Impact of AlphaFold on structure prediction of protein complexes: The CASP15-CAPRI experiment.

We present the results for CAPRI Round 54, the 5th joint CASP-CAPRI protein assembly prediction challenge. The Round offered 37 targets, including 14 homodimers, 3 homo-trimers, 13 heterodimers including 3 antibody-antigen complexes, and 7 large assemblies. On average ~70 CASP and CAPRI predictor groups, including more than 20 automatics servers, submitted models for each target. A total of 21 941 models submitted by these groups and by 15 CAPRI scorer groups were evaluated using the CAPRI model quality measures and the DockQ score consolidating these measures. The prediction performance was quantified by a weighted score based on the number of models of acceptable quality or higher submitted by each group among their five best models. Results show substantial progress achieved across a significant fraction of the 60+ participating groups. High-quality models were produced for about 40% of the targets compared to 8% two years earlier. This remarkable improvement is due to the wide use of the AlphaFold2 and AlphaFold2-Multimer software and the confidence metrics they provide. Notably, expanded sampling of candidate solutions by manipulating these deep learning inference engines, enriching multiple sequence alignments, or integration of advanced modeling tools, enabled top performing groups to exceed the performance of a standard AlphaFold2-Multimer version used as a yard stick. This notwithstanding, performance remained poor for complexes with antibodies and nanobodies, where evolutionary relationships between the binding partners are lacking, and for complexes featuring conformational flexibility, clearly indicating that the prediction of protein complexes remains a challenging problem.

Synthesis and Biological Activity of a New Indenoisoquinoline Copper Derivative as a Topoisomerase I Inhibitor.

Topoisomerases are interesting targets in cancer chemotherapy. Here, we describe the design and synthesis of a novel copper(II) indenoisoquinoline complex, WN198. The new organometallic compound exhibits a cytotoxic effect on five adenocarcinoma cell lines (MCF-7, MDA-MB-231, HeLa, HT-29, and DU-145) with the lowest IC50 (0.37 ± 0.04 µM) for the triple-negative MDA-MB-231 breast cancer cell line. Below 5 µM, WN198 was ineffective on non-tumorigenic epithelial breast MCF-10A cells and Xenopus oocyte G2/M transition or embryonic development. Moreover, cancer cell lines showed autophagy markers including Beclin-1 accumulation and LC3-II formation. The DNA interaction of this new compound was evaluated and the dose-dependent topoisomerase I activity starting at 1 µM was confirmed using in vitro tests and has intercalation properties into DNA shown by melting curves and fluorescence measurements. Molecular modeling showed that the main interaction occurs with the aromatic ring but copper stabilizes the molecule before binding and so can putatively increase the potency as well. In this way, copper-derived indenoisoquinoline topoisomerase I inhibitor WN198 is a promising antitumorigenic agent for the development of future DNA-damaging treatments.

N-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sda Synthase B4GALNT2.

The Sda carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human B4GALNT2 gene drives the expression of a long and a short protein isoform (LF-B4GALNT2 and SF-B4GALNT2) sharing identical transmembrane and luminal domains. Both isoforms are trans-Golgi proteins and the LF-B4GALNT2 also localizes to post-Golgi vesicles thanks to its extended cytoplasmic tail. Control mechanisms underpinning Sda and B4GALNT2 expression in the gastrointestinal tract are complex and not fully understood. This study reveals the existence of two unusual N-glycosylation sites in B4GALNT2 luminal domain. The first atypical N-X-C site is evolutionarily conserved and occupied by a complex-type N-glycan. We explored the influence of this N-glycan using site-directed mutagenesis and showed that each mutant had a slightly decreased expression level, impaired stability, and reduced enzyme activity. Furthermore, we observed that the mutant SF-B4GALNT2 was partially mislocalized in the endoplasmic reticulum, whereas the mutant LF-B4GALNT2 was still localized in the Golgi and post-Golgi vesicles. Lastly, we showed that the formation of homodimers was drastically impaired in the two mutated isoforms. An AlphaFold2 model of the LF-B4GALNT2 dimer with an N-glycan on each monomer corroborated these findings and suggested that N-glycosylation of each B4GALNT2 isoform controlled their biological activity.

Prediction of protein assemblies, the next frontier: The CASP14-CAPRI experiment.

