Refinement of Protein Structures into Low-Resolution Density Maps Using Rosetta

We describe a method based on Rosetta structure refinement for generating high-resolution, all-atom protein models from electron cryomicroscopy density maps. A local measure of the fit of a model to the density is used to directly guide structure refinement and to identify regions incompatible with the density that are then targeted for extensive rebuilding. Over a range of test cases using both simulated and experimentally generated data, the method consistently increases the accuracy of starting models generated either by comparative modeling or by hand-tracing the density. The method can achieve near-atomic resolution starting from density maps at 4-6 Å resolution.     

基于超滤膜辅助的糖蛋白全N-连接糖链的富集和质谱解析

糖基化作为一种常见的蛋白质翻译后修饰,对蛋白质的空间结构、生物功能等具有重要的影响.解析糖蛋白糖链结构有助于更清楚地认识糖蛋白及其功能.本研究建立了一种基于超滤膜富集血清中糖蛋白全N-连接糖链,并利用质谱技术对糖链结构进行分析的方法.根据糖蛋白及其糖链结构之间的分子质量差异,利用Millipore公司的10 ku超滤膜富集血清糖蛋白上酶解(PNGase F)释放的全N-连接糖链,并使用MALDI-TOF/TOF-MS解析糖链结构.通过该技术可以从血清中富集并鉴定到23种独特的N-连接的糖链结构,并且利用二级质谱进行了结构确认.该方法可以被用于从大量生物样本中富集糖蛋白全N-连接糖链,可以达到快速、高通量地解析糖蛋白N-连接糖链的目的.     

An alpha-numeric code for representing N-linked glycan structures in secreted glycoproteins

Advances in high-throughput techniques have led to the creation of increasing amounts of glycome data. The storage and analysis of this data would benefit greatly from a compact notation for describing glycan structures that can be easily stored and interpreted by computers. Towards this end, we propose a fixed-length alpha-numeric code for representing N-linked glycan structures commonly found in secreted glycoproteins from mammalian cell cultures. This code, GlycoDigit, employs a pre-assigned alpha-numeric index to represent the monosaccharides attached in different branches to the core glycan structure. The present branch-centric representation allows us to visualize the structure while the numerical nature of the code makes it machine readable. In addition, a difference operator can be defined to quantitatively differentiate between glycan structures for further analysis. The usefulness and applicability of GlycoDigit were demonstrated by constructing and visualizing an N-linked glycosylation network.     

Modeling of protein conformational changes with Rosetta guided by limited experimental data

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Glycoprotein changes in the rat sperm plasma membrane during maturation in the epididymis.

To investigate surface glycoprotein changes during post-testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS-PAGE fractionated samples using biotinylated lectins and Vecta-stain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation-dependent sperm surface glycoproteins co-migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation-related surface changes occurring within the epididymis than are noted with surface labeling procedures.     

黏蛋白型O-聚糖在人类肿瘤中的结构异常及生物学功能

黏蛋白是细胞表面的或分泌的、具有高度O-糖基化修饰的糖蛋白.在黏蛋白中,O-聚糖(O-glycan)是通过N-乙酰氨基半乳糖与丝氨酸或苏氨酸之间形成α连接,该结构即被称为黏蛋白型O-聚糖.黏蛋白型O-聚糖是由多肽∶N-乙酰氨基半乳糖转移酶(ppGalNAc-T)家族催化起始合成的,近年来,该酶的催化机制及结构特点已成为糖基转移酶研究的热点.在肿瘤中常常伴随着黏蛋白型O-聚糖结构上和数量上的改变,形成肿瘤特异聚糖结构(cancer-associated glycans),如肿瘤Tn和T抗原等.肿瘤特异聚糖使肿瘤细胞的抗原性和黏附能力发生改变,促进肿瘤细胞的恶性增生与转移.而这些肿瘤特异聚糖结构,也为肿瘤的诊断与抗肿瘤药物或疫苗开发提供了理论基础.     

Characterization of gp93, a Novel, Highly Heterogeneous Glycoprotein Present in Growth Cone Membranes

