Victor L. Hsu

HADDOCK versus HADDOCK: New features and performance of HADDOCK2.0 on the CAPRI targets

Here we present version 2.0 of HADDOCK, which incorporates considerable improvements and new features. HADDOCK is now able to model not only protein–protein complexes but also other kinds of biomolecular complexes and multi‐component (N > 2) systems. In the absence of any experimental and/or predicted information to drive the docking, HADDOCK now offers two additional ab initio docking modes based on either random patch definition or center‐of‐mass restraints. The docking protocol has been considerably improved, supporting among other solvated docking, automatic definition of semi‐flexible regions, and inclusion of a desolvation energy term in the scoring scheme. The performance of HADDOCK2.0 is evaluated on the targets of rounds 4‐11, run in a semi‐automated mode using the original information we used in our CAPRI submissions. This enables a direct assessment of the progress made since the previous versions. Although HADDOCK performed very well in CAPRI (65% and 71% success rates, overall and for unbound targets only, respectively), a substantial improvement was achieved with HADDOCK2.0. Proteins 2007.

Prenylflavonoids from Humulus lupulus

Abstract Five flavonoids were isolated from the resin part of the female inflorescences of Humulus lupulus , together with four known hop flavonoids, i.e. xanthohumol, 2′,4′,6′,4-tetrahydroxy-3′- C -prenylchalcone, iso-xanthohumol and 6-prenylnaringenin. The new hop compounds were identified as 2′,4′,6′,4-tetrahydroxy-3′- C -geranylchalcone, 5′-prenylxanthohumol, 6″,6″-dimethylpyrano (2″,3″: 3′,4′)-2′,4-dihydroxy-6′-methoxychalcone, its hydrate and 8-prenylnaringenin; apart from 8-prenylnaringenin, these are new flavonoids. Their mass fragmentation patterns were studied by mass spectrometry using atmospheric pressure chemical ionization in combination with collision-activated decomposition. Loss of the isoprenoid substituent in the positive ion mode and retro Diels-Alder fission in both the positive and negative ion modes provided useful information on the substitution patterns of the A and B rings. Nine hop varieties were qualitatively and quantatively characterized by HPLC-mass spectrometry. The flavonoid profiles of the samples examined were uniform and proved to be of little value in hop variety identification. Xanthohumol was the principal flavonoid in all samples (80–90% of the total of flavonoids) and was accompanied by minor amounts of the other eight flavonoids in virtually all samples.

Information-driven protein–DNA docking using HADDOCK: it is a matter of flexibility

Intrinsic flexibility of DNA has hampered the development of efficient protein−DNA docking methods. In this study we extend HADDOCK (High Ambiguity Driven DOCKing) [C. Dominguez, R. Boelens and A. M. J. J. Bonvin (2003) J. Am. Chem. Soc. 125, 1731–1737] to explicitly deal with DNA flexibility. HADDOCK uses non-structural experimental data to drive the docking during a rigid-body energy minimization, and semi-flexible and water refinement stages. The latter allow for flexibility of all DNA nucleotides and the residues of the protein at the predicted interface. We evaluated our approach on the monomeric repressor−DNA complexes formed by bacteriophage 434 Cro, the Escherichia coli Lac headpiece and bacteriophage P22 Arc. Starting from unbound proteins and canonical B-DNA we correctly predict the correct spatial disposition of the complexes and the specific conformation of the DNA in the published complexes. This information is subsequently used to generate a library of pre-bent and twisted DNA structures that served as input for a second docking round. The resulting top ranking solutions exhibit high similarity to the published complexes in terms of root mean square deviations, intermolecular contacts and DNA conformation. Our two-stage docking method is thus able to successfully predict protein−DNA complexes from unbound constituents using non-structural experimental data to drive the docking.

Community-wide assessment of protein-interface modeling suggests improvements to design methodology

The CAPRI (Critical Assessment of Predicted Interactions) and CASP (Critical Assessment of protein Structure Prediction) experiments have demonstrated the power of community-wide tests of methodology in assessing the current state of the art and spurring progress in the very challenging areas of protein docking and structure prediction. We sought to bring the power of community-wide experiments to bear on a very challenging protein design problem that provides a complementary but equally fundamental test of current understanding of protein-binding thermodynamics. We have generated a number of designed protein-protein interfaces with very favorable computed binding energies but which do not appear to be formed in experiments, suggesting that there may be important physical chemistry missing in the energy calculations. A total of 28 research groups took up the challenge of determining what is missing: we provided structures of 87 designed complexes and 120 naturally occurring complexes and asked participants to identify energetic contributions and/or structural features that distinguish between the two sets. The community found that electrostatics and solvation terms partially distinguish the designs from the natural complexes, largely due to the nonpolar character of the designed interactions. Beyond this polarity difference, the community found that the designed binding surfaces were, on average, structurally less embedded in the designed monomers, suggesting that backbone conformational rigidity at the designed surface is important for realization of the designed function. These results can be used to improve computational design strategies, but there is still much to be learned; for example, one designed complex, which does form in experiments, was classified by all metrics as a nonbinder.

Determination of proanthocyanidin A2 content in phenolic polymer isolates by reversed-phase high-performance liquid chromatography

This article summarizes the development of an analytical method for the determination of proanthocyanidin (PAC) A2 in phenolic polymer isolates following acid-catalyzed degradation in the presence of excess phloroglucinol. Isolates from concentrated cranberry juice (CCJ) were extensively characterized and molar extinction coefficients were determined for the terminal A2 and phloroglucinol adduct of the extension A2 unit. Peanuts were also found to contain both extension and terminal A-type PACs and therefore a total peanut system (TPS) was chosen to test the effectiveness of the HPLC method that was developed with the CCJ system. Kinetic studies were conducted and reaction conditions were optimized for the A2 units in both CCJ and TPS. The optimized method provides quantitative and reproducible information on the A2 content of proanthocyanidin isolates.

