XPD helicase structures and activities: insights into the cancer and aging phenotypes from XPD mutations.

Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

Combining H/D Exchange Mass Spectrometry and Computational Docking To Derive the Structure of Protein-Protein Complexes.

Protein-protein interactions are essential for biological function, but structures of protein-protein complexes are difficult to obtain experimentally. To derive the protein complex of the DNA-repair enzyme human uracil-DNA-glycosylase (hUNG) with its protein inhibitor (UGI), we combined rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). Computational docking of the unbound protein structures provides a list of possible three-dimensional models of the complex; DXMS identifies solvent-protected protein residues. DXMS showed that unbound hUNG is compactly folded, but unbound UGI is loosely packed. An increased level of solvent protection of hUNG in the complex was localized to four regions on the same face. The decrease in the number of incorporated deuterons was quantitatively interpreted as the minimum number of main-chain hUNG amides buried in the protein-protein interface. The level of deuteration of complexed UGI decreased throughout the protein chain, indicating both tighter packing and direct solvent protection by hUNG. Three UGI regions showing the greatest decreases were best interpreted leniently, requiring just one main-chain amide from each in the interface. Applying the DXMS constraints as filters to a list of docked complexes gave the correct complex as the largest favorable energy cluster. Thus, identification of approximate protein interfaces was sufficient to distinguish the protein complex. Surprisingly, incorporating the DXMS data as added favorable potentials in the docking calculation was less effective in finding the correct complex. The filtering method has greater flexibility, with the capability to test each constraint and enforce simultaneous contact by multiple regions, but with the caveat that the list from the unbiased docking must include correct complexes.

Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformedNicotiana tabacum Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently inN. tabacumand demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

Combining H/D exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase.

X-ray crystallography provides excellent structural data on protein-DNA interfaces, but crystallographic complexes typically contain only small fragments of large DNA molecules. We present a new approach that can use longer DNA substrates and reveal new protein-DNA interactions even in extensively studied systems. Our approach combines rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). DXMS identifies solvent-exposed protein surfaces; docking is used to create a 3-dimensional model of the protein-DNA interaction. We investigated the enzyme uracil-DNA glycosylase (UNG), which detects and cleaves uracil from DNA. UNG was incubated with a 30 bp DNA fragment containing a single uracil, giving the complex with the abasic DNA product. Compared with free UNG, the UNG-DNA complex showed increased solvent protection at the UNG active site and at two regions outside the active site: residues 210-220 and 251-264. Computational docking also identified these two DNA-binding surfaces, but neither shows DNA contact in UNG-DNA crystallographic structures. Our results can be explained by separation of the two DNA strands on one side of the active site. These non-sequence-specific DNA-binding surfaces may aid local uracil search, contribute to binding the abasic DNA product and help present the DNA product to APE-1, the next enzyme on the DNA-repair pathway.

Predicting protein-DNA interactions by full search computational docking.

Protein-DNA interactions are essential for many biological processes. X-ray crystallography can provide high-resolution structures, but protein-DNA complexes are difficult to crystallize and typically contain only small DNA fragments. Thus, there is a need for computational methods that can provide useful predictions to give insights into mechanisms and guide the design of new experiments. We used the program DOT, which performs an exhaustive, rigid-body search between two macromolecules, to investigate four diverse protein-DNA interactions. Here, we compare our computational results with subsequent experimental data on related systems. In all cases, the experimental data strongly supported our structural hypotheses from the docking calculations: a mechanism for weak, nonsequence-specific DNA binding by a transcription factor, a large DNA-binding footprint on the surface of the DNA-repair enzyme uracil-DNA glycosylase (UNG), viral and host DNA-binding sites on the catalytic domain of HIV integrase, and a three-DNA-contact model of the linker histone bound to the nucleosome. In the case of UNG, the experimental design was based on the DNA-binding surface found by docking, rather than the much smaller surface observed in the crystallographic structure. These comparisons demonstrate that the DOT electrostatic energy gives a good representation of the distinctive electrostatic properties of DNA and DNA-binding proteins. The large, favourably ranked clusters resulting from the dockings identify active sites, map out large DNA-binding sites, and reveal multiple DNA contacts with a protein. Thus, computational docking can not only help to identify protein-DNA interactions in the absence of a crystal structure, but also expand structural understanding beyond known crystallographic structures.