We present the results for CAPRI Round 50, the fourth joint CASP-CAPRI protein assembly prediction challenge. The Round comprised a total of twelve targets, including six dimers, three trimers, and three higher-order oligomers. Four of these were easy targets, for which good structural templates were available either for the full assembly, or for the main interfaces (of the higher-order oligomers). Eight were difficult targets for which only distantly related templates were found for the individual subunits. Twenty-five CAPRI groups including eight automatic servers submitted ~1250 models per target. Twenty groups including six servers participated in the CAPRI scoring challenge submitted ~190 models per target. The accuracy of the predicted models was evaluated using the classical CAPRI criteria. The prediction performance was measured by a weighted scoring scheme that takes into account the number of models of acceptable quality or higher submitted by each group as part of their five top-ranking models. Compared to the previous CASP-CAPRI challenge, top performing groups submitted such models for a larger fraction (70-75%) of the targets in this Round, but fewer of these models were of high accuracy. Scorer groups achieved stronger performance with more groups submitting correct models for 70-80% of the targets or achieving high accuracy predictions. Servers performed less well in general, except for the MDOCKPP and LZERD servers, who performed on par with human groups. In addition to these results, major advances in methodology are discussed, providing an informative overview of where the prediction of protein assemblies currently stands.

Assembly of B4GALT1/ST6GAL1 heteromers in the Golgi membranes involves lateral interactions via highly charged surface domains.

ß-1,4-Galactosyltransferase 1 (B4GALT1) and ST6 ß-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) catalyze the successive addition of terminal ß-1,4-linked galactose and α-2,6-linked sialic acid to N-glycans. Their exclusive interaction in the Golgi compartment is a prerequisite for their full catalytic activity, whereas a lack of this interaction is associated with cancers and hypoxia. To date, no structural information exists that shows how glycosyltransferases functionally assemble with each other. Using molecular docking simulations to predict interaction surfaces, along with mutagenesis screens and high-throughput FRET analyses in live cells to validate these predictions, we show here that B4GALT1 and ST6GAL1 interact via highly charged noncatalytic surfaces, leaving the active sites exposed and accessible for donor and acceptor substrate binding. Moreover, we found that the assembly of ST6GAL1 homomers in the endoplasmic reticulum before ST6GAL1 activation in the Golgi utilizes the same noncatalytic surface, whereas B4GALT1 uses its active-site surface for assembly, which silences its catalytic activity. Last, we show that the homomeric and heteromeric B4GALT1/ST6GAL1 complexes can assemble laterally in the Golgi membranes without forming cross-cisternal contacts between enzyme molecules residing in the opposite membranes of each Golgi cisterna. Our results provide detailed mechanistic insights into the regulation of glycosyltransferase interactions, the transitions between B4GALT1 and ST6GAL1 homo- and heteromers in the Golgi, and cooperative B4GALT1/ST6GAL1 function in N-glycan synthesis.

Modeling protein-protein, protein-peptide, and protein-oligosaccharide complexes: CAPRI 7th edition.

We present the seventh report on the performance of methods for predicting the atomic resolution structures of protein complexes offered as targets to the community-wide initiative on the Critical Assessment of Predicted Interactions. Performance was evaluated on the basis of 36 114 models of protein complexes submitted by 57 groups-including 13 automatic servers-in prediction rounds held during the years 2016 to 2019 for eight protein-protein, three protein-peptide, and five protein-oligosaccharide targets with different length ligands. Six of the protein-protein targets represented challenging hetero-complexes, due to factors such as availability of distantly related templates for the individual subunits, or for the full complex, inter-domain flexibility, conformational adjustments at the binding region, or the multi-component nature of the complex. The main challenge for the protein-peptide and protein-oligosaccharide complexes was to accurately model the ligand conformation and its interactions at the interface. Encouragingly, models of acceptable quality, or better, were obtained for a total of six protein-protein complexes, which included four of the challenging hetero-complexes and a homo-decamer. But fewer of these targets were predicted with medium or higher accuracy. High accuracy models were obtained for two of the three protein-peptide targets, and for one of the protein-oligosaccharide targets. The remaining protein-sugar targets were predicted with medium accuracy. Our analysis indicates that progress in predicting increasingly challenging and diverse types of targets is due to closer integration of template-based modeling techniques with docking, scoring, and model refinement procedures, and to significant incremental improvements in the underlying methodologies.

Blind prediction of homo- and hetero-protein complexes: The CASP13-CAPRI experiment.

