that could be exploited to increase the powerstroke of yet to be characterized plus-end-directed myosin classes.

Ab initio molecular-replacement phasing for symmetric helical membrane proteins.

Obtaining phases for X-ray diffraction data can be a rate-limiting step in structure determination. Taking advantage of constraints specific to membrane proteins, an ab initio molecular-replacement method has been developed for phasing X-ray diffraction data for symmetric helical membrane proteins without prior knowledge of their structure or heavy-atom derivatives. The described method is based on generating all possible orientations of idealized transmembrane helices and using each model in a molecular-replacement search. The number of models is significantly reduced by taking advantage of geometrical and structural restraints specific to membrane proteins. The top molecular-replacement results are evaluated based on noncrystallographic symmetry (NCS) map correlation, OMIT map correlation and R(free) value after refinement of a polyalanine model. The feasibility of this approach is illustrated by phasing the mechanosensitive channel of large conductance (MscL) with only 4 A diffraction data. No prior structural knowledge was used other than the number of transmembrane helices. The search produced the correct spatial organization and the position in the asymmetric unit of all transmembrane helices of MscL. The resulting electron-density maps were of sufficient quality to automatically build all helical segments of MscL including the cytoplasmic domain. The method does not require high-resolution diffraction data and can be used to obtain phases for symmetrical helical membrane proteins with one or two helices per monomer.

Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 1

Crystals of baculovirus-expressed adeno-associated virus serotype 1 (AAV1) capsids have been grown in the rhombohedral space group R32 (unit-cell parameters a = 254.7 A, alpha = 62.3 degrees) and shown to diffract X-rays to at least 2.5 A resolution. The diffraction data were subsequently processed and reduced with an overall R(sym) of 12.3% and a completeness of 89.0%. Based on the unit-cell volume, rotation-function and translation-function results and packing considerations, there is one virus capsid (60 viral proteins) per unit cell and there are ten viral proteins per crystallographic asymmetric unit. The AAV1 capsid shares both the twofold and threefold crystallographic symmetry operators. The AAV1 data have been initially phased using a polyalanine model (based on the crystal structure of AAV4) to 4.0 A resolution and the structure determination and refinement is in progress using tenfold noncrystallographic symmetry electron-density averaging.

Structural Basis for Activation of the Thiamin Diphosphate-dependent Enzyme Oxalyl-CoA Decarboxylase by Adenosine Diphosphate

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate-dependent enzyme that plays an important role in the catabolism of the highly toxic compound oxalate. We have determined the crystal structure of the enzyme from Oxalobacter formigenes from a hemihedrally twinned crystal to 1.73 A resolution and characterized the steady-state kinetic behavior of the decarboxylase. The monomer of the tetrameric enzyme consists of three alpha/beta-type domains, commonly seen in this class of enzymes, and the thiamin diphosphate-binding site is located at the expected subunit-subunit interface between two of the domains with the cofactor bound in the conserved V-conformation. Although oxalyl-CoA decarboxylase is structurally homologous to acetohydroxyacid synthase, a molecule of ADP is bound in a region that is cognate to the FAD-binding site observed in acetohydroxyacid synthase and presumably fulfils a similar role in stabilizing the protein structure. This difference between the two enzymes may have physiological importance since oxalyl-CoA decarboxylation is an essential step in ATP generation in O. formigenes, and the decarboxylase activity is stimulated by exogenous ADP. Despite the significant degree of structural conservation between the two homologous enzymes and the similarity in catalytic mechanism to other thiamin diphosphate-dependent enzymes, the active site residues of oxalyl-CoA decarboxylase are unique. A suggestion for the reaction mechanism of the enzyme is presented.

The Crystal Structure of the Outer Membrane Protein VceC from the Bacterial Pathogen Vibrio cholerae at 1.8 Å Resolution

Multidrug resistance in Gram-negative bacteria arises in part from the activities of tripartite drug efflux pumps. In the pathogen Vibrio cholerae, one such pump comprises the inner membrane proton antiporter VceB, the periplasmic adaptor VceA, and the outer membrane channel VceC. Here, we report the crystal structure of VceC at 1.8 A resolution. The trimeric VceC is organized in the crystal lattice within laminar arrays that resemble membranes. A well resolved detergent molecule within this array interacts with the transmembrane beta-barrel domain in a fashion that may mimic protein-lipopolysaccharide contacts. Our analyses of the external surfaces of VceC and other channel proteins suggest that different classes of efflux pumps have distinct architectures. We discuss the implications of these findings for mechanisms of drug and protein export.