DOT2: Macromolecular docking with improved biophysical models.

Computational docking is a useful tool for predicting macromolecular complexes, which are often difficult to determine experimentally. Here, we present the DOT2 software suite, an updated version of the DOT intermolecular docking program. DOT2 provides straightforward, automated construction of improved biophysical models based on molecular coordinates, offering checkpoints that guide the user to include critical features. DOT has been updated to run more quickly, allow flexibility in grid size and spacing, and generate an infinitive complete list of favorable candidate configurations. Output can be filtered by experimental data and rescored by the sum of electrostatic and atomic desolvation energies. We show that this rescoring method improves the ranking of correct complexes for a wide range of macromolecular interactions and demonstrate that biologically relevant models are essential for biologically relevant results. The flexibility and versatility of DOT2 accommodate realistic models of complex biological systems, improving the likelihood of a successful docking outcome.

MDB: the Metalloprotein Database and Browser at The Scripps Research Institute.

The Metalloprotein Database and Browser (MDB; http://metallo.scripps.edu) at The Scripps Research Institute is a web-accessible resource for metalloprotein research. It offers the scientific community quantitative information on geometrical parameters of metal-binding sites in protein structures available from the Protein Data Bank (PDB). The MDB also offers analytical tools for the examination of trends or patterns in the indexed metal-binding sites. A user can perform interactive searches, metal-site structure visualization (via a Java applet), and analysis of the quantitative data by accessing the MDB through a web browser without requiring an external application or platform-dependent plugin. The MDB also has a non-interactive interface with which other web sites and network-aware applications can seamlessly incorporate data or statistical analysis results from metal-binding sites. The information contained in the MDB is periodically updated with automated algorithms that find and index metal sites from new protein structures released by the PDB.

Self-Assembly of the Cephalopod Protein Reflectin.

Films from the cephalopod protein reflectin demonstrate multifaceted functionality as infrared camouflage coatings, proton transport media, and substrates for growth of neural stem cells. A detailed study of the in vitro formation, structural characteristics, and stimulus response of such films is presented. The reported observations hold implications for the design and development of advanced cephalopod-inspired functional materials.

C-Terminal Domain of Integrase Binds between the Two Active Sites.

HIV integrase (HIV-IN), one of three HIV enzymes, is a target for the treatment of AIDS, but the full biological assembly has been difficult to characterize, hampering inhibitor design. The recent crystallographic structures of integrase from prototype foamy virus (PFV-IN) with bound DNA were a breakthrough, revealing how viral DNA organizes two integrase dimers into a tetramer that has the two active sites appropriately spaced for insertion of the viral DNA into host DNA. The organization of domains within each PFV-IN protein chain, however, varies significantly from that found in HIV-IN structures. With the goal of identifying shared structural characteristics, the interactions among components of the PFV-IN and HIV-IN assemblies were investigated with the macromolecular docking program DOT. DOT performs an exhaustive, rigid-body search between two macromolecules. Computational docking reproduced the crystallographic interactions of the PFV-IN catalytic and N-terminal domains with viral DNA and found similar viral DNA interactions for HIV-IN. Computational docking did not reproduce the crystallographic interactions of the PFV-IN C-terminal domain (CTD). Instead, two symmetry-related positions were found for the PFV-IN CTD that indicate formation of a CTD dimer between the two active sites. Our predicted CTD dimer is consistent with cross-linking studies showing interactions of the CTD with viral DNA that appear to be blocked in the PFV-IN structures. The CTD dimer can insert two arginine-rich loops between the two bound vDNA molecules and the host DNA, a region that is unoccupied in the PFV-IN crystallographic structures. The positive potential from these two loops would alleviate the large negative potential created by the close proximity of two viral vDNA ends, helping to bring together the two active sites and assisting host DNA binding. This study demonstrates the ability of computational docking to evaluate complex crystallographic assemblies, identify interactions that are influenced by the crystal environment, and provide plausible alternatives.