We present the results for CAPRI Round 46, the third joint CASP-CAPRI protein assembly prediction challenge. The Round comprised a total of 20 targets including 14 homo-oligomers and 6 heterocomplexes. Eight of the homo-oligomer targets and one heterodimer comprised proteins that could be readily modeled using templates from the Protein Data Bank, often available for the full assembly. The remaining 11 targets comprised 5 homodimers, 3 heterodimers, and two higher-order assemblies. These were more difficult to model, as their prediction mainly involved "ab-initio" docking of subunit models derived from distantly related templates. A total of ~30 CAPRI groups, including 9 automatic servers, submitted on average ~2000 models per target. About 17 groups participated in the CAPRI scoring rounds, offered for most targets, submitting ~170 models per target. The prediction performance, measured by the fraction of models of acceptable quality or higher submitted across all predictors groups, was very good to excellent for the nine easy targets. Poorer performance was achieved by predictors for the 11 difficult targets, with medium and high quality models submitted for only 3 of these targets. A similar performance "gap" was displayed by scorer groups, highlighting yet again the unmet challenge of modeling the conformational changes of the protein components that occur upon binding or that must be accounted for in template-based modeling. Our analysis also indicates that residues in binding interfaces were less well predicted in this set of targets than in previous Rounds, providing useful insights for directions of future improvements.

RINspector: a Cytoscape app for centrality analyses and DynaMine flexibility prediction.

MOTIVATION: Protein function is directly related to amino acid residue composition and the dynamics of these residues. Centrality analyses based on residue interaction networks permit to identify key residues in a protein that are important for its fold or function. Such central residues and their environment constitute suitable targets for mutagenesis experiments. Predicted flexibility and changes in flexibility upon mutation provide valuable additional information for the design of such experiments. RESULTS: We combined centrality analyses with DynaMine flexibility predictions in a Cytoscape app called RINspector. The app performs centrality analyses and directly visualizes the results on a graph of predicted residue flexibility. In addition, the effect of mutations on local flexibility can be calculated. AVAILABILITY AND IMPLEMENTATION: The app is publicly available in the Cytoscape app store. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The challenge of modeling protein assemblies: the CASP12-CAPRI experiment.

We present the quality assessment of 5613 models submitted by predictor groups from both CAPRI and CASP for the total of 15 most tractable targets from the second joint CASP-CAPRI protein assembly prediction experiment. These targets comprised 12 homo-oligomers and 3 hetero-complexes. The bulk of the analysis focuses on 10 targets (of CAPRI Round 37), which included all 3 hetero-complexes, and whose protein chains or the full assembly could be readily modeled from structural templates in the PDB. On average, 28 CAPRI groups and 10 CASP groups (including automatic servers), submitted models for each of these 10 targets. Additionally, about 16 groups participated in the CAPRI scoring experiments. A range of acceptable to high quality models were obtained for 6 of the 10 Round 37 targets, for which templates were available for the full assembly. Poorer results were achieved for the remaining targets due to the lower quality of the templates available for the full complex or the individual protein chains, highlighting the unmet challenge of modeling the structural adjustments of the protein components that occur upon binding or which must be accounted for in template-based modeling. On the other hand, our analysis indicated that residues in binding interfaces were correctly predicted in a sizable fraction of otherwise poorly modeled assemblies and this with higher accuracy than published methods that do not use information on the binding partner. Lastly, the strengths and weaknesses of the assessment methods are evaluated and improvements suggested.

Computational characterization of the binding mode between oncoprotein Ets-1 and DNA-repair enzymes.

The Ets-1 oncoprotein is a transcription factor that promotes target gene expression in specific biological processes. Typically, Ets-1 activity is low in healthy cells, but elevated levels of expression have been found in cancerous cells, specifically related to tumor progression. Like the vast majority of the cellular effectors, Ets-1 does not act alone but in association with partners. Given the important role that is attributed to Ets-1 in major human diseases, it is crucial to identify its partners and characterize their interactions. In this context, two DNA-repair enzymes, PARP-1 and DNA-PK, have been identified recently as interaction partners of Ets-1. We here identify their binding mode by means of protein docking. The results identify the interacting surface between Ets-1 and the two DNA-repair enzymes centered on the α-helix H1 of the ETS domain, leaving α-helix H3 available to bind DNA. The models highlight a hydrophobic patch on Ets-1 at the center of the interaction interface that includes three tryptophans (Trp338, Trp356, and Trp361). We rationalize the binding mode using a series of computational analyses, including alanine scanning, molecular dynamics simulation, and residue centrality analysis. Our study constitutes a first but important step in the characterization, at the molecular level, of the interaction between an oncoprotein and DNA-repair enzymes.

