Chiara Pallara

Community-wide evaluation of methods for predicting the effect of mutations on protein-protein interactions

Community‐wide blind prediction experiments such as CAPRI and CASP provide an objective measure of the current state of predictive methodology. Here we describe a community‐wide assessment of methods to predict the effects of mutations on protein–protein interactions. Twenty‐two groups predicted the effects of comprehensive saturation mutagenesis for two designed influenza hemagglutinin binders and the results were compared with experimental yeast display enrichment data obtained using deep sequencing. The most successful methods explicitly considered the effects of mutation on monomer stability in addition to binding affinity, carried out explicit side‐chain sampling and backbone relaxation, evaluated packing, electrostatic, and solvation effects, and correctly identified around a third of the beneficial mutations. Much room for improvement remains for even the best techniques, and large‐scale fitness landscapes should continue to provide an excellent test bed for continued evaluation of both existing and new prediction methodologies. Proteins 2013; 81:1980–1987.

Blind prediction of interfacial water positions in CAPRI.

We report the first assessment of blind predictions of water positions at protein–protein interfaces, performed as part of the critical assessment of predicted interactions (CAPRI) community‐wide experiment. Groups submitting docking predictions for the complex of the DNase domain of colicin E2 and Im2 immunity protein (CAPRI Target 47), were invited to predict the positions of interfacial water molecules using the method of their choice. The predictions—20 groups submitted a total of 195 models—were assessed by measuring the recall fraction of water‐mediated protein contacts. Of the 176 high‐ or medium‐quality docking models—a very good docking performance per se—only 44% had a recall fraction above 0.3, and a mere 6% above 0.5. The actual water positions were in general predicted to an accuracy level no better than 1.5 Å, and even in good models about half of the contacts represented false positives. This notwithstanding, three hotspot interface water positions were quite well predicted, and so was one of the water positions that is believed to stabilize the loop that confers specificity in these complexes. Overall the best interface water predictions was achieved by groups that also produced high‐quality docking models, indicating that accurate modelling of the protein portion is a determinant factor. The use of established molecular mechanics force fields, coupled to sampling and optimization procedures also seemed to confer an advantage. Insights gained from this analysis should help improve the prediction of protein–water interactions and their role in stabilizing protein complexes. Proteins 2014; 82:620–632.

Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5.

Significance A long-standing question in the field of microbial pathogenesis is how virulence factors are regulated within host cells and how their activity is specifically directed toward a particular host cell compartment. Legionella pneumophila resolves this dilemma by tightly coupling the phospholipase A1 activity of one of its effectors, vacuolar protein sorting inhibitor protein D (VipD), to this protein’s interaction with endosomal host GTPases. We now present the crystal structure of VipD in complex with host cell Rab5c, providing a detailed look into the ingenious molecular mechanisms underlying the allosteric activation of a virulence factor by a host protein and its spatiotemporal regulation. These results open the path for the development of novel therapeutics aimed at blocking the VipD activation process rather than the enzyme’s active site. A challenge for microbial pathogens is to assure that their translocated effector proteins target only the correct host cell compartment during infection. The Legionella pneumophila effector vacuolar protein sorting inhibitor protein D (VipD) localizes to early endosomal membranes and alters their lipid and protein composition, thereby protecting the pathogen from endosomal fusion. This process requires the phospholipase A1 (PLA1) activity of VipD that is triggered specifically on VipD binding to the host cell GTPase Rab5, a key regulator of endosomes. Here, we present the crystal structure of VipD in complex with constitutively active Rab5 and reveal the molecular mechanism underlying PLA1 activation. An active site-obstructing loop that originates from the C-terminal domain of VipD is repositioned on Rab5 binding, thereby exposing the catalytic pocket within the N-terminal PLA1 domain. Substitution of amino acid residues located within the VipD–Rab5 interface prevented Rab5 binding and PLA1 activation and caused a failure of VipD mutant proteins to target to Rab5-enriched endosomal structures within cells. Experimental and computational analyses confirmed an extended VipD-binding interface on Rab5, explaining why this L. pneumophila effector can compete with cellular ligands for Rab5 binding. Together, our data explain how the catalytic activity of a microbial effector can be precisely linked to its subcellular localization.

