UCSF Chimera Research Articles

Neonicotinoids in vector control: In silico approach

Insecticide based intervention remains the primary control tool for vector borne diseases. Many mosquito vectors have developed resistance to the insecticides which are in use in public health programmes. There is a need to search for alternative insecticides to manage the insecticide resistance. Neonicotinoids are one of the emerging new promising insecticides with novel mode of action against agricultural pests. To understand the neonicotinoids efficacy in vector control, In silico docking study has been explored between the neonicotinoids and nicotinic acetylcholine receptor of Anopheles gambiae Giles (Diptera: Culicidae). The format of nicotinic acetylcholine receptor (nAChR) protein subunits sequence of the An. gambiae was retrieved from NCBI database. Three dimensional structure of nAChR protein subunits has been predicted using Modeller 9.10. The predicted protein structure was energy minimized by Swiss Pdb viewer 4.1. Stereo chemical analysis of a structure was performed using PROCHECK Ramachandran plots from SAVES server. Validated protein structure was visualized by UCSF Chimera 1.5.3 and models were submitted in Protein Model Database (PMDB). Molegro virtual docker 5.0 was used for nAChR protein subunits and neonicotinoid compounds docking study. Nicotinic acetylcholine receptor subunit’s three dimensional structure predicted models were submitted in Protein Model Database having PMID PM0079637, PM0079638, PM0079639, PM0079640, PM0079641, PM0079642, PM0079643, PM0079644, PM0079645 and PM0079646. Each neonicotinoid compound selectively bound to the receptor subunits individually. The best conformation with the least binding energy was selected. Out of seven compounds, dinotefuran has the least binding energy value of –110 Mol Dock score and Re rank score of –94.63 with more number of H-bond interactions showing the highest activity in nAChR α3 subunit. The docking scores of neonicotinoid compounds against nicotinic acetylcholine receptor subunit of An. gambiae are in the order of dinotefuran >thiamethoxam >clothianidin >imidacloprid >nitenpyram >acetamiprid> thiacloprid. Among the seven neonicotinoids, dinotefuran has shown the least docking score with highest activity and possibly this molecule can be used in control of mosquito vectors.

RRDistMaps: a UCSF Chimera tool for viewing and comparing protein distance maps.

Contact maps are a convenient method for the structural biologists to identify structural features through two-dimensional simplification. Binary (yes/no) contact maps with a single cutoff distance can be generalized to show continuous distance ranges. We have developed a UCSF Chimera tool, RRDistMaps, to compute such generalized maps in order to analyze pairwise variations in intramolecular contacts. An interactive utility, RRDistMaps, visualizes conformational changes, both local (e.g. binding-site residues) and global (e.g. hinge motion), between unbound and bound proteins through distance patterns. Users can target residue pairs in RRDistMaps for further navigation in Chimera. The interface contains the unique features of identifying long-range residue motion and aligning sequences to simultaneously compare distance maps. RRDistMaps was developed as part of UCSF Chimera release 1.10, which is freely available at http://rbvi.ucsf.edu/chimera/download.html, and operates on Linux, Windows, and Mac OS. conrad@cgl.ucsf.edu.

Homology modeling and molecular docking studies of ArnA protein from Erwinia amylovora: role in polymyxin antibiotic resistance

The resistivity of plant pathogen Erwinia amylovora against the polymyxin group of antibiotics is enhanced by modification of lipid A from lipopolysaccharide with 4-amino-4-deoxy L-arabinose (Ara4N) catalyzed by a bifunctional protein ArnA. ArnA is the first enzyme in the lipid A modification pathway with distinct dehydrogenase and transformylase domains which has been known in development of resistivity to polymyxin group of antibiotics. Thus, three dimensional structure of ArnA protein from Erwinia amylovora was constructed using homology modeling technique. The quality and reliability of the generated 3-D model was then assessed by different online available programs such as What if, PROCHECK, QMEAN, ProSA along with superimposition by UCSF Chimera. Sequence analysis study of ArnA protein from E. coli, Erwinia amylovora, Yersinia pestis, Ps. aeruginosa and Salmonella showed conserved domains with exact active site residues. Molecular docking study of ArnA protein with substrate UDP-GlcA and different inhibitors such as 5-formyl-5,6,7,8 tetrahydrofolate, leucovorin and 5-methyl tetrahydrofolate revealed similar binding pocket. The residues ASN492, ARG510, SER433 and ARG619 of ArnA protein are involved in interactions with inhibitors. Thus, this study could be useful to understand the proper binding mode of inhibitors to inhibit the lipid A modification pathway of ArnA protein from plant pathogen Erwinia amylovora.