Structure and dynamics of a human myelin protein P2 portal region mutant indicate opening of the ß barrel in fatty acid binding proteins.

BACKGROUND: Myelin is a multilayered proteolipid sheath wrapped around selected axons in the nervous system. Its constituent proteins play major roles in forming of the highly regular membrane structure. P2 is a myelin-specific protein of the fatty acid binding protein (FABP) superfamily, which is able to stack lipid bilayers together, and it is a target for mutations in the human inherited neuropathy Charcot-Marie-Tooth disease. A conserved residue that has been proposed to participate in membrane and fatty acid binding and conformational changes in FABPs is Phe57. This residue is thought to be a gatekeeper for the opening of the portal region upon ligand entry and egress. RESULTS: We performed a structural characterization of the F57A mutant of human P2. The mutant protein was crystallized in three crystal forms, all of which showed changes in the portal region and helix α2. In addition, the behaviour of the mutant protein upon lipid bilayer binding suggested more unfolding than previously observed for wild-type P2. On the other hand, membrane binding rendered F57A heat-stable, similarly to wild-type P2. Atomistic molecular dynamics simulations showed opening of the side of the discontinuous ß barrel, giving important indications on the mechanism of portal region opening and ligand entry into FABPs. The results suggest a central role for Phe57 in regulating the opening of the portal region in human P2 and other FABPs, and the F57A mutation disturbs dynamic cross-correlation networks in the portal region of P2. CONCLUSIONS: Overall, the F57A variant presents similar properties to the P2 patient mutations recently linked to Charcot-Marie-Tooth disease. Our results identify Phe57 as a residue regulating conformational changes that may accompany membrane surface binding and ligand exchange in P2 and other FABPs.

Targeting Dynamical Binding Processes in the Design of Non-Antibiotic Anti-Adhesives by Molecular Simulation-The Example of FimH.

Located at the tip of type I fimbria of Escherichia coli, the bacterial adhesin FimH is responsible for the attachment of the bacteria to the (human) host by specifically binding to highly-mannosylated glycoproteins located on the exterior of the host cell wall. Adhesion represents a necessary early step in bacterial infection and specific inhibition of this process represents a valuable alternative pathway to antibiotic treatments, as such anti-adhesive drugs are non-intrusive and are therefore unlikely to induce bacterial resistance. The currently available anti-adhesives with the highest affinities for FimH still feature affinities in the nanomolar range. A prerequisite to develop higher-affinity FimH inhibitors is a molecular understanding of the FimH-inhibitor complex formation. The latest insights in the formation process are achieved by combining several molecular simulation and traditional experimental techniques. This review summarizes how molecular simulation contributed to the current knowledge of the molecular function of FimH and the importance of dynamics in the inhibitor binding process, and highlights the importance of the incorporation of dynamical aspects in (future) drug-design studies.

A Novel Integrated Way for Deciphering the Glycan Code for the FimH Lectin.

The fimbrial lectin FimH from uro- and enteropathogenic Escherichia coli binds with nanomolar affinity to oligomannose glycans exposing Manα1,3Man dimannosides at their non-reducing end, but only with micromolar affinities to Manα1,2Man dimannosides. These two dimannoses play a significantly distinct role in infection by E. coli. Manα1,2Man has been described early on as shielding the (Manα1,3Man) glycan that is more relevant to strong bacterial adhesion and invasion. We quantified the binding of the two dimannoses (Manα1,2Man and Manα1,3Man to FimH using ELLSA and isothermal microcalorimetry and calculated probabilities of binding modes using molecular dynamics simulations. Our experimentally and computationally determined binding energies confirm a higher affinity of FimH towards the dimannose Manα1,3Man. Manα1,2Man displays a much lower binding enthalpy combined with a high entropic gain. Most remarkably, our molecular dynamics simulations indicate that Manα1,2Man cannot easily take its major conformer from water into the FimH binding site and that FimH is interacting with two very different conformers of Manα1,2Man that occupy 42% and 28% respectively of conformational space. The finding that Manα1,2Man binding to FimH is unstable agrees with the earlier suggestion that E. coli may use the Manα1,2Man epitope for transient tethering along cell surfaces in order to enhance dispersion of the infection.

Modeling protein-protein and protein-peptide complexes: CAPRI 6th edition.