Abstract: gp93 was first described in growth cones from fetal rat brain as a 90–97-kDa glycoprotein family that binds wheat-germ agglutinin and consists of at least 12 different isoelectric variants (pl range ∼4.9–6.4). Of particular interest is that different sets of gp93 variants are expressed in growth cones isolated from different brain regions. The preparation of a polyclonal antibody to gp93 allowed further characterization of this glycoprotein. The carbohydrate groups of gp93 were partially characterized by digestion with different glycosidases. The results indicate that most or all oligosaccharide units are N-linked (asparagine-linked) and contain sialic acid. Two-dimensional polyacrylamide gel electrophoresis and western blot with anti-gp93 show that deglycosylated gp93 is an only slightly heterogeneous polypeptide of 66 kDa, indicating that gp93 heterogeneity is due, primarily or exclusively, to differential glycosylation. Analysis of the tissue distribution in fetal rat showed gp93 to be highly enriched in the brain. Immunoblots and immunostaining of cross sections of developing cerebellum revealed that gp93 is developmentally regulated in this tissue, associated primarily with growing parallel fibers and Purkinje dendrites. Immunostaining of neurons in culture shows significant amounts of gp93 in elongating neurites and growth cones. Our results indicate that gp93 is a developmentally regulated glycoprotein of the brain that is most prominent in growth cones and growing neurites and that appears to be glycosylated differentially by different neurons.     

Sampling Native-like Structures of RNA-Protein Complexes through Rosetta Folding and Docking

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Purification and Characterization of a Major Cell Surface Glycoprotein in Zajdela Ascites Hepatoma Cells Which Displays a Potent Concanavalin A Receptor Activity

A major cell surface sialoglycoprotein with Concanavalin A receptor activity has been isolated from rat Zajdela ascites hepatoma cells. The sialic acid residues of the plasma membrane glycoproteins were specifically labeled by oxidation with NaIO₄ followed by reduction with NaB³H₄. Surface-labeled glycoproteins were released by short incubations with TPCK-trypsin at 37°C and then separated by gel filtration on Sepharose 6B column. The predominantly labeled fraction, GP II₂, was then purified by chromatography on DEAE-cellulose equilibrated with 0.05 M phosphate buffer, pH 7.5, and eluted with increasing molarities of NaCl. It was shown to be homogeneous by protein and carbohydrate staining on SDS-polyacrylamide gels, isoelectric focusing, rechromatography on DEAE-cellulose and immunoelectrophoresis. It has an apparent molecular weight of 110,000 daltons. The location of GP II₂on the cell surface was confirmed by the fact that it could be labeled metabolically with, D-(³H) glucosamine and externally through the nonpenetrating periodate-NaB³H₄ system. GP II₂could not be removed from the cell surface by high salt concentrations, chelator, or chaotropic agents but was released from the membrane by detergents. This suggests that GP II₂could be an integral protein. Analysis of the carbohydrate composition of GP II₂ revealed galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid as major constituents and mannose as a minor one. This suggests that it contains carbohydrate chains both O- and N-linked to the polypeptide chain, most of them being O-linked. Finally, GP II₂has a potent Concanavalin A receptor activity. It inhibits the interaction between Concanavalin A and hepatoma cells and suppresses its effects on hepatoma cell proliferation.     

人类疱疹病毒7型糖蛋白gB、gH、gL、gO介导细胞融合

人类疱疹病毒 7 型(HHV-7)的感染依赖于包膜糖蛋白在病毒生命周期的多个阶段发挥功能. 这些蛋白质可以介导病毒吸附,病毒包膜和宿主细胞膜融合以及病毒在细胞间的接触传播. 将表达 HHV-7 糖蛋白的 293T 细胞与 HHV-7 易感的SupT1 细胞共培养,检测虫荧光素酶报告基因的表达,以鉴定介导膜融合的 HHV-7 糖蛋白. 研究发现,HHV-7 糖蛋白 gB、gH、gL、gO 能介导 293T 细胞与 SupT1 细胞的融合,且融合可被抗 CD4 单抗所抑制. 结果表明,糖蛋白 gB、gH、gL、gO对于 HHV-7 引发的膜融合是必需的,其中某个蛋白质或所形成的蛋白质复合物可能是 CD4 的配体.     

Crystal Structures of Fungal Tectonin in Complex with O-Methylated Glycans Suggest Key Role in Innate Immune Defense

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Glycoprotein differences among cells of the 14-day embryonic chick neural retina

In order to test the hypothesis that the progressive layering and differentiation of cell types during the development of the neural retina are associated with cell surface alterations we have separated distinct cell populations from the 14-day embryonic chick retina. Cells of these populations have been shown to differ in associative behavior and intramembrane particle content. We now report that these cells differ in cell surface glycoproteins. Proteins were labeled with two different extrinsic labels and one metabolic label. We used enzymatic transfer of galactose from UDP-gal to cellular acceptors, and borotritide reduction after galactose oxidation as extrinsic labels. Glucosamine incorporation was used as the metabolic label. In all these cases, we were able to identify bands on electrophoretic gels which were unique to individual populations.     

