Mesoscope: A Web-based Tool for Mesoscale Data Integration and Curation.

Interest is growing for 3D models of the biological mesoscale, the intermediate scale between the nanometer scale of molecular structure and micrometer scale of cellular biology. However, it is currently difficult to gather, curate and integrate all the data required to define such models. To address this challenge we developed Mesoscope (mesoscope.scripps.edu/beta), a web-based data integration and curation tool. Mesoscope allows users to begin with a listing of molecules (such as data from proteomics), and to use resources at UniProt and the PDB to identify, prepare and validate appropriate structures and representations for each molecule, ultimately producing a portable output file used by CellPACK and other modeling tools for generation of 3D models of the biological mesoscale. The availability of this tool has proven essential in several exploratory applications, given the high complexity of mesoscale models and the heterogeneity of the available data sources.

Soluble proteins: size, shape and function.

Why are proteins so big? Why do cells build oligomeric proteins? A visual survey of the protein structures available in the Protein Data Bank sheds new light on these questions.

Cuttlefish: Color Mapping for Dynamic Multi-Scale Visualizations.

Visualizations of hierarchical data can often be explored interactively. For example, in geographic visualization, there are continents, which can be subdivided into countries, states, counties and cities. Similarly, in models of viruses or bacteria at the highest level are the compartments, and below that are macromolecules, secondary structures (such as α-helices), amino-acids, and on the finest level atoms. Distinguishing between items can be assisted through the use of color at all levels. However, currently, there are no hierarchical and adaptive color mapping techniques for very large multi-scale visualizations that can be explored interactively. We present a novel, multi-scale, color-mapping technique for adaptively adjusting the color scheme to the current view and scale. Color is treated as a resource and is smoothly redistributed. The distribution adjusts to the scale of the currently observed detail and maximizes the color range utilization given current viewing requirements. Thus, we ensure that the user is able to distinguish items on any level, even if the color is not constant for a particular feature. The coloring technique is demonstrated for a political map and a mesoscale structural model of HIV. The technique has been tested by users with expertise in structural biology and was overall well received.

Morphology of protein-protein interfaces.

BACKGROUND: Most soluble proteins are active as low-number oligomers. Statistical surveys of oligomeric proteins have defined the roles of hydrophobicity and complementarity in the stability of protein interfaces, but tend to average structural features over a diverse set of protein-protein interfaces, blurring information on how individual interfaces are stabilized. RESULTS: We report a visual survey of 136 homodimeric proteins from the Brookhaven Protein Data Bank, with images that highlight the major structural features of each protein-protein interaction surface. Nearly all of these proteins have interfaces formed between two globular subunits. Surprisingly, the pattern of hydrophilicity over the surface of these interfaces is quite variable. Approximately one-third of the interfaces show a recognizable hydrophobic core, with a single large, contiguous, hydrophobic patch surrounded by a ring of intersubunit polar interactions. The remaining two-thirds of the proteins show a varied mixture of small hydrophobic patches, polar interactions and water molecules scattered over the entire interfacial area. Ten proteins in the survey have intertwined interfaces formed by extensive interdigitation of the two subunit chains. These interfaces are very hydrophobic and are associated with proteins that require both stability and internal symmetry. CONCLUSIONS: The archetypal protein interface, with a defined hydrophobic core, is present in only a minority of the surveyed homodimeric proteins. Most homodimeric proteins are stabilized by a combination of small hydrophobic patches, polar interactions and a considerable number of bridging water molecules. The presence or absence of a hydrophobic core within these interfaces does not correlate with specific protein functions.

Defining GC-specificity in the minor groove: side-by-side binding of the di-imidazole lexitropsin to C-A-T-G-G-C-C-A-T-G.

