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Abstract Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) is a critical suppressor of proinflammatory cytokine production that acts as a regulator of innate immune signalling and inflammation. However, our current understanding about the molecular properties that enable N4BP1 to exert its suppressive potential remain limited. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NFκB signalling by its unique dimerization-dependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure preferential recognition of linear ubiquitin. Under proinflammatory conditions, N4BP1 is recruited to the nascent TNFR1 signalling complex, where it regulates duration of proinflammatory signalling in LUBIN-dependent manner. N4BP1 deficiency accelerates TNFα-induced cell death by increasing complex II assembly. Under proapoptotic conditions, caspase-8 mediates proteolytic processing of N4BP1, resulting in rapid degradation of N4BP1 by the 26 S proteasome, and acceleration of apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a novel type of ubiquitin reader that selectively recognises linear ubiquitin which enables the timely and coordinated regulation of TNFR1-mediated inflammation and cell death.

Legionella pneumophila is a Gram-negative bacterium that is able to replicate within a broad range of aquatic protozoan hosts. L. pneumophila is also an opportunistic human pathogen that can infect macrophages and epithelia in the lung and lead to Legionnaires’ disease. The type II secretion system is a key virulence factor of L. pneumophila and is used to promote bacterial growth at low temperatures, regulate biofilm formation, modulate host responses to infection, facilitate bacterial penetration of mucin gels and is necessary for intracellular growth during the initial stages of infection. The L. pneumophila type II secretion system exports at least 25 substrates out of the bacterium and several of these, including NttA to NttG, contain unique amino acid sequences that are generally not observed outside of the Legionella genus. NttA, NttC, and NttD are required for infection of several amoebal species but it is unclear what influence other novel substrates have within their host. In this study, we show that NttE is required for optimal infection of Acanthamoeba castellanii and Vermamoeba vermiformis amoeba and is essential for the typical colony morphology of L. pneumophila. In addition, we report the atomic structures of NttA, NttC, and NttE and through a combined biophysical and biochemical hypothesis driven approach we propose novel functions for these substrates during infection. This work lays the foundation for future studies into the mechanistic understanding of novel type II substrate functions and how these relate to L. pneumophila ecology and disease.

The calcium calmodulin-dependent protein kinase II (CaMKII) is a dodecameric holoenzyme important for encoding memory. Its activation, triggered by binding of calcium-calmodulin, persists autonomously after calmodulin dissociation. One (receiver) kinase captures and subsequently phosphorylates the regulatory domain peptide of a donor kinase forming a chained dimer as the first stage of autonomous activation. Protein dynamics simulations examined the conformational changes triggered by dimer formation and phosphorylation, aimed to provide a molecular rationale for human mutations that result in learning disabilities. Ensembles generated from X-ray crystal structures were characterized by network centrality and community analysis. Mutual information related collective motions to local fragment dynamics encoded with a structural alphabet. Implicit solvent tCONCOORD conformational ensembles revealed the dynamic architecture of inactive kinase domains was co-opted in the activated dimer but the network hub shifted from the nucleotide binding cleft to the captured peptide. Explicit solvent molecular dynamics (MD) showed nucleotide and substrate binding determinants formed coupled nodes in long-range signal relays between regulatory peptides in the dimer. Strain in the extended captured peptide was balanced by reduced flexibility of the receiver kinase C-lobe core. The relays were organized around a hydrophobic patch between the captured peptide and a key binding helix. The human mutations aligned along the relays. Thus, these mutations could disrupt the allosteric network alternatively, or in addition, to altered binding affinities. Non-binding protein sectors distant from the binding sites mediated the allosteric signalling; providing possible targets for inhibitor design. Phosphorylation of the peptide modulated the dielectric of its binding pocket to strengthen the patch, non-binding sectors, domain interface and temporal correlations between parallel relays. These results provide the molecular details underlying the reported positive kinase cooperativity to enrich the discussion on how autonomous activation by phosphorylation leads to long-term behavioural effects.