A Glycoprotein Expressed by Human Fibrous Astrocytes is a Hyaluronate-Binding Protein and a Member of the CD44 Family

We have isolated and characterized an antigen from normal human brain called p80, so called because it migrated with an Mr of 80 kDa on SDS PAGE. The Mr of 80 kDa consists of a protein of about 55-60 kDa and carbohydrate (20-25 kDa). The carbohydrate is almost entirely of the N-linked type, although a small amount of O-linked carbohydrate was detected. Cross-reactivity with monoclonal antibodies A3D8 and A1G3 showed that p80 could therefore be considered an isoform of the CD44 adhesion molecules. In addition, specific binding to hyaluronate which was not competed for by proteoglycan demonstrated that it involved different sites than the proteoglycan binding sites. We also observed that fucoidan and dextran sulphate increased the binding by 200-250% while chondroitin sulphate C also increased the binding but to a lesser extent. Heparin, heparan sulphate and chondroitin sulphates A and B did not have such an effect. The binding of p80 to hyaluronate was pH dependent with a maximum at pH 6.4. We concluded that p80 was an astrocyte specific adhesion molecule.     

Improving NMR protein structure quality by Rosetta refinement: a molecular replacement study

The structure of human protein HSPC034 has been determined by both solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. Refinement of the NMR structure ensemble, using a Rosetta protocol in the absence of NMR restraints, resulted in significant improvements not only in structure quality, but also in molecular replacement (MR) performance with the raw X-ray diffraction data using MOLREP and Phaser. This method has recently been shown to be generally applicable with improved MR performance demonstrated for eight NMR structures refined using Rosetta (Qian et al., Nature 2007;450:259-264). Additionally, NMR structures of HSPC034 calculated by standard methods that include NMR restraints have improvements in the RMSD to the crystal structure and MR performance in the order DYANA, CYANA, XPLOR-NIH, and CNS with explicit water refinement (CNSw). Further Rosetta refinement of the CNSw structures, perhaps due to more thorough conformational sampling and/or a superior force field, was capable of finding alternative low energy protein conformations that were equally consistent with the NMR data according to the Recall, Precision, and F-measure (RPF) scores. On further examination, the additional MR-performance shortfall for NMR refined structures as compared with the X-ray structure were attributed, in part, to crystal-packing effects, real structural differences, and inferior hydrogen bonding in the NMR structures. A good correlation between a decrease in the number of buried unsatisfied hydrogen-bond donors and improved MR performance demonstrates the importance of hydrogen-bond terms in the force field for improving NMR structures. The superior hydrogen-bond network in Rosetta-refined structures demonstrates that correct identification of hydrogen bonds should be a critical goal of NMR structure refinement. Inclusion of nonbivalent hydrogen bonds identified from Rosetta structures as additional restraints in the structure calculation results in NMR structures with improved MR performance.     

Bacteriophage T5 structure reveals similarities with HK97 and T4 suggesting evolutionary relationships

Evolutionary relationships between viruses may be obscure by protein sequence but unmasked by structure. Analysis of bacteriophage T5 by cryo-electron microscopy and protein sequence analysis reveals analogies with HK97 and T4 that suggest a mosaic of such connections. The T5 capsid is consistent with the HK97 capsid protein fold but has a different geometry, incorporating three additional hexamers on each icosahedral facet. Similarly to HK97, the T5 major capsid protein has an N-terminal extension, or Delta-domain that is missing in the mature capsid, and by analogy with HK97, may function as an assembly or scaffold domain. This Delta-domain is predicted to be largely coiled-coil, as for that of HK97, but is approximately 70% longer correlating with the larger capsid. Thus, capsid architecture appears likely to be specified by the Delta-domain. Unlike HK97, the T5 capsid binds a decoration protein in the center of each hexamer similarly to the "hoc" protein of phage T4, suggesting a common role for these molecules. The tail-tube has unusual trimeric symmetry that may aid in the unique two-stage DNA-ejection process, and joins the tail-tip at a disk where tail fibers attach. This intriguing mix of characteristics embodied by phage T5 offers insights into virus assembly, subunit function, and the evolutionary connections between related viruses.     

Structures of Gα Proteins in Complex with Their Chaperone Reveal Quality Control Mechanisms

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Involvement of a highly polyvalent glycan in the cell-binding of the aggregation factor from the marine sponge Microciona prolifera

A proteoglycan-like aggregation factor from the marine sponge Microciona prolifera (MAF) mediates cell-cell recognition via a cell-binding and a self-association domain. After repetitive and prolonged treatment of MAF with glycopeptide-N-glycosidase (PNGase) the specific binding of MAF to homotypic cells was decreased by 72%. Polyacrylamide gel electrophoresis and gel filtration analysis of such PNGase digests showed that: 1) the enzyme released a single glycan type of Mr = 6 X 10(3) (G-6) from MAF, 2) 1 mole of MAF contains at least 830 moles of N-linked chains of G-6 glycan. The correlation between the loss of the binding activity of MAF and the extent of the release of the repetitive G-6 polysaccharide strongly suggests its involvement in MAF-cell association via highly polyvalent interactions.     