BACKGROUND: Polyamide drugs, such as netropsin, distamycin and their lexitropsin derivatives, can be inserted into a narrow B-DNA minor groove to form 1:1 complexes that can distinguish AT base pairs from GC, but cannot detect end-for-end base-pair reversals such as TA for AT. In contrast, 2:1 side-by-side polyamide drug complexes potentially are capable of such discrimination. Imidazole (Im) and pyrrole (Py) rings side-by-side read a GC base pair with the Im ring recognizing the guanine side. But the reason for this specific G-Im association is unclear because the guanine NH2 group sits in the center of the groove. A 2:1 drug:DNA complex that presents Im at both ends of a GC base pair should help unscramble the issue of imidazole reading specificity. RESULTS: We have determined the crystal structure of a 2:1 complex of a di-imidazole lexitropsin (DIM), an analogue of distamycin, and a DNA decamer with the sequence C-A-T-G-G-C-C-A-T-G. The two DIM molecules sit antiparallel to one another in a broad minor groove, with their cationic tails widely separated. Im rings of one drug molecule stack against amide groups of the other. DIM1 rests against nucleotides C7A8T9G10 of strand 1 of the helix, whereas DIM2 rests against G14G15C16C17 on strand 2. All DIM amide nitrogens donate hydrogen bonds to N and O atoms on the floor of the DNA groove and, in addition, the two Im rings on DIM2 accept hydrogen bonds from guanine N2 amines, thereby providing specific reading. The guanine N2 amine can bond to Im on its own side of the groove, but not on the cytosine side, because of limits on close approach of the two Im rings and the geometry of sp2 hybridization about the amide nitrogen. CONCLUSIONS: Im and Py rings distinguish AT from GC base pairs because of steric factors involving the bulk of the guanine amine, and the ability of Im to form a hydrogen bond with the amine. Side-by-side Im and Py rings differentiate GC from CG base pairs because of tight steric contacts and sp2 hybridization at the amine nitrogen atom, with the favored conformations being G/Im,Py/C and C/Py,Im/G. Discrimination between AT and TA base pairs may be possible using bulkier rings, such as thiazole to select the A end of the base pair.

The crystal structure of C-C-A-T-T-A-A-T-G-G. Implications for bending of B-DNA at T-A steps.

The single-crystal X-ray analysis of trigonal C-C-A-T-T-A-A-T-G-G, and its comparison with orthorhombic C-G-A-T-T-A-A-T-C-G, have shown that the A-T-T-A-A-T sequence has limited polymorphism under the influence of packing forces from neighboring molecules in the crystal. The T-A step is intrinsically variable. It is not inconsistent with a large propeller twist, a narrow minor groove, and a single spine of hydration, as has sometimes been claimed on theoretical grounds. The T-A step does show a persistent positive roll, in a direction that compresses the major groove, and this may be a significant factor in macroscopic DNA curvature induced by phased A-tracts. A-tracts, as understood in this paper, include A-A and A-T steps, but not the T-A step, which is disruptive. Three conclusions regarding A-tract-induced curvature can be drawn from this and other X-ray crystal structure analyses, and from key gel retardation experiments: (1) The A-tract bending model is disqualified on two grounds: (i) tilt-wedge bending within A-tracts is incompatible with the observed direction of curvature; (ii) roll-wedge bending within A-tracts is contradicted by every crystal structure analysis, and is inconsistent with gel retardation results for (G-C-A-A-A-A-T-T-T-T)n and for (A-A-A-A-A-T-T-T-T-T)n. (2) The junction bend model is contradicted by crystallography because: (i) the inclination of base-pairs does not change between A-tract and non-A-tract regions of helix; and (ii) the observed bends at GC/AT junctions are roll-wedge bends, not tilt-wedge as the junction bend model demands. (3) The non-A-tract bending model is consistent with both gel retardation data and with X-ray crystallography, and must be regarded as the only consistent model for A-tract bending.

Recognition templates for predicting adenylate-binding sites in proteins.

