Bilayer lipid membrane formation on surface assemblies with sparsely distributed tethers.

A combined computational and experimental study of small unilamellar vesicle (SUV) fusion on mixed self-assembled monolayers (SAMs) terminated with different deuterated tether moieties (-(CD2