We present the sixth report evaluating the performance of methods for predicting the atomic resolution structures of protein complexes offered as targets to the community-wide initiative on the Critical Assessment of Predicted Interactions (CAPRI). The evaluation is based on a total of 20,670 predicted models for 8 protein-peptide complexes, a novel category of targets in CAPRI, and 12 protein-protein targets in CAPRI prediction Rounds held during the years 2013-2016. For two of the protein-protein targets, the focus was on the prediction of side-chain conformation and positions of interfacial water molecules. Seven of the protein-protein targets were particularly challenging owing to their multicomponent nature, to conformational changes at the binding site, or to a combination of both. Encouragingly, the very large multiprotein complex with the nucleosome was correctly predicted, and correct models were submitted for the protein-peptide targets, but not for some of the challenging protein-protein targets. Models of acceptable quality or better were obtained for 14 of the 20 targets, including medium quality models for 13 targets and high quality models for 8 targets, indicating tangible progress of present-day computational methods in modeling protein complexes with increased accuracy. Our evaluation suggests that the progress stems from better integration of different modeling tools with docking procedures, as well as the use of more sophisticated evolutionary information to score models. Nonetheless, adequate modeling of conformational flexibility in interacting proteins remains an important area with a crucial need for improvement. Proteins 2017; 85:359-377. © 2016 Wiley Periodicals, Inc.

FlexPepDock lessons from CAPRI peptide-protein rounds and suggested new criteria for assessment of model quality and utility.

CAPRI rounds 28 and 29 included, for the first time, peptide-receptor targets of three different systems, reflecting increased appreciation of the importance of peptide-protein interactions. The CAPRI rounds allowed us to objectively assess the performance of Rosetta FlexPepDock, one of the first protocols to explicitly include peptide flexibility in docking, accounting for peptide conformational changes upon binding. We discuss here successes and challenges in modeling these targets: we obtain top-performing, high-resolution models of the peptide motif for cases with known binding sites but there is a need for better modeling of flanking regions, as well as better selection criteria, in particular for unknown binding sites. These rounds have also provided us the opportunity to reassess the success criteria, to better reflect the quality of a peptide-protein complex model. Using all models submitted to CAPRI, we analyze the correlation between current classification criteria and the ability to retrieve critical interface features, such as hydrogen bonds and hotspots. We find that loosening the backbone (and ligand) RMSD threshold, together with a restriction on the side chain RMSD measure, allows us to improve the selection of high-accuracy models. We also suggest a new measure to assess interface hydrogen bond recovery, which is not assessed by the current CAPRI criteria. Finally, we find that surprisingly much can be learned from rather inaccurate models about binding hotspots, suggesting that the current status of peptide-protein docking methods, as reflected by the submitted CAPRI models, can already have a significant impact on our understanding of protein interactions. Proteins 2017; 85:445-462. © 2016 Wiley Periodicals, Inc.

Virulence regulation with Venus flytrap domains: structure and function of the periplasmic moiety of the sensor-kinase BvgS.

Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions.

Sites for Dynamic Protein-Carbohydrate Interactions of O- and C-Linked Mannosides on the E. coli FimH Adhesin.

Antagonists of the Escherichia coli type-1 fimbrial adhesin FimH are recognized as attractive alternatives for antibiotic therapies and prophylaxes against acute and recurrent bacterial infections. In this study α-d-mannopyranosides O- or C-linked with an alkyl, alkene, alkyne, thioalkyl, amide, or sulfonamide were investigated to fit a hydrophobic substituent with up to two aryl groups within the tyrosine gate emerging from the mannose-binding pocket of FimH. The results were summarized into a set of structure-activity relationships to be used in FimH-targeted inhibitor design: alkene linkers gave an improved affinity and inhibitory potential, because of their relative flexibility combined with a favourable interaction with isoleucine-52 located in the middle of the tyrosine gate. Of particular interest is a C-linked mannoside, alkene-linked to an ortho-substituted biphenyl that has an affinity similar to its O-mannosidic analog but superior to its para-substituted analog. Docking of its high-resolution NMR solution structure to the FimH adhesin indicated that its ultimate, ortho-placed phenyl ring is able to interact with isoleucine-13, located in the clamp loop that undergoes conformational changes under shear force exerted on the bacteria. Molecular dynamics simulations confirmed that a subpopulation of the C-mannoside conformers is able to interact in this secondary binding site of FimH.

Signal Transduction by BvgS Sensor Kinase: BINDING OF MODULATOR NICOTINATE AFFECTS THE CONFORMATION AND DYNAMICS OF THE ENTIRE PERIPLASMIC MOIETY.