Issue Cover (September 2014)

Cover legend: Lipid transfer proteins (LTPs) begin to act on the stage center. X-ray -crystallography -information of lipid transfer proteins in -complex with lipid ligands was obtained from the RCSB protein -database (PDB). With the UCSF Chimera program, proteins with ligands were three-dimensionally viewed. From top left to bottom right, human CERT START domain with C16-ceramide (PDB code: 2E3O), yeast Osh3p -OSBP-related domain with phosphatidylinositol 4-monophosphate (PDB code: 4INQ), human α-tocopherol transfer -protein with α-tocopherol (PDB code: 1OIP), and human glycolipid transfer protein with -lactosylceramide, (PDB code: 1SX6). Lipid ligands are shown as the sphere model. For detail, see the -thematic review series that starts from this issue. (Image courtesy of Kentaro Hanada) See -Hurlock et al. Traffic 15(9): 915–932 Read the full article on doi:10.1111/tra.12187

The α-Subunit Regulates Stability of the Metal Ion at the Ligand-associated Metal Ion-binding Site in β3 Integrins

The aspartate in the prototypical integrin-binding motif Arg-Gly-Asp binds the integrin βA domain of the β-subunit through a divalent cation at the metal ion-dependent adhesion site (MIDAS). An auxiliary metal ion at a ligand-associated metal ion-binding site (LIMBS) stabilizes the metal ion at MIDAS. LIMBS contacts distinct residues in the α-subunits of the two β3 integrins αIIbβ3 and αVβ3, but a potential role of this interaction on stability of the metal ion at LIMBS in β3 integrins has not been explored. Equilibrium molecular dynamics simulations of fully hydrated β3 integrin ectodomains revealed strikingly different conformations of LIMBS in unliganded αIIbβ3 versus αVβ3, the result of stronger interactions of LIMBS with αV, which reduce stability of the LIMBS metal ion in αVβ3. Replacing the αIIb-LIMBS interface residue Phe(191) in αIIb (equivalent to Trp(179) in αV) with Trp strengthened this interface and destabilized the metal ion at LIMBS in αIIbβ3; a Trp(179) to Phe mutation in αV produced the opposite but weaker effect. Consistently, an F191/W substitution in cellular αIIbβ3 and a W179/F substitution in αVβ3 reduced and increased, respectively, the apparent affinity of Mn(2+) to the integrin. These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in αVβ3 structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation.

Enhancing UCSF Chimera through web services

Integrating access to web services with desktop applications allows for an expanded set of application features, including performing computationally intensive tasks and convenient searches of databases. We describe how we have enhanced UCSF Chimera (http://www.rbvi.ucsf.edu/chimera/), a program for the interactive visualization and analysis of molecular structures and related data, through the addition of several web services (http://www.rbvi.ucsf.edu/chimera/docs/webservices.html). By streamlining access to web services, including the entire job submission, monitoring and retrieval process, Chimera makes it simpler for users to focus on their science projects rather than data manipulation. Chimera uses Opal, a toolkit for wrapping scientific applications as web services, to provide scalable and transparent access to several popular software packages. We illustrate Chimera's use of web services with an example workflow that interleaves use of these services with interactive manipulation of molecular sequences and structures, and we provide an example Python program to demonstrate how easily Opal-based web services can be accessed from within an application. Web server availability: http://webservices.rbvi.ucsf.edu/opal2/dashboard?command=serviceList.