Structural Basis for Rab1 De-AMPylation by the Legionella pneumophila Effector SidD

The covalent attachment of adenosine monophosphate (AMP) to proteins, a process called AMPylation (adenylylation), has recently emerged as a novel theme in microbial pathogenesis. Although several AMPylating enzymes have been characterized, the only known virulence protein with de-AMPylation activity is SidD from the human pathogen Legionella pneumophila. SidD de-AMPylates mammalian Rab1, a small GTPase involved in secretory vesicle transport, thereby targeting the host protein for inactivation. The molecular mechanisms underlying Rab1 recognition and de-AMPylation by SidD are unclear. Here, we report the crystal structure of the catalytic region of SidD at 1.6 Å resolution. The structure reveals a phosphatase-like fold with additional structural elements not present in generic PP2C-type phosphatases. The catalytic pocket contains a binuclear metal-binding site characteristic of hydrolytic metalloenzymes, with strong dependency on magnesium ions. Subsequent docking and molecular dynamics simulations between SidD and Rab1 revealed the interface contacts and the energetic contribution of key residues to the interaction. In conjunction with an extensive structure-based mutational analysis, we provide in vivo and in vitro evidence for a remarkable adaptation of SidD to its host cell target Rab1 which explains how this effector confers specificity to the reaction it catalyses.

Expanding the frontiers of protein–protein modeling: From docking and scoring to binding affinity predictions and other challenges

In addition to protein–protein docking, this CAPRI edition included new challenges, like protein–water and protein–sugar interactions, or the prediction of binding affinities and ΔΔG changes upon mutation. Regarding the standard protein–protein docking cases, our approach, mostly based on the pyDock scheme, submitted correct models as predictors and as scorers for 67% and 57% of the evaluated targets, respectively. In this edition, available information on known interface residues hardly made any difference for our predictions. In one of the targets, the inclusion of available experimental small‐angle X‐ray scattering (SAXS) data using our pyDockSAXS approach slightly improved the predictions. In addition to the standard protein–protein docking assessment, new challenges were proposed. One of the new problems was predicting the position of the interface water molecules, for which we submitted models with 20% and 43% of the water‐mediated native contacts predicted as predictors and scorers, respectively. Another new problem was the prediction of protein–carbohydrate binding, where our submitted model was very close to being acceptable. A set of targets were related to the prediction of binding affinities, in which our pyDock scheme was able to discriminate between natural and designed complexes with area under the curve = 83%. It was also proposed to estimate the effect of point mutations on binding affinity. Our approach, based on machine learning methods, showed high rates of correctly classified mutations for all cases. The overall results were highly rewarding, and show that the field is ready to move forward and face new interesting challenges in interactomics. Proteins 2013; 81:2192–2200.

Tetramerization-defects of p53 result in aberrant ubiquitylation and transcriptional activity.

The tumor suppressor p53 regulates the expression of genes involved in cell cycle progression, senescence and apoptosis. Here, we investigated the effect of single point mutations in the oligomerization domain (OD) on tetramerization, transcription, ubiquitylation and stability of p53. As predicted by docking and molecular dynamics simulations, p53 OD mutants show functional defects on transcription, Mdm2‐dependent ubiquitylation and 26S proteasome‐mediated degradation. However, mutants unable to form tetramers are well degraded by the 20S proteasome. Unexpectedly, despite the lower structural stability compared to WT p53, p53 OD mutants form heterotetramers with WT p53 when expressed transiently or stably in cells wild type or null for p53. In consequence, p53 OD mutants interfere with the capacity of WT p53 tetramers to be properly ubiquitylated and result in changes of p53‐dependent protein expression patterns, including the pro‐apoptotic proteins Bax and PUMA under basal and adriamycin‐induced conditions. Importantly, the patient derived p53 OD mutant L330R (OD1) showed the more severe changes in p53‐dependent gene expression. Thus, in addition to the well‐known effects on p53 stability, ubiquitylation defects promote changes in p53‐dependent gene expression with implications on some of its functions.

Conformational Heterogeneity of Unbound Proteins Enhances Recognition in Protein-Protein Encounters.