The two-component sensory transduction system BvgAS controls the virulence regulon of the whooping-cough agent Bordetella pertussis. The periplasmic moiety of the homodimeric sensor kinase BvgS is composed of four bilobed Venus flytrap (VFT) perception domains followed by α helices that extend into the cytoplasmic membrane. In the virulent phase, the default state of B. pertussis, the cytoplasmic enzymatic moiety of BvgS acts as kinase by autophosphorylating and transferring the phosphoryl group to the response regulator BvgA. Under laboratory conditions, BvgS shifts to phosphatase activity in response to modulators, notably nicotinate ions. Here we characterized the effects of nicotinate and related modulators on the BvgS periplasmic moiety by using site-directed mutagenesis and in silico and biophysical approaches. Modulators bind with low affinity to BvgS in the VFT2 cavity. Electron paramagnetic resonance shows that their binding globally affects the conformation and dynamics of the periplasmic moiety. Specific amino acid substitutions designed to slacken interactions within and between the VFT lobes prevent BvgS from responding to nicotinate, showing that BvgS shifts from kinase to phosphatase activity in response to this modulator via a tense transition state that involves a large periplasmic structural block. We propose that this transition enables the transmembrane helices to adopt a distinct conformation that sets the cytoplasmic enzymatic moiety in the phosphatase mode. The bona fide, in vivo VFT ligands that remain to be identified are likely to trigger similar effects on the transmembrane and cytoplasmic moieties. This mechanism may be relevant to the other VFT-containing sensor kinases homologous to BvgS.

Prediction of homoprotein and heteroprotein complexes by protein docking and template-based modeling: A CASP-CAPRI experiment.

We present the results for CAPRI Round 30, the first joint CASP-CAPRI experiment, which brought together experts from the protein structure prediction and protein-protein docking communities. The Round comprised 25 targets from amongst those submitted for the CASP11 prediction experiment of 2014. The targets included mostly homodimers, a few homotetramers, and two heterodimers, and comprised protein chains that could readily be modeled using templates from the Protein Data Bank. On average 24 CAPRI groups and 7 CASP groups submitted docking predictions for each target, and 12 CAPRI groups per target participated in the CAPRI scoring experiment. In total more than 9500 models were assessed against the 3D structures of the corresponding target complexes. Results show that the prediction of homodimer assemblies by homology modeling techniques and docking calculations is quite successful for targets featuring large enough subunit interfaces to represent stable associations. Targets with ambiguous or inaccurate oligomeric state assignments, often featuring crystal contact-sized interfaces, represented a confounding factor. For those, a much poorer prediction performance was achieved, while nonetheless often providing helpful clues on the correct oligomeric state of the protein. The prediction performance was very poor for genuine tetrameric targets, where the inaccuracy of the homology-built subunit models and the smaller pair-wise interfaces severely limited the ability to derive the correct assembly mode. Our analysis also shows that docking procedures tend to perform better than standard homology modeling techniques and that highly accurate models of the protein components are not always required to identify their association modes with acceptable accuracy. Proteins 2016; 84(Suppl 1):323-348. © 2016 Wiley Periodicals, Inc.

The Antiadhesive Strategy in Crohn's Disease: Orally Active Mannosides to Decolonize Pathogenic Escherichia coli from the Gut.

Blocking the adherence of bacteria to cells is an attractive complementary approach to current antibiotic treatments, which are faced with increasing resistance. This strategy has been particularly studied in the context of urinary tract infections (UTIs), in which the adhesion of pathogenic Escherichia coli strains to uroepithelial cells is prevented by blocking the FimH adhesin expressed at the tips of bacteria organelles called fimbriae. Recently, we extended the antiadhesive concept, showing that potent FimH antagonists can block the attachment of adherent-invasive E. coli (AIEC) colonizing the intestinal mucosa of patients with Crohn's disease (CD). In this work, we designed a small library of analogues of heptyl mannoside (HM), a previously identified nanomolar FimH inhibitor, but one that displays poor antiadhesive effects in vivo. The anomeric oxygen atom was replaced by a sulfur or a methylene group to prevent hydrolysis by intestinal glycosidases, and chemical groups were attached at the end of the alkyl tail. Importantly, a lead compound was shown to reduce AIEC levels in the feces and in the colonic and ileal mucosa after oral administration (10 mg kg(-1) ) in a transgenic mouse model of CD. The compound showed a low bioavailability, preferable in this instance, thus suggesting the possibility of setting up an innovative antiadhesive therapy, based on the water-soluble and non-cytotoxic FimH antagonists developed here, for the CD subpopulation in which AIEC plays a key role.