Expanding the spectrum of HEXA mutations in Indian patients with Tay–Sachs disease

Tay–Sachs disease is an autosomal recessive neurodegenerative disorder occurring due to impaired activity of β-hexosaminidase-A (EC 3.2.1.52), resulting from the mutation in HEXA gene. Very little is known about the molecular pathology of TSD in Indian children except for a few mutations identified by us. The present study is aimed to determine additional mutations leading to Tay–Sachs disease in nine patients confirmed by the deficiency of β-hexosaminidase-A (<2% of total hexosaminidase activity for infantile patients) in leucocytes. The enzyme activity was assessed by using substrates 4-methylumbelliferyl-N-acetyl-β-d-glucosamine and 4-methylumbelliferyl-N-acetyl-β-d-glucosamine-6-sulfate for total-hexosaminidase and hexosaminidase-A respectively, and heat inactivation method for carrier detection. The exons and exon–intron boundaries of the HEXA gene were bi-directionally sequenced on an automated sequencer. ‘In silico’ analyses for novel mutations were carried out using SIFT, Polyphen2 and MutationT@ster software programs. The structural study was carried out by UCSF Chimera software using the crystallographic structure of β-hexosaminidase-A (PDB-ID: 2GJX) as the template. Our study identified four novel mutations in three cases. These include a compound heterozygous missense mutation c.524A>C (D175A) and c.805G>C (p.G269R) in one case; and one small 1bp deletion c.426delT (p.F142LfsX57) and one splice site mutation c.459+4A>C in the other two cases respectively. None of these mutations were detected in 100 chromosomes from healthy individuals of the same ethnic group. Three previously reported missense mutations, (i) c.532C>T (p.R178C), (ii) c.964G>T (p.D322Y), and (iii) c.1385A>T (p.E462V); two nonsense mutations (i) c.709C>T (p.Q237X) and (ii) c.1528C>T (p.R510X), one 4bp insertion c.1277_1278insTATC (p.Y427IfsX5) and one splice site mutation c.459+5G>A were also identified in six cases. We observe from this study that novel mutations are more frequently observed in Indian patients with Tay–Sachs disease with clustering of ~73% of disease causing mutations in exons 5 to 12. This database can be used for a carrier rate screening in the larger population of the country.

Assessment of template‐free modeling in CASP10 and ROLL

We present the assessment of predictions for Template-Free Modeling in CASP10 and a report on the first ROLL experiment wherein predictions are collected year round for review at the regular CASP season. Models were first clustered so that duplicated or very similar ones were grouped together and represented by one model in the cluster. The representatives were then compared with targets using GDT_TS, QCS, and three additional superposition-independent score functions newly developed for CASP10. For each target, the top 15 representatives by each score were pooled to form the Top15Union set. All models in this set were visually inspected by four of us independently using the new plugin, EvalScore, which we developed with the UCSF Chimera group. The best models were selected for each target after extensive debate among the four examiners. Groups were ranked by the number of targets (hits) for which a group's model was selected as one of the best models. The Keasar group had most hits in both categories, with four of 19 FM and eight of 36 ROLL targets. The most successful prediction servers were QUARK from Zhang's group for FM category with three hits and Zhang-server for the ROLL category with seven hits. As observed in CASP9, many successful groups were not true "template-free" modelers but used remote templates and/or server models to obtain their winning models. The results of the first ROLL experiment were broadly similar to those of the CASP10 FM exercise.

Characterization of Three Full-length Human Nonmuscle Myosin II Paralogs

Nonmuscle myosin IIs (NM IIs) are a group of molecular motors involved in a wide variety of cellular processes including cytokinesis, migration, and control of cell morphology. There are three paralogs of the NM II heavy chain in humans (IIA, IIB, and IIC), each encoded by a separate gene. These paralogs are expressed at different levels according to cell type and have different roles and intracellular distributions in vivo. Most previous studies on NM II used tissue-purified protein or expressed fragments of the molecule, which presents potential drawbacks for characterizing individual paralogs of the intact protein in vitro. To circumvent current limitations and approach their native properties, we have successfully expressed and purified the three full-length human NM II proteins with their light chains, using the baculovirus/Sf9 system. The enzymatic and structural properties of the three paralogs were characterized. Although each NM II is capable of forming bipolar filaments, those formed by IIC tend to contain fewer constituent molecules than those of IIA and IIB. All paralogs adopt the compact conformation in the presence of ATP. Phosphorylation of the regulatory light chain leads to assembly into filaments, which bind to actin in the presence of ATP. The nature of interactions with actin filaments is shown with different paralogs exhibiting different actin binding behaviors under equivalent conditions. The data show that although NM IIA and IIB form filaments with similar properties, NM IIC forms filaments that are less well suited to roles such as tension maintenance within the cell.