Effects of buffering conditions and culture pH on production rates and glycosylation of clinical phase I anti-melanoma mouse IgG3 monoclonal antibody R24

R24, a mouse IgG3 monoclonal antibody (MAb) against ganglioside GD3 (Neu5Acalpha8Neu5Acalpha3Gal beta4Glcbeta1Cer), can block tumor growth as reported in a series of clinical trials in patients with metastatic melanoma. The IgG molecule basically contains an asparagine-linked biantennary complex type oligosaccharide on the C(H)2 domain of each heavy chain, which is necessary for its in vivo effector function. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important MAb in CO(2)/HCO(3) (-) (pH 7.4, 7.2, and 6.9) and HEPES buffered serum-free medium. Growth, metabolism, and IgG production of hybridoma cells (ATCC HB-8445) were analyzed on a 2-L bioreactor scale using fed-batch mode. Specific growth rates (mu) and MAb production rates (q(IgG)) varied significantly with maximum product yields at pH 6.9 (q(IgG) = 42.9 microg 10(-6) cells d(-1), mu = 0.30 d(-1)) and lowest yields in pH 7.4 adjusted batches (q(IgG) = 10.8 microg 10(-6) cells d(-1), mu = 0.40 d(-1)). N-glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray-ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry (MS). The highest relative amounts of agalacto and monogalacto biantennary complex type oligosaccharides were detected in the pH 7.2 (46% and 38%, respectively) and pH 6.9 (44% and 40%, respectively) cultivations and the uppermost quantities of digalacto (fully galactosylated) structures in the pH 7.4 (32%) and the HEPES (26%) buffered fermentation. In the experiments with HEPES buffering, antibodies with a molar Neu5Ac/Neu5Gc ratio of 3.067 were obtained. The fermentations at pH 7.2 and 6.9 resulted in almost equal molar Neu5Ac/Neu5Gc ratios of 1.008 and 0.985, respectively, while the alkaline shift caused a moderate overexpression of Neu5Ac deduced from the Neu5Ac/Neu5Gc quotient of 1.411. Different culture buffering gave rise to altered glycosylation pattern of the MAb R24. Consequently, a detailed molecular characterization of MAb glycosylation is generally recommended as a part of the development of MAbs for targeted in vivo immunotherapy to assure biochemical consistency of product lots and oligosaccharide-dependent biological activity.     

Structure-based prediction of protein-peptide specificity in Rosetta

Protein-peptide interactions mediate many of the connections in intracellular signaling networks. A generalized computational framework for atomically precise modeling of protein-peptide specificity may allow for predicting molecular interactions, anticipating the effects of drugs and genetic mutations, and redesigning molecules for new interactions. We have developed an extensible, general algorithm for structure-based prediction of protein-peptide specificity as part of the Rosetta molecular modeling package. The algorithm is not restricted to any one peptide-binding domain family and, at minimum, does not require an experimentally characterized structure of the target protein nor any information about sequence specificity; although known structural data can be incorporated when available to improve performance. We demonstrate substantial success in specificity prediction across a diverse set of peptide-binding proteins, and show how performance is affected when incorporating varying degrees of input structural data. We also illustrate how structure-based approaches can provide atomic-level insight into mechanisms of peptide recognition and can predict the effects of point mutations on peptide specificity. Shortcomings and artifacts of our benchmark predictions are explained and limits on the generality of the method are explored. This work provides a promising foundation upon which further development of completely generalized, de novo prediction of peptide specificity may progress.     

High-accuracy refinement using Rosetta in CASP13

Because proteins generally fold to their lowest free energy states, energy-guided refinement in principle should be able to systematically improve the quality of protein structure models generated using homologous structure or co-evolution derived information. However, because of the high dimensionality of the search space, there are far more ways to degrade the quality of a near native model than to improve it, and hence, refinement methods are very sensitive to energy function errors. In the 13th Critial Assessment of techniques for protein Structure Prediction (CASP13), we sought to carry out a thorough search for low energy states in the neighborhood of a starting model using restraints to avoid straying too far. The approach was reasonably successful in improving both regions largely incorrect in the starting models as well as core regions that started out closer to the correct structure. Models with GDT-HA over 70 were obtained for five targets and for one of those, an accuracy of 0.5 å backbone root-mean-square deviation (RMSD) was achieved. An important current challenge is to improve performance in refining oligomers and larger proteins, for which the search problem remains extremely difficult.