Crystal Structure of Vinorine Synthase, the First Representative of the BAHD Superfamily

Vinorine synthase is an acetyltransferase that occupies a central role in the biosynthesis of the antiarrhythmic monoterpenoid indole alkaloid ajmaline in the plant Rauvolfia. Vinorine synthase belongs to the benzylalcohol acetyl-, anthocyanin-O-hydroxy-cinnamoyl-, anthranilate-N-hydroxy-cinnamoyl/benzoyl-, deacetylvindoline acetyltransferase (BAHD) enzyme superfamily, members of which are involved in the biosynthesis of several important drugs, such as morphine, Taxol, or vindoline, a precursor of the anti-cancer drugs vincaleucoblastine and vincristine. The x-ray structure of vinorine synthase is described at 2.6-angstrom resolution. Despite low sequence identity, the two-domain structure of vinorine synthase shows surprising similarity with structures of several CoA-dependent acyltransferases such as dihydrolipoyl transacetylase, polyketide-associated protein A5, and carnitine acetyltransferase. All conserved residues typical for the BAHD family are found in domain 1. His160 of the HXXXD motif functions as a general base during catalysis. It is located in the center of the reaction channel at the interface of both domains and is accessible from both sides. The channel runs through the entire molecule, allowing the substrate and co-substrate to bind independently. Asp164 points away from the catalytic site and seems to be of structural rather than catalytic importance. Surprisingly, the DFGWG motif, which is indispensable for the catalyzed reaction and unique to the BAHD family, is located far away from the active site and seems to play only a structural role. Vinorine synthase represents the first solved protein structure of the BAHD superfamily.

Mechanism for the alpha-helix to beta-hairpin transition.

The aggregation of alpha-helix-rich proteins into beta-sheet-rich amyloid fibrils is associated with fatal diseases, such as Alzheimer's disease and prion disease. During an aggregation process, protein secondary structure elements-alpha-helices-undergo conformational changes to beta-sheets. The fact that proteins with different sequences and structures undergo a similar transition on aggregation suggests that the sequence nonspecific hydrogen bond interaction among protein backbones is an important factor. We perform molecular dynamics simulations of a polyalanine model, which is an alpha-helix in its native state and observe a metastable beta-hairpin intermediate. Although a beta-hairpin has larger potential energy than an alpha-helix, the entropy of a beta-hairpin is larger because of fewer constraints imposed by the hydrogen bonds. In the vicinity of the transition temperature, we observe the interconversion of the alpha-helix and beta-sheet states via a random coil state. We also study the effect of the environment by varying the relative strength of side-chain interactions for a designed peptide-an alpha-helix in its native state. For a certain range of side-chain interaction strengths, we find that the intermediate beta-hairpin state is destabilized and even disappears, suggesting an important role of the environment in the aggregation propensity of a peptide.

Automatic model building based on flexible fragment formalism. The case of high-resolution protein structures.

A concept of flexible fragments has been developed for automatic building of crystal structures. Six monopeptides were designed as search fragments in a phased rotation and translation function for protein building. Electron density in crystal and in molecular fragments is expanded in spherical harmonics and normalized spherical Bessel functions. A fast rotation function, which is calculated at each grid point of the asymmetric unit, is used to find the fragment orientation. Position, orientation and internal torsion angles are refined. An algorithm for chain building is simplified using generalized atoms and virtual bonds. The structure is built from molecular structure units rather than from individual atoms. A polyalanine model is built with a high accuracy at resolutions 1.2-2.1 A.