Ab Initio prediction of mycobacteriophages protein structure and function

* Correspondence: claire.rinehart@wku.edu Department of Biology, Western Kentucky University, Bowling Green, KY, 42101, USA Figure 1 Seven predicted pham 6714 structures superimposed into a single model matching methyltransferase structures. PDB files were rendered using UCSF Chimera (http://www.cgl.ucsf.edu/chimera/). Proteins include MeeZee gp 78, JN243856.1; Backyardigan gp 74, JF704093.1; and ShiLan gp 70, JN020143.1 Kapadia and Rinehart BMC Bioinformatics 2013, 14(Suppl 17):A10 http://www.biomedcentral.com/1471-2105/14/S17/A10

Inhibition of VEGF: a novel mechanism to control angiogenesis by Withania somnifera's key metabolite Withaferin A.

PurposeAngiogenesis, or new blood vessel formation from existing one, plays both beneficial and detrimental roles in living organisms in different aspects. Vascular endothelial growth factor (VEGF), a signal protein, well established as key regulator of vasculogenesis and angiogenesis. VEGF ensures oxygen supply to the tissues when blood supply is not adequate, or tissue environment is in hypoxic condition. Limited expression of VEGF is necessary, but if it is over expressed, then it can lead to serious disease like cancer. Cancers that have ability to express VEGF are more efficient to grow and metastasize because solid cancers cannot grow larger than a limited size without adequate blood and oxygen supply. Anti-VEGF drugs are already available in the market to control angiogenesis, but they are often associated with severe side-effects like fetal bleeding and proteinuria in the large number of patients. To avoid such side-effects, new insight is required to find potential compounds as anti-VEGF from natural sources. In the present investigation, molecular docking studies were carried out to find the potentiality of Withaferin A, a key metabolite of Withania somnifera, as an inhibitor of VEGF.MethodsMolecular Docking studies were performed in DockingServer and SwissDock. Bevacizumab, a commercial anti-VEGF drug, was used as reference to compare the activity of Withaferin A. X-ray crystallographic structure of VEGF, was retrieved from Protein Data Bank (PDB), and used as drug target protein. Structure of Withaferin A and Bevacizumab was obtained from PubChem and ZINC databases. Molecular visualization was performed using UCSF Chimera.ResultsWithaferin A showed favorable binding with VEGF with low binding energy in comparison to Bevacizumab. Molecular Docking studies also revealed potential protein-ligand interactions for both Withaferin A and Bevacizumab.ConclusionsConclusively our results strongly suggest that Withaferin A is a potent anti-VEGF agent as ascertained by its potential interaction with VEGF. This scientific hypothesis might provide a better insight to control angiogenesis as well as to control solid cancer growth and metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-9616-1-11) contains supplementary material, which is available to authorized users.

Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain.

Abstract 2891: Comparative analyses of human estrogen receptor- EF-hand protein complexes: molecular basis for hormone independent activation.