Recognition templates encapsulate the structural and energetic features for the specific recognition of a given ligand by a protein active site. These templates identify the major interactions used for specific recognition and may be used to find specific binding sites in proteins of unknown function. We present a grid-based method for deriving recognition templates for adenylate groups from a set of diverse nucleotide-binding proteins. The templates reveal the basis of specific binding of adenylate, including tight shape complementarity, specific hydrogen bonds, and underscoring the importance of a key steric contact for excluding guanylate from adenylate-specific sites. We demonstrate the utility of recognition templates in identifying specific adenylate-binding sites in a diverse set of dinucleotide-binding proteins.

Coevolution and subsite decomposition for the design of resistance-evading HIV-1 protease inhibitors.

Drug resistance sharply limits the effectiveness of human immunodeficiency virus (HIV) protease inhibitors in acquired immunodeficiency syndrome therapy. In previous work, we presented methods for design of resistance-evading inhibitors using a computational coevolution technique. Here, we report subsite decomposition experiments that examine the relative importance and roles of each subsite in HIV protease, and the constraints on robust inhibitor design that are imposed by possible resistance mutations in each subsite. The results identify several structural features of robust resistance-evading inhibitors for use in drug design, and show their basis in the constraints imposed by the range of allowable mutation in the protease. In particular, the results identify the P3 and P3' sites as being particularly sensitive to protease mutation: inhibitors designed to fill the S3 and S3' sites of the wild-type protease will be susceptible to viral resistance, but inhibitors with side-chains smaller than a phenylalanine residue at P3 and P3', preferably medium-sized amino acids in the range from valine to leucine and isoleucine residues, will be more robust in the face of protease resistance mutation.

Sequence recognition of DNA by lexitropsins.

Lexitropsins are modular polyamide molecules that are designed to "read" the base sequence of DNA. Lexitropsins constructed of three types of subunits--pyrrole, imidazole and hydroxypyrrole--allow full recognition of DNA base sequences. Structural studies have revealed the atomic basis of this specificity. Theoretical studies have explored the effectiveness of lexitropsins in targeting a given sequence within a genome, and have been used to analyze and improve lexitropsin design.

An analysis of a class of DNA sequence reading molecules.

Linked polyamides are a class of designed molecules that bind in the minor groove of double-stranded DNA in a partially sequence-specific manner but have limited sequence discriminatory abilities. This suggests a need for design alternatives to create molecules with enhanced sequence specificity. In this report we present formal proofs of the theoretical limits of the DNA sequence specificity of hypothetical sequence reading molecules as a function of their base recognition properties and sequence content and length of their target sequence. We prove that molecules containing nonspecific readers at critical positions within the molecule may have enhanced sequence specificity over molecules composed entirely of base specific reading elements. We also determine optimal patterns of base recognition for molecules in order to optimize their target sequence specificity. We also examine the effect of the length of a polyamide (i.e., the number of base pairs it binds) on its sequence discriminatory ability and determine necessary concentration dependent constraints on the binding free energies in order for longer polyamides to have greater sequence specificity than shorter ones. We show that unless the discriminatory ability of a ring for its preferred base is very strong, longer polyamides do not necessarily have greater sequence specificity over shorter ones when compared at the same molar concentration.

Interactive modeling of supramolecular assemblies.

The modeling of supramolecular structure presents two major challenges: (1) managing the large amount of sequence, structural and biochemical data, and (2) presenting the data to the user in a flexible and comprehensible manner that addresses these problems. We describe a visualization environment for the creation and analysis of supramolecular models. A set of modular symmetry tools, collectively called SymGen, has been created, providing a flexible platform for the creation of complex assemblies, with interactive control of all symmetry elements and their parameters. A second tool, SymSearch, allows a range of parameters defined within SymGen to be sampled and the resulting conformations to be evaluated. The environment avoids information overload, caused by the large number of atoms in supramolecular complexes, by using a multiresolution spherical harmonic representation that allows the user to display only essential features. Spherical harmonics also enables control of the triangulation level, allowing the user to reduce the complexity of the geometric description to retain interactive speed. The visual fidelity of the surface data is retained by using texture maps that are independent of the resolution of the underlying triangulation. We describe the design and implementation of this environment, and three illustrative examples of its utility.

The effect of crystal packing on oligonucleotide double helix structure.