Studies of the Monodipole-macrodipole Interactions within α-Helices Using the Point-charge Systems for Alanine

Our previous quantum mechanical calculations using polyalanine model systems showed that the monodipolemacrodipoleinteractions selectively stabilize α-helices and make it possible for α-helices to be formed inhydrophobic environment where the solvent effect is not available. The monodipole-macrodipole interactionsin α-helices were studied molecular mechanically using various point-charge systems available. The resultsshow that all the point-charge systems used in the calculations produce the monodipole-macrodipoleinteractions up to about 60% compared to the results of the quantum mechanical calculations. The results ofmolecular mechanical calculations are explained and discussed compared to the results of the quantummechanical calculations.

Expression, purification, crystallization and preliminary crystallographic analysis of Trypanosoma brucei phosphofructokinase.

Phosphofructokinase from Trypanosoma brucei (TbPFK) was purified from a recombinant expression system in Escherichia coli by metal-affinity chromatography via its N-terminal His tag. The yield was 15-20 mg of pure enzyme per litre of culture. M(r) was shown to be 55 585 by mass spectrometry. Crystals suitable for X-ray diffraction analysis were obtained by the hanging-drop method of vapour diffusion with sodium formate as the precipitating agent. Monoclinic crystals of the apoenzyme grew within one week, as did orthorhombic crystals of PFK in the presence of enzymic reaction products or an active-site inhibitor. Initial attempts to solve the structure by molecular replacement with bacterial PFK structures as search models proved unrewarding, but a multiple-copy search with a polyalanine model was successful. In addition, heavy-atom soaking with platinum and mercury has yielded derivatives suitable for X-ray diffraction. A combination of the phase information from the molecular-replacement solution and the heavy-atom derivatives should allow structure solution of TbPFK. The availability of this first eukaryotic PFK structure will be of particular significance for structure-based drug design and will also provide important additional structural evidence for the allosteric control of PFK activity.

A rapid method for positioning small flexible molecules, nucleic acids, and large protein fragments in experimental electron density maps

A Monte Carlo procedure, encoded in the program Blob, has been developed and tested for the purpose of positioning large molecular fragments or small flexible molecules in electron density maps. The search performed by the algorithm appears to be sufficiently thorough to accurately position a small flexible ligand in well-defined density while remaining sufficiently random to offer interesting alternate suggestions for density representing disordered binding modes of a ligand. Furthermore, the algorithm is shown to be efficient enough to accurately position large rigid molecular fragments. In the first of the test cases with large molecular fragments, Blob was surprisingly effective in positioning a poly-alanine model of a 53-residue domain in poor electron density resulting from molecular replacement with a partial model. At 3.0 A resolution the domain was positioned consistently within 0.2 A of its experimentally determined position. Even at 6.0 A resolution Blob could consistently position the domain to within 0.75 A of its actual position. A second set of tests with large molecular fragments revealed that Blob could correctly position large molecular fragments with quite significant deviations from the actual structure. In this test case, fragments ranging from a 170-residue protein domain with a 3.8 A rms deviation from the actual structure to a 22-base pair ideal B-form DNA duplex were positioned accurately in a 3.2 A electron density map derived from multiple isomorphous replacement methods. Even when decreasing the quality of the maps, from a figure of merit of 0.57 to as low as 0. 35, Blob could still effectively position the large protein domain and the DNA duplex. Since it is efficient, can handle large molecular fragments, and works in poor and low resolution maps, Blob could be a useful tool for interpreting electron density maps in de novo structure determinations and in molecular replacement studies. Proteins 1999;36:512-525.

Meaningful refinement of polyalanine models using rigid-body simulated annealing: application to the structure determination of the A31P Rop mutant.

Conventional refinement methods, when applied to even correctly positioned polyalanine models of a target structure, result in a systematic distortion of the molecular geometry and to a concomitant increase in the mean phase difference from the correct phase set. Here, it is shown that iterative rigid-body simulated-annealing refinement of polyalanine models employing successively fewer residues per rigid body (down to one alanine residue per body) at a very high initial temperature (of the order of T0 = 10000 K) and with the geometric energy terms switched on, not only preserves the geometry of the model but can also converge to an essentially correct polyalanine trace of the target structure, even when the starting model deviates systematically and significantly from the sought structure. As an example of the application of the method, details are presented of the structure determination of the Ala31Pro mutant of the Rop protein, where an initial roughly positioned polyalanine model (giving an average phase difference of 78.2 degrees from the final phase set) was successfully refined against a 1.8 A resolution native data set, leading to an essentially correct model of the main chain with an average displacement of its atomic positions from the final model of 0.275 A. The phases calculated from this refined polyalanine model had an average difference of 43.8 degrees from the final phase set (corresponding to a mean figure of merit of 0.72) and gave a readily interpretable electron-density map.