Abstract Estrogen Receptor (ER) belongs to the nuclear receptor super family of ligand-triggered transcription factors. ER is present in about 70% of breast cancers and plays a critical role in the biology of breast carcinoma and improves prognosis. Estrogen binds to ER and mediates a cascade of cellular signaling events. It has been shown that several proteins including calcium binding proteins with EF-hand motifs, which control cellular calcium and calcium mediated signaling pathways, including calmodulin (CaM) could bind to unliganded ER and activate ER. CaM has four EF-hand motifs that change conformation upon binding Ca2+ ions. Ca2+-CaM complex binds to the target proteins and initiates various signaling cascades. Each EF-hand motif contains two alpha helices connected by a 12-residue loop. Both X-ray and NMR studies show that the alpha helices of the EF-hand motifs change their position relative to each other, forming an almost perpendicular conformation. This allows calmodulin to increase its binding affinity for a number of target proteins. CaM forms a compact globular conformation by bending its central helix upon binding its target peptides. S100, a soluble protein that is recently reported to interact with ER has only two EF-hand motifs. S100 protein is overexpressed in several cancers and shares a high degree of sequence homology with calmodulin. Methods: Protein-protein interactions of CaM, S100 with ER alpha were simulated using HADDOCK and HEX. Docking results were analyzed in UCSF Chimera and integrated with data from in vitro proteomics experiments to determine the conformations of ER bound EF-hand protein complexes. Proteomic experiments consisted of chemical crosslinking of the corresponding protein complexes followed by tryptic digestion, analyzing the resulting peptide peaks using FTICR- Mass Spectrometry. Results/Discussion: Using the fold information and contact regions obtained from the Mass spectrometry, 3D structures of ER-EF hand protein complexes were reconstructed. These structures of ER best represent the conformational state sensed by the specific interacting partner. ER-S100 complex is distinct from CaM. The lack of the connective peptidic region between the EF-hand pairs in S100 attributed to less interaction coverage indicative of only inducing a partial agonist-like conformation. We believe that the ensemble of ER-EF hand protein complexes generated by our integrated proteomics-assisted protein interaction profiling will shed light on the lingering issue of hormone independent activation of ER at the molecular level. Acknowledgements: This research was supported via funds from the New York State, National Institutes of Health (5P20MD002725), through the NCMHD -RIMI program (RVR) Citation Format: David H. Lee, Bethany K. Asare, Suchitra Singh, Rajendram V. Rajnarayanan. Comparative analyses of human estrogen receptor- EF-hand protein complexes: molecular basis for hormone independent activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2891. doi:10.1158/1538-7445.AM2013-2891

Crystal Structures of the DNA-binding Domain Tetramer of the p53 Tumor Suppressor Family Member p73 Bound to Different Full-site Response Elements

How cells choose between developmental pathways remains a fundamental biological question. In the case of the p53 protein family, its three transcription factors (p73, p63, and p53) each trigger a gene expression pattern that leads to specific cellular pathways. At the same time, these transcription factors recognize the same response element (RE) consensus sequences, and their transactivation of target genes overlaps. We aimed to understand target gene selectivity at the molecular level by determining the crystal structures of the p73 DNA-binding domain (DBD) in complex with full-site REs that vary in sequence. We report two structures of the p73 DBD bound as a tetramer to 20-bp full-site REs based on two distinct quarter-sites: GAACA and GAACC. Our study confirms that the DNA-binding residues are conserved within the p53 family, whereas the dimerization and tetramerization interfaces diverge. Moreover, a conserved lysine residue in loop L1 of the DBD senses the presence of guanines in positions 2 and 3 of the quarter-site RE, whereas a conserved arginine in loop 3 adapts to changes in position 5. Sequence variations in the RE elicit a p73 conformational response that might explain target gene specificity.

A LiTat 1.5 variant surface glycoprotein‐derived peptide with diagnostic potential for Trypanosoma brucei gambiense

To evaluate the accuracy of a peptide, corresponding to the variant surface glycoprotein (VSG) LiTat 1.5 amino acid (AA) sequence 268-281 and identified through alignment of monoclonal antibody selected mimotopes, for diagnosis of Trypanosoma brucei gambiense sleeping sickness. A synthetic biotinylated peptide (peptide 1.5/268-281), native VSG LiTat 1.3 and VSG LiTat 1.5 were tested in an indirect ELISA with 102 sera from patients with HAT and 102 endemic HAT-negative controls. The area under the curve (AUC) of peptide 1.5/268-281 was 0.954 (95% confidence interval 0.918-0.980), indicating diagnostic potential. The areas under the curve of VSG LiTat 1.3 and LiTat 1.5 were 1.000 (0.982-1.000) and 0.997 (0.973-1.000), respectively, and significantly higher than the AUC of peptide 1.5/268-281. On a model of VSG LiTat 1.5, peptide 1.5/268-281 was mapped near the top of the VSG. A biotinylated peptide corresponding to AA 268-281 of VSG LiTat 1.5 may replace the native VSG in serodiagnostic tests, but the diagnostic accuracy is lower than for the full-length native VSG LiTat 1.3 and VSG LiTat 1.5.