Predicting interactions of winged-helix transcription factors with DNA.

Determining protein-DNA interactions is important for understanding gene regulation, DNA repair and chromatin structure. Unfortunately, the structures of DNA-bound complexes are often difficult to obtain experimentally, so the development of computational methods that provide good models of these complexes would be valuable. Here, we present a rigid-body docking approach using the computer program DOT. DOT performs a complete, six-dimensional search of all orientations for two rigid molecules and calculates the interaction energy as the sum of electrostatic and van der Waals terms. DOT was applied to three winged-helix transcription factors that share similar DNA-binding structural motifs but bind DNA in different ways. Docking with linear B-form DNA models accomplished several objectives; it (1) distinguished the different ways the transcription factors bind DNA, (2) identified each protein's DNA-binding site and the DNA orientation at the site and (3) gave at least one solution among the three best-ranked that shows the protein side chain-DNA base interactions responsible for recognition. Furthermore, the ensemble of top-ranked, docked linear B-DNA fragments indicated the DNA bending induced upon protein binding. Docking linear B-DNA to structures of the transcription factor FadR suggests that the allosteric, conformational change induced upon effector binding results in loss of the ability to bend DNA as well as loss of sequence-specific interactions with DNA. The electrostatic energy term calculated by DOT is comparable to the electrostatic binding energy calculated by Poisson-Boltzmann methods. Our results show rigid-body docking that includes a rigorous treatment of the electrostatic interaction energy can be effective in predicting protein-DNA interactions.

Finding needles in haystacks: Reranking DOT results by using shape complementarity, cluster analysis, and biological information.

We present an evaluation of our results for the first Critical Assessment of PRedicted Interaction (CAPRI). The methods used include the molecular docking program DOT, shape analysis tool FADE, cluster analysis and filtering based on biological data. Good results were obtained for most of the seven CAPRI targets, and for two systems, submissions having the highest number of correctly predicted contacts were produced.

Prediction of HIV-1 integrase/viral DNA interactions in the catalytic domain by fast molecular docking.

This study details the separate analyses of binding specificity of HIV-1 integrase (IN) and viral B-DNA forms through ligand-receptor docking studies by means of a fast molecular docking method. The application of solvated electrostatics with the University of Houston Brownian Dynamics Program (UHBD) and configurational sampling by the Daughter of Turnip (DOT) docking program resulted in the computation of energies of more than 113 billion configurations for each ligand-receptor docking study, a procedure considered computationally intractable a few years ago. A specific binding pattern of viral DNA to the IN catalytic domain region has been predicted as a result of these calculations. In a representative docked configuration, we observe the 3'-hydroxyl of the conserved deoxyadenosine to be close to one of the two divalent metal ions that are necessary for catalysis. A superimposition of our energy-minimized docked complex on representative structures from a molecular dynamics (MD) simulation of a crystallographically resolved IN/inhibitor complex revealed an overlap of viral DNA with the inhibitor, indicating that the bound inhibitor might operate by blocking substrate binding. The DOT docking calculation also identified a second, adjacent DNA-binding site, which we believe is the nonspecific host DNA binding site. The binding pattern predicted by DOT complements previous electrostatics, MD simulation, photo-cross-linking, and mutagenesis studies and also provides a further refinement of the IN/viral DNA binding interaction as a basis for new structure-based design efforts.

Computational reconstruction of multidomain proteins using atomic force microscopy data.

Classical structural biology techniques face a great challenge to determine the structure at the atomic level of large and flexible macromolecules. We present a novel methodology that combines high-resolution AFM topographic images with atomic coordinates of proteins to assemble very large macromolecules or particles. Our method uses a two-step protocol: atomic coordinates of individual domains are docked beneath the molecular surface of the large macromolecule, and then each domain is assembled using a combinatorial search. The protocol was validated on three test cases: a simulated system of antibody structures; and two experimentally based test cases: Tobacco mosaic virus, a rod-shaped virus; and Aquaporin Z, a bacterial membrane protein. We have shown that AFM-intermediate resolution topography and partial surface data are useful constraints for building macromolecular assemblies. The protocol is applicable to multicomponent structures connected in the polypeptide chain or as disjoint molecules. The approach effectively increases the resolution of AFM beyond topographical information down to atomic-detail structures.