Polyalanine reconstruction from Calpha positions using the program CALPHA can aid initial phasing of data by molecular replacement procedures.

The Calpha positions for a protein can provide a scaffold for the reconstruction of more complete models. Reconstructions can be by manual rebuilding, from geometric solutions to the constraints on main-chain torsion angles or from databases of known protein structures. The last method is usually the most convenient and reliable. This paper describes a database reconstruction program, CALPHA, and assesses its accuracy and reliability by test reconstructions of well refined structures. Typically, backbone atoms are repositioned to within 0.3 A of their original positions. This corresponds to regenerating main-chain torsion angles to within 15 degrees. Uses of CALPHA for automating refinement procedures are discussed. In particular, the uses of Calpha-only and rcconstructed polyalanine models of HIV-1 reverse transcriptase for cross-rotation and translation function searches are compared. The CALPHA polyalanine model is found to provide more selectivity for approximately correct orientations. The effect on the translation function is dependent on the resolution shell employed. It is expected that these observations will be applicable in other cases.

The crystal structure of seleno-glutathione peroxidase from human plasma at 2.9 Å resolution

Glutathione peroxidase belongs to the family of selenoproteins and plays an important role in the defense mechanisms of mammals, birds and fish against oxidative damage by catalyzing the reduction of a variety of hydroperoxides, using glutathione as the reducing substrate. However, the physiological role of human plasma glutathione peroxidase remains unclear due to the low levels of reduced glutathione in human plasma and the low reactivity of this enzyme. The crystal structure of human plasma glutathione peroxidase was determined by Patterson search methods using a polyalanine model modified from the known structure of bovine erythrocyte glutathione peroxidase. The structure was refined to a crystallographic R-factor of 0.228 ( R free = 0.335) with I > 2σ( I) reflections in the resolution range of 8 to 2.9 Å. The asymmetric unit contains a dimer. Tetramers are built up from dimers by crystallographic symmetry. The subunit structure of the plasma enzyme shows the typical structure motif of the thioredoxin fold consisting of a central β-sheet and several flanking α-helices. The active site selenocysteine residue is situated in the loop between β1 and α1 and is located in a pocket on the protein surface. The overall structure of the human plasma enzyme is similar to that of the bovine erythrocyte enzyme. The main differences in their subunit structures are an extended N terminus and the possible existence of a disulfide bridge in the plasma enzyme. Compared to the bovine erythrocyte enzyme, a number of residues in the active site are mutated or deleted in the plasma enzyme, including all the residues that were previously suggested to be involved in glutathione binding. The observed structural differences between the two enzymes suggest differences in substrate binding and specificity.

Differences in peptide-binding specificity of two ankylosing spondylitis-associated HLA-B27 subtypes.

Two HLA-B27 subtypes, B*2702 and B*2705, both associated with ankylosing spondylitis, were tested for binding affinity with a panel of polyalanine model nonapeptides carrying Arg at position 2 (P2) and a series of different amino acids at position 9 (P9). The alpha chains were isolated from BTB(B*2705), C1R/B*2702 (a B*2702 transfectant cell line) and from the NW (B*2702) cell line that has a peculiar peptide presentation behavior. Peptide binding was measured by the HLA alpha chain refolding assay. The results obtained show that: 1) Peptides with basic residues (Arg and Lys) and also aliphatic (Leu) and aromatic (Phe and Tyr) peptides at P9 have a similar high affinity in the binding to B*2705; 2) B*2702 binds well to P9 aliphatic and aromatic peptides but only very weakly to P9 basic peptides. Since both B*2702 and B*2705 are associated with AS the presumed arthritogenic peptide is hypothesized to have an aromatic or aliphatic residue at position 9. Peptides with basic residues in this position would be excluded as candidates because of their low binding affinity with B*2702.