The Native Structure and Composition of the Cruciferin Complex in Brassica napus

Globulins are an important group of seed storage proteins in dicotyledonous plants. They are synthesized during seed development, assembled into very compact protein complexes, and finally stored in protein storage vacuoles (PSVs). Here, we report a proteomic investigation on the native composition and structure of cruciferin, the 12 S globulin of Brassica napus. PSVs were directly purified from mature seeds by differential centrifugations. Upon analyses by blue native (BN) PAGE, two major types of cruciferin complexes of ∼ 300-390 kDa and of ∼470 kDa are resolved. Analyses by two-dimensional BN/SDS-PAGE revealed that both types of complexes are composed of several copies of the cruciferin α and β polypeptide chains, which are present in various isoforms. Protein analyses by two-dimensional isoelectric focusing (IEF)/SDS-PAGE not only revealed different α and β isoforms but also several further versions of the two polypeptide chains that most likely differ with respect to posttranslational modifications. Overall, more than 30 distinct forms of cruciferin were identified by mass spectrometry. To obtain insights into the structure of the cruciferin holocomplex, a native PSV fraction was analyzed by single particle electron microscopy. More than 20,000 images were collected, classified, and used for the calculation of detailed projection maps of the complex. In contrast to previous reports on globulin structure in other plant species, the cruciferin complex of Brassica napus has an octameric barrel-like structure, which represents a very compact building block optimized for maximal storage of amino acids within minimal space.

Characterization of the Molecular Architecture of Human Caveolin-3 and Interaction with the Skeletal Muscle Ryanodine Receptor

Caveolin-3 facilitates both caveolae formation and a range of cell signaling pathways, including Ca(2+) homeostasis. Caveolin-3 forms a disc-shaped nonamer that binds the Ca(2+)-release channel, RyR1. Multiple caveolin-3 nonamers bind to a single RyR1 homotetramer. First three-dimensional structural insights into caveolin-3 assembly, interactions with RyR1 suggest a novel role in muscle contraction and/or for channel localization within the membrane. Caveolin-3 (cav-3), an integral membrane protein, is a building block of caveolae as well as a regulator of a number of physiological processes by facilitating the formation of multiprotein signaling complexes. We report that the expression of cav-3 in insect (Sf9) cells induces caveola formation, comparable in size with those observed in native tissue. We have also purified the recombinant cav-3 determining that it forms an oligomer of ∼220 kDa. We present the first three-dimensional structure for cav-3 (using transmission electron microscopy and single particle analysis methods) and show that nine cav-3 monomers assemble to form a complex that is toroidal in shape, ~16.5 nm in diameter and ~ 5.5 nm in height. Labeling experiments and reconstitution of the purified cav-3 into liposomes have allowed a proposal for the orientation of the protein with respect to the membrane. We have identified multiple caveolin-binding motifs within the ryanodine receptor (RyR1) sequence employing a bioinformatic analysis. We have then shown experimentally that there is a direct interaction between recombinant cav-3 nonamers and purified RyR1 homotetramers that would imply that at least one of the predicted cav-3-binding sites is exposed within the fully assembled RyR1 structure. The cav-3 three-dimensional model provides new insights as to how a cav-3 oligomer can bind multiple partners in close proximity to form signaling complexes. Furthermore, a direct interaction with RyR1 suggests a possible role for cav-3 as a modifier of muscle excitation-contraction coupling and/or for localization of the receptor to regions of the sarcoplasmic reticulum.

Maturation in Action: CryoEM Study of a Viral Capsid Caught during Expansion

Bacteriophage HK97 maturation involves discrete intermediate particle forms, comparable to transitional states in protein folding, before reaching its mature form. The process starts by formation of a metastable prohead, poised for exothermic expansion triggered by DNA packaging. During maturation, the capsid subunit transitions from a strained to a canonical tertiary conformation and this has been postulated to be the driving mechanism for initiating expansion via switching hexameric capsomer architecture from skewed to 6-fold symmetric. We report the subnanometer electron-cryomicroscopy reconstruction of the HK97 first expansion intermediate before any crosslink formation. This form displays 6-fold symmetric hexamers, but capsid subunit tertiary structures exhibit distortions comparable to the prohead forms. We propose that coat subunit strain release acts in synergy with the first crosslinks to drive forward maturation. Finally, we speculate that the energetic features of this transition may result from increased stability of intermediates during maturation via enhanced inter-subunit interactions.