To understand cellular processes at the molecular level we need to improve our knowledge of protein-protein interactions, from a structural, mechanistic, and energetic point of view. Current theoretical studies and computational docking simulations show that protein dynamics plays a key role in protein association and support the need for including protein flexibility in modeling protein interactions. Assuming the conformational selection binding mechanism, in which the unbound state can sample bound conformers, one possible strategy to include flexibility in docking predictions would be the use of conformational ensembles originated from unbound protein structures. Here we present an exhaustive computational study about the use of precomputed unbound ensembles in the context of protein docking, performed on a set of 124 cases of the Protein-Protein Docking Benchmark 3.0. Conformational ensembles were generated by conformational optimization and refinement with MODELLER and by short molecular dynamics trajectories with AMBER. We identified those conformers providing optimal binding and investigated the role of protein conformational heterogeneity in protein-protein recognition. Our results show that a restricted conformational refinement can generate conformers with better binding properties and improve docking encounters in medium-flexible cases. For more flexible cases, a more extended conformational sampling based on Normal Mode Analysis was proven helpful. We found that successful conformers provide better energetic complementarity to the docking partners, which is compatible with recent views of binding association. In addition to the mechanistic considerations, these findings could be exploited for practical docking predictions of improved efficiency.

Intrinsically active MEK variants are differentially regulated by proteinases and phosphatases

MAPK/ERK kinase (MEK) 1/2 are central signaling proteins that serve as specificity determinants of the MAPK/ERK cascade. More than twenty activating mutations have been reported for MEK1/2, and many of them are known to cause diseases such as cancers, arteriovenous malformation and RASopathies. Changes in their intrinsic activity do not seem to correlate with the severity of the diseases. Here we studied four MEK1/2 mutations using biochemical and molecular dynamic methods. Although the studied mutants elevated the activating phosphorylation of MEK they had no effect on the stimulated ERK1/2 phosphorylation. Studying the regulatory mechanism that may explain this lack of effect, we found that one type of mutation affects MEK stability and two types of mutations demonstrate a reduced sensitivity to PP2A. Together, our results indicate that some MEK mutations exert their function not only by their elevated intrinsic activity, but also by modulation of regulatory elements such as protein stability or dephosphorylation.

pyDock scoring for the new modeling challenges in docking: protein-peptide, homo-multimers and domain-domain interactions

The sixth CAPRI edition included new modeling challenges, such as the prediction of protein–peptide complexes, and the modeling of homo‐oligomers and domain–domain interactions as part of the first joint CASP–CAPRI experiment. Other non‐standard targets included the prediction of interfacial water positions and the modeling of the interactions between proteins and nucleic acids. We have participated in all proposed targets of this CAPRI edition both as predictors and as scorers, with new protocols to efficiently use our docking and scoring scheme pyDock in a large variety of scenarios. In addition, we have participated for the first time in the servers section, with our recently developed webserver, pyDockWeb. Excluding the CASP–CAPRI cases, we submitted acceptable models (or better) for 7 out of the 18 evaluated targets as predictors, 4 out of the 11 targets as scorers, and 6 out of the 18 targets as servers. The overall success rates were below those in past CAPRI editions. This shows the challenging nature of this last edition, with many difficult targets for which no participant submitted a single acceptable model. Interestingly, we submitted acceptable models for 83% of the evaluated protein–peptide targets. As for the 25 cases of the CASP–CAPRI experiment, in which we used a larger variety of modeling techniques (template‐based, symmetry restraints, literature information, etc.), we submitted acceptable models for 56% of the targets. In summary, this CAPRI edition showed that pyDock scheme can be efficiently adapted to the increasing variety of problems that the protein interactions field is currently facing. Proteins 2017; 85:487–496.

Interaction of photosystem I from Phaeodactylum tricornutum with plastocyanins as compared with its native cytochrome c6: Reunion with a lost donor

In the Phaeodactylum tricornutum alga, as in most diatoms, cytochrome c6 is the only electron donor to photosystem I, and thus they lack plastocyanin as an alternative electron carrier. We have investigated, by using laser-flash absorption spectroscopy, the electron transfer to Phaeodactylum photosystem I from plastocyanins from cyanobacteria, green algae and plants, as compared with its own cytochrome c6. Diatom photosystem I is able to effectively react with eukaryotic acidic plastocyanins, although with less efficiency than with Phaeodactylum cytochrome c6. This efficiency, however, increases in some green alga plastocyanin mutants mimicking the electrostatics of the interaction site on the diatom cytochrome. In addition, the structure of the transient electron transfer complex between cytochrome c6 and photosystem I from Phaeodactylum has been analyzed by computational docking and compared to that of green lineage and mixed systems. Taking together, the results explain why the Phaeodactylum system shows a lower efficiency than the green systems, both in the formation of the properly arranged [cytochrome c6-photosystem I] complex and in the electron transfer itself.