Hypoxia inducible factors (HIFs) are transcription factors belonging to the basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) protein family with a role in sensing oxygen levels in the cell. Under hypoxia, the HIF-α degradation pathway is blocked and dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT) makes HIF-α transcriptionally active. Due to the common hypoxic environment of tumors, inhibition of this mechanism by destabilization of HIF-α:ARNT dimerization has been proposed as a promising therapeutic strategy. Following the discovery of a druggable cavity within the PAS-B domain of HIF-2α, research efforts have been directed to identify artificial ligands that can impair heterodimerization. Although the crystallographic structures of the HIF-2α:ARNT complex have elucidated the dimer architecture and the 0X3-inhibitor placement within the HIF-2α PAS-B, unveiling the inhibition mechanism requires investigation of how ligand-induced perturbations could dynamically propagate through the structure and affect dimerization. To this end, we compared evolutionary features, intrinsic dynamics and energetic properties of the dimerization interfaces of HIF-2α:ARNT in both the apo and holo forms. Residue conservation analysis highlighted inter-domain connecting elements that have a role in dimerization. Analysis of domain contributions to the dimerization energy demonstrated the importance of bHLH and PAS-A of both partners and of HIF-2α PAS-B domain in dimer stabilization. Among quaternary structure oscillations revealed by Molecular Dynamics simulations, the hinge-bending motion of the ARNT PAS-B domain around the flexible PAS-A/PAS-B linker supports a general model for ARNT dimerization in different heterodimers. Comparison of the HIF-2α:ARNT dynamics in the apo and 0X3-bound forms indicated a model of inhibition where the HIF-2α-PAS-B interfaces are destabilised as a result of water-bridged ligand-protein interactions and these local effects allosterically propagate to perturb the correlated motions of the domains and inter-domain communication. These findings will guide the design of improved inhibitors to contrast cell survival in tumor masses.

Proteins are large, complex molecules with crucial roles in the functioning of living organisms. Understanding the underlying mechanisms by which proteins achieve their structures and substructures, as well as those involved in the conformational transitions may contribute to a deeper comprehension of the involved biological processes. This paper investigates a new machine learning perspective upon analyzing protein conformational transitions and introduces a new formalization for the problem, with the more general goal of uncovering interesting patterns in protein conformational transitions. This study represents the starting point of a research which is being conducted in order to obtain a better comprehension of proteins’ structures and, implicitly, functions, by investigating computational intelligence methods for analyzing and deducing proteins conformational transitions.

Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC) "torque" helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM) domains (amino-terminal (FliGN), middle (FliGM) and FliGC) as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM) has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6). FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C) and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM-C could be the convertor element that provides mechanistic and species diversity.

Motivation: The size of current protein databases is a challenge for many Bioinformatics applications, both in terms of processing speed and information redundancy. It may be therefore desirable to efficiently reduce the database of interest to a maximally representative subset.Results: The MinSet method employs a combination of a Suffix Tree and a Genetic Algorithm for the generation, selection and assessment of database subsets. The approach is generally applicable to any type of string-encoded data, allowing for a drastic reduction of the database size whilst retaining most of the information contained in the original set. We demonstrate the performance of the method on a database of protein domain structures encoded as strings. We used the SCOP40 domain database by translating protein structures into character strings by means of a structural alphabet and by extracting optimized subsets according to an entropy score that is based on a constant-length fragment dictionary. Therefore, optimized subsets are maximally representative for the distribution and range of local structures. Subsets containing only 10% of the SCOP structure classes show a coverage of >90% for fragments of length 1–4.Availability:http://mathbio.nimr.mrc.ac.uk/~jkleinj/MinSetContact:jkleinj@nimr.mrc.ac.ukSupplementary information: Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Aim of this work is to assess the informativeness of protein dynamics in the detection of similarities among distant homologous proteins. To this end, an approach to perform large-scale comparisons of protein domain flexibilities is proposed. CONCOORD is confirmed as a reliable method for fast conformational sampling. The root mean square fluctuation of alpha carbon positions in the essential dynamics subspace is employed as a measure of local flexibility and a synthetic index of similarity is presented. The dynamics of a large collection of protein domains from ASTRAL/SCOP40 is analyzed and the possibility to identify relationships, at both the family and the superfamily levels, on the basis of the dynamical features is discussed. The obtained picture is in agreement with the SCOP classification, and furthermore suggests the presence of a distinguishable familiar trend in the flexibility profiles. The results support the complementarity of the dynamical and the structural information, suggesting that information from dynamics analysis can arise from functional similarities, often partially hidden by a static comparison. On the basis of this first test, flexibility annotation can be expected to help in automatically detecting functional similarities otherwise unrecoverable. [ABSTRACT FROM AUTHOR]