A Novel p.E311K Mutation of Thyroid Receptor Beta Gene in Resistance to Thyroid Hormone Syndrome, Inherited in Autosomal Recessive Trait

Resistance to thyroid hormone (RTH) syndrome is caused by mutations in THRB gene and is inherited mainly as an autosomal dominant trait with dominant negative effect. Most of up-to-now described RTH cases were heterozygous. We studied a 19-year-old woman presenting severe mental impairment, hyperkinetic behavior, learning disability, hearing loss, tachycardia, goiter, strabismus, nystagmus, and normal stature. The laboratory findings revealed elevated TSH, T3, and T4 serum levels. Her parents were healthy with normal serum level of TSH, fT3, and fT4. Sequence based prediction of a substitution was analyzed by SDM, PolPhen, and SNAP software whereas structural visualizations were performed in UCSF Chimera. We found a novel mutation in THRB gene in position 1216 (G to A transition, codon 311) resulting in novel Glu-311-Lys (p.E311K) substitution, homozygous in proband presenting with severe symptoms of RTH and heterozygous in both of her healthy parents, thus suggesting autosomal recessive mode of inheritance. p.E311K substitution was not found in 50 healthy, unrelated individuals. p.E311K was shown to be deleterious by SDM, PolPhen, and SNAP software. Structural visualizations of mutated protein performed by UCSF Chimera software disclosed a loss of hydrogen bonds between E311, R383, and R429 along with abnormal residue-residue contact between K311 and L377. This is a very rare case of a homozygous mutation in a patient with severe symptoms of RTH and lack of symptoms in both heterozygous parents. Although, computational analyses have provided the evidence that p.E311K substitution may affect THRB function, lack of dominant negative effect typical for THRB mutations could not be explained by structure-based modeling. Further in vitro analysis is required to assess the functional consequences of this substitution.

Abstract 3892: Anti-cooperative allosteric inhibitors of thymidylate synthase as novel antineoplastic agents

Abstract Thymidylate synthase (TS) inhibitors are a class of antineoplastic agents widely used for the treatment of gastrointestinal, breast, ovarian and other cancers. TS catalyzes reductive methylation of dUMP to dTMP and serves as the sole de novo source of thymidylate inside the cell. While TS is essential for DNA biosynthesis and normal cell proliferation, TS overexpression has been associated with resistant tumor phenotype and poor prognosis. Although TS inhibitor 5-flourouracil (5-FU) continues to be one of the most frequently used chemo-therapeutic agents, 5-FU and its metabolites can cause significant liver, cardiac and bone marrow toxicity and are associated with development of drug resistance. In this study, we developed a new strategy that targets TS subunit cooperativity by freezing isoform interconversions and structural dynamics of the enzyme. In TS catalytic mechanism, conserted motion of amino acid residues 181-197 brings the catalytic Cys195 in and out of the active site. These small scale motions that translate across the dimer interface upon substrate binding and product release are integral to TS enzymatic function and can be taken advantage of in structure-based design of its inhibitors. For this purpose, we selected TS crystal structure with high asymmetry between the subunits (PDBID:1HVY) and used it as a template for molecular docking. The structure was prepared by removing heteroatoms and liganded molecules, followed by energy minimization in UCSF Chimera (1.5.2) and testing resulting geometries in Coot (0.6.2). We identified 5 potential docking sites and selected the site at the dimer interface specific to inactive conformation of TS. National Cancer Institute (NCI) small molecule library of 139,735 compounds was extracted from ZINC and pruned for redundant structures and molecules with molecular weight less than 200 Da. Resulting compound library was docked (DOCK6.4) into the selected site in 1000 orientations with flexible bond parameters enabled. The top 1000 hits were re-scored using AMBER and visualized in PyMOL to check for consistency and geometric fit. 837 out of 1000 molecules were further selected and re-ranked based on predicted physicochemical characteristics (cLogP, pKa, solubility, etc.). The twenty-seven highest ranked compounds were requested from the NCI and tested for their effects on viability in HeLa, K562 and A549 human cancer cell lines. Five out of 27 compounds reduced viability by more than 50% at a concentration of 10uM; one of the compounds was effective below 1uM. In conclusion, by using this new approach that targets TS subunit cooperativity, we identified small molecules with potential anti-cancer activity. 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 3892. doi:1538-7445.AM2012-3892