Complex of linker histone H5 with the nucleosome and its implications for chromatin packing.

Linker histones are essential for chromatin filament formation, and they play key roles in the regulation of gene expression. Despite the determination of structures of the nucleosome and linker histones, the location of the linker histone on the nucleosome is still a matter of debate. Here we show by computational docking that the globular domain of linker histone variant H5 (GH5) has three distinct DNA-binding sites, through which GH5 contacts the DNA at the nucleosome dyad and the linker DNA strands entering and exiting the nucleosome. Our results explain the extensive mutagenesis and crosslinking data showing that side chains spread throughout the GH5 surface interact with nucleosomal DNA. The nucleosome DNA contacts positively charged side chains that are conserved within the linker histone family, indicating that our model extends to linker histone-nucleosome interactions in general. Furthermore, our model provides a structural mechanism for formation of a dinucleosome complex specific to the linker histone H5, explaining its efficiency in chromatin compaction and transcription regulation. Thus, this work provides a basis for understanding how structural differences within the linker histone family result in functional differences, which in turn are important for gene regulation.

Structural basis for preferential binding of non-ortho-substituted polychlorinated biphenyls by the monoclonal antibody S2B1.

Polychlorinated biphenyls (PCBs) are a family of 209 isomers (congeners) with a wide range of toxic effects. In structural terms, they are of two types: those with and those without chlorines at the ortho positions (2, 2', 6 and 6'). Only 20 congeners have no ortho chlorines. Three of these are bound by the aryl hydrocarbon receptor and are one to four orders of magnitude more toxic than all others. A monoclonal antibody, S2B1, and its recombinant Fab have high selectivity and nanomolar binding affinities for two of the most toxic non-ortho-chlorinated PCBs, 3,4,3',4'-tetrachlorobiphenyl and 3,4,3',4',5'-pentachlorobiphenyl. To investigate the basis for these properties, we built a three-dimensional structure model of the S2B1 variable fragment (Fv) based on the high-resolution crystallographic structures of antibodies 48G7 and N1G9. Two plausible conformations for the complementarity-determining region (CDR) H3 loop led to two putative PCB-binding pockets with very different shapes (models A and B). Docking studies using molecular mechanics and potentials of mean force (PMF) indicated that model B was most consistent with the selectivity observed for S2B1 in competition ELISAs. The binding site in model B had a deep, narrow pocket between V(L) and V(H), with a slight constriction at the top that opened into a wider pocket between CDRs H1 and H3 on the antibody surface. This binding site resembles those of esterolytic antibodies that bind haptens with phenyl rings. One phenyl ring of the PCB fits into the deep pocket, and the other ring is bound in the shallower one. The bound PCB is surrounded by the side chains of TyrL91, TyrL96 and TrpH98, and it has a pi-cation interaction with ArgL46. The tight fit of the binding pocket around the ortho positions of the bound PCBs indicates that steric hindrance of ortho chlorines in the binding site, rather than induced conformational change of the PCBs, is responsible for the selectivity of S2B1.

Identification of a new cryptochrome class. Structure, function, and evolution.

Cryptochrome flavoproteins, which share sequence homology with light-dependent DNA repair photolyases, function as photoreceptors in plants and circadian clock components in animals. Here, we coupled sequencing of an Arabidopsis cryptochrome gene with phylogenetic, structural, and functional analyses to identify a new cryptochrome class (cryptochrome DASH) in bacteria and plants, suggesting that cryptochromes evolved before the divergence of eukaryotes and prokaryotes. The cryptochrome crystallographic structure, reported here for Synechocystis cryptochrome DASH, reveals commonalities with photolyases in DNA binding and redox-dependent function, despite distinct active-site and interaction surface features. Whole genome transcriptional profiling together with experimental confirmation of DNA binding indicated that Synechocystis cryptochrome DASH functions as a transcriptional repressor.