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Cardiovascular disease (CVD) is a leading cause of death worldwide. Approximately 60% of patients treated with low‐density lipoprotein (LDL)‐lowering drug treatments, with on‐target plasma cholesterol levels, are still suffering clinical acute ischemic events. Mechanisms, such as LDL aggregation, underlie extracellular and intracellular cholesterol accumulation in the vasculature. A peptide sequence (P3) of the low‐density lipoprotein receptor‐related protein 1 (LRP1) efficiently protects LDL from sphingomyelinase (SMase‐) and phospholipase A2 (PLA2)‐induced LDL aggregation. The aim is to design families of peptide derivatives from P3 with enhanced potency and proteolytic stability. New peptides are designed through in silico conformational sampling and ApoB‐100 molecular docking, and are tested in dual (biochemical‐cellular) screening assays. A total of 46 new peptides including linear, fragment, cyclic, and alanine scanning derivatives are generated through two consecutive optimization rounds. Structurally and functionally optimized peptides contain hotspot residues that are replaced by alanine. This strategy confers an increased capacity to form prone alpha‐helix conformations crucial for the electrostatic interaction with ApoB‐100. These new compounds are highly efficient at inhibiting LDL aggregation and human coronary vascular smooth muscle cell‐cholesteryl ester loading and should be studied in preclinical models of atherosclerosis., A.B.-A. and C.P. contributed equally to this work. The authors thank Dr. Ignasi Gich, Professor of Clinical Pharmacology and Therapeutics and Researcher of Sant Pau Biomedical Research Institute (IIB-SantPau) and CIBER Epidemiología y Salud Pública (CIBERESP), for this help in statistical analysis and graph representations. The Ministry of Science and Innovation of Spain, in the framework of the State Plan of Scientific and Technical Innovation Investigation 2013–2016, awarded funding to the project “DEVELOPMENT OF AN INNOVATIVE THERAPY FOR THE TREATMENT OF THE ATHEROSCLEROSIS THROUGH INHIBITION OF CHOLESTEROL VASCULAR ACCUMULATION” led by IPROTEOS SL with File No. RTC-2016-5078-1. Support was also received from the Fundació MARATÓ TV3 Project 201521-10 (to VLl-C), and FIS PI18/01584 (to VLl-C) from the Instituto de Salud Carlos III (ISCIII) and cofinanced with ERDFs. Support was also received from Ministerio de Economía y Competitividad to DdG-C (IJCI-2016-29393). CIBER Enfermedades Cardiovasculares (CIBERCV; CB16/1100403 (DdG-C, VLl-C) are projects run by the Instituto de Salud Carlos III (ISCIII). The authors also acknowledge the support from “Secretaria d’Universitats i Recerca del Departament d’Economia I Coneixement de la Generalitat de Catalunya” (2017SGR946 to VLl-C).

A huge amount of genetic information is available thanks to the recent advances in sequencing technologies and the larger computational capabilities, but the interpretation of such genetic data at phenotypic level remains elusive. One of the reasons is that proteins are not acting alone, but are specifically interacting with other proteins and biomolecules, forming intricate interaction networks that are essential for the majority of cell processes and pathological conditions. Thus, characterizing such interaction networks is an important step in understanding how information flows from gene to phenotype. Indeed, structural characterization of protein–protein interactions at atomic resolution has many applications in biomedicine, from diagnosis and vaccine design, to drug discovery. However, despite the advances of experimental structural determination, the number of interactions for which there is available structural data is still very small. In this context, a complementary approach is computational modeling of protein interactions by docking, which is usually composed of two major phases: (i) sampling of the possible binding modes between the interacting molecules and (ii) scoring for the identification of the correct orientations. In addition, prediction of interface and hot-spot residues is very useful in order to guide and interpret mutagenesis experiments, as well as to understand functional and mechanistic aspects of the interaction. Computational docking is already being applied to specific biomedical problems within the context of personalized medicine, for instance, helping to interpret pathological mutations involved in protein–protein interactions, or providing modeled structural data for drug discovery targeting protein–protein interactions. Spanish Ministry of Economy grant number BIO2016-79960-R; D.B.B. is supported by a predoctoral fellowship from CONACyT; M.R. is supported by an FPI fellowship from the Severo Ochoa program. We are grateful to the Joint BSC-CRG-IRB Programme in Computational Biology.

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.

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.

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.

Two errors were made in this article. First, a reference was omitted from the second sentence of the fourth paragraph in the Introduction section. The sentence should read: SidD lacks any obvious sequence homology with the AT-N or other known proteins, although fold recognition analysis of the N-terminal portion of SidD predicted limited resemblance with members of the metal-dependent protein phosphatase (PPM) family (Rigden 2011). The reference is: Rigden DJ. (2011) Identification and modelling of a PPM protein phosphatase fold in the Legionella pneumophila deAMPylase SidD. FEBS Lett. 585:2749-54. doi: 10.1016/j.febslet.2011.08.006. Second, there were some omissions from the acknowledgements section, which should instead read as follows: We thank K. B. Decker, Y. Lin, and A.H. Gaspar for comments on the manuscript, and A. Vidaurrazaga for assistance with biochemical experiments. This study made use of the European Synchrotron Radiation Facility (ESRF, Grenoble, France) under the Block Allocation Group (BAG) MX1420, the Diamond Light Source (Oxfordshire, UK) under the rapid access mode MX7512 and BAG MX8302, the beamline PROXIMA1 at the SOLEIL synchrotron (Saint-Aubin, France), the X-ray crystallography platform and proteomics platform (member of CIBERehd and ProteoRed-ISCIII) at CIC bioGUNE (Derio, Spain), and the SGIker analytical facility at UPV/EHU (Leioa, Spain). We thank all the staff from these facilities for technical and human support. Access to synchrotron facilities was part funded by the the BioStruct-X project (proposal number 2460).

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., Author Summary The covalent attachment of adenosine monophosphate (AMP) to proteins, a process called AMPylation (adenylylation), has recently emerged as a novel theme in microbial pathogenesis. While AMPylases from various pathogenic microorganisms have recently been characterized, the only virulence protein with de-AMPylation activity known to date is the Legionella pneumophila effector SidD which catalyzes AMP removal from the host GTPase Rab1. Thus, both AMPylation and de-AMPylation constitute a novel catalytic mechanism to precisely control the function and membrane dynamics of a host Rab GTPase. In spite of this pivotal role, the molecular mechanism of AMP removal and the structural determinants for Rab1 recognition by SidD have remained largely unexplored. Here, we present the crystal structure of the de-adenylylation domain of SidD and reveal the catalytic mechanism of Rab1 de-adenylylation. Surprisingly, the structure of SidD is not related to the other known enzyme with de-AMPylation activity, the Escherichia coli GS-ATase. Instead, the catalitic domain of SidD is remarkably similar to that of the metal-dependent protein phosphatases (PPMs), however with distinctive structural features to distinguish AMPylated Rab1 from similarly modified substrates. Importantly, we provide a model for the SidD-Rab1 complex which sheds light into the specific details of substrate recognition and catalysis by this virulence factor.