Leaf surface flavonoids of Chrysothamnus

Twenty-six flavonoid aglycones have been identified from eight plants covering three species of Chrysothamnus that were collected in eastern Oregon. The flavonoids were identified by NMR spectroscopy, tandem mass spectrometry and co-TLC with authentic markers. Chrysothamnus nauseosus yielded methyl ethers of apigenin, isoscutellarein, luteolin, kaempferol, herbacetin and quercetin. O-Methylated kaempferol and quercetin derivatives were isolated from the leaf exudate of C. humilis. The flavonoid chemistry of C. viscidiflorus was found different from the other two species by the presence of methyl ethers of quercetin, eriodictyol and taxifolin-3-acetate. Although the flavonoid profiles proved of diagnostic value at the species level, they provided little further evidence in favour of inclusion of Chrysothamnus into Ericameria as proposed earlier on the basis of morphological similarities.

The extended left-handed helix: a simple nucleic acid-binding motif.

The poly‐proline type II extended left‐handed helical structure is well represented in proteins. In an effort to determine the helixs role in nucleic acid recognition and binding, a survey of 258 nucleic acid‐binding protein structures from the Protein Data Bank was conducted. Results indicate that left‐handed helices are commonly found at the nucleic acid interfacial regions. Three examples are used to illustrate the utility of this structural element as a recognition motif. The third K homology domain of NOVA‐2, the Epstein‐Barr nuclear antigen‐1, and the Drosophila paired protein homeodomain all contain left‐handed helices involved in nucleic acid interactions. In each structure, these helices were previously unidentified as left‐handed helices by secondary structure algorithms but, rather, were identified as either having small amounts of hydrogen bond patterns to the rest of the protein or as being “unstructured.” Proposed mechanisms for nucleic acid interactions by the extended left‐handed helix include both nonspecific and specific recognition. The observed interactions indicate that this secondary structure utilizes an increase in protein backbone exposure for nucleic acid recognition. Both main‐chain and side‐chain atoms are involved in specific and nonspecific hydrogen bonding to nucleobases or sugar‐phosphates, respectively. Our results emphasize the need to classify the left‐handed helix as a viable nucleic acid recognition and binding motif, similar to previously identified motifs such as the helix‐turn‐helix, zinc fingers, leucine zippers, and others. Proteins 2004.

Conformational dynamics of human FXR-LBD ligand interactions studied by hydrogen/deuterium exchange mass spectrometry: insights into the antagonism of the hypolipidemic agent Z-guggulsterone.

Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily of transcription factors that plays a key role in the regulation of bile acids, lipid and glucose metabolisms. The regulative function of FXR is governed by conformational changes of the ligand binding domain (LBD) upon ligand binding. Although FXR is a highly researched potential therapeutic target, only a limited number of FXR-agonist complexes have been successfully crystallized and subsequently yielded high resolution structures. There is currently no structural information of any FXR-antagonist complexes publically available. We therefore explored the use of amide hydrogen/deuterium exchange (HDX) coupled with mass spectrometry for characterizing conformational changes in the FXR-LBD upon ligand binding. Ligand-specific deuterium incorporation profiles were obtained for three FXR ligand chemotypes: GW4064, a synthetic non-steroidal high affinity agonist; the bile acid chenodeoxycholic acid (CDCA), the endogenous low affinity agonist of FXR; and Z-guggulsterone (GG), an in vitro antagonist of the steroid chemotype. A comparison of the HDX profiles of their ligand-bound FXR-LBD complexes revealed a unique mode of interaction for GG. The conformational features of the FXR-LBD-antagonist interaction are discussed.

Conformational modulation of the farnesoid X receptor by prenylflavonoids: Insights from hydrogen deuterium exchange mass spectrometry (HDX-MS), fluorescence titration and molecular docking studies.

We report on the molecular interactions of the farnesoid X receptor (FXR) with prenylflavonoids, an emerging class of FXR modulators. FXR is an attractive therapeutic target for mitigating metabolic syndromes (MetS) because FXR activates the inhibitory nuclear receptor, small heterodimer partner (SHP), thereby inhibiting both gluconeogenesis and de novo lipogenesis. We and others have shown that xanthohumol (XN), the principal prenylflavonoid of the hop plant (Humulus lupulus L.), is a FXR agonist based on its ability to affect lipid and glucose metabolism in vivo and to induces FXR target genes in biliary carcinoma cells and HEK293 cells. However, studies are currently lacking to rationalize the molecular mechanisms of FXR modulation by prenylflavonoids. We addressed this deficiency and report the first systematic study of FXR prenylflavonoid interactions. We combined hydrogen deuterium exchange mass spectrometry (HDX-MS) with computational studies for dissecting molecular recognition and conformational impact of prenylflavonoid interactions on the ligand binding domain (LBD) of human FXR. Four prenylflavonoids were tested: xanthohumol, a prenylated chalcone, two prenylated flavonones, namely isoxanthohumol (IX) and 8-prenylnaringenin (8PN), and a semisynthetic prenylflavonoid derivative, tetrahydroxanthohumol (TX). Enhancement of the HDX protection profile data by in silico predicted models of FXR prenylflavonoid complexes resulted in mapping of the prenylflavonoid interactions within the canonical ligand binding pocket. Our findings provide a foundation for the exploration of the chemical scaffolds of prenylated chalcones and flavanones as leads for future structure activity studies of this important nuclear receptor with potential relevance for ameliorating lipid metabolic disorders associated with obesity and MetS.