One of the questions that constantly is asked regarding x-ray crystal structure analyses of macromolecules is: To what extent is the observed crystal structure representative of the molecular conformation when free in solution, and to what degree is the structure perturbed by intermolecular crystal forces? This can be assessed with DNA oligomers because of an unusual aspect of crystallization self-complementary oligomers should possess a twofold symmetry axis normal to their helix axis, yet more often than not crystal of such oligomers do not use this internal symmetry. The two ends of the helix are crystallographically distinct though chemically identical. Complexes of DNA oligomers with intercalating drugs such as triostin A tend to use their twofold symmetry when they crystallize, whereas complexes with non-intercalating, groove-binding drugs ignore this symmetry unless the drug molecule is very small. A detailed examination of crystal packing in the dodecamer C-G-C-G-A-A-T-T-C-G-C-G provides an explanation of all of the foregoing behavior in terms of the mechanism of nucleation of DNA or DNA-drug complexes on the surface of a growing crystal. Asymmetry of the ends of the DNA helix is the price that is paid for efficient lateral packing of helices within the crystal. The actual end-for-end variation in standard helix parameters is compared with the experimental noise level as gauged by independent re-refinement of the same oligonucleotide structure where available, and with the observed extent of variation of these same parameters along the helix. Oligomers analyzed are the B-DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G, the A-DNA octamer G-G-T-A-T-A-C-C, and the phosphorothioate analogue of the B-DNA hexamer G-C-G-C-G-C. End-for-end variation, presumably the result of crystal packing is typically double the experimental noise level, and half the variation in the same parameter along the helix. Analysis of crystal packing in the phosphorothioate hexamer, which uses the same P212121 space group as the dodecamer, shows that the highly unsymmetrical B1 vs. BII backbone conformation probably is to be ascribed to crystal packing forces, and not to the sequence of the hexamer.

The structure of DAPI bound to DNA.

The structure of the DNA fluorochrome 4'-6-diamidine-2-phenyl indole (DAPI) bound to the synthetic B-DNA oligonucleotide C-G-C-G-A-A-T-T-C-G-C-G has been solved by single crystal x-ray diffraction methods, at a resolution of 2.4 A. The structure is nearly isomorphous with that of the native DNA molecule alone. With one DAPI and 25 waters per DNA double helix, the residual error is 21.5% for the 2428 reflections above the 2-sigma level. DAPI inserts itself edgewise into the narrow minor groove, displacing the ordered spine of hydration. DAPI and a single water molecule together span the four AT base pairs at the center of the duplex. The indole nitrogen forms a bifurcated hydrogen bond with the thymine O2 atoms of the two central base pairs, as with netropsin and Hoechst 33258. The preference of all three of these drugs for AT regions of B-DNA is a consequence of three factors: (1) The intrinsically narrower minor groove in AT regions than in GC regions of B-DNA, leading to a snug fit of the flat aromatic drug rings between the walls of the groove. (2) The more negative electrostatic potential within the minor groove in AT regions, attributable in part to the absence of electropositive-NH2 groups along the floor of the groove, and (3) The steric advantage of the absence of those same guanine-NH2 groups, thus permitting the drug molecule to sink deeper into the groove. Groove width and electrostatic factors are regional, and define the relative receptiveness of a section of DNA since they operate over several contiguous base pairs. The steric factor is local, varying from one base pair to the next, and hence is the means of fine-tuning sequence specificity.

Automated docking and the search for HIV protease inhibitors.

This article will discuss the motivations, technologies, and future directions of computational automated docking in the context of the structure-based rational design of HIV-1 protease inhibitors. Docking simulations are widely used for screening of compound libraries to identify new drug leads, employing a simple model for rapid testing of thousands of compounds. Docking simulations are also useful for lead enhancement, using more detailed models to analyze the atomic interactions between inhibitors and target macromolecules. Major advances have been reported in the development of empirical force fields, which now allow assessment of relative binding strength and drug specificity, and extensions of automated docking techniques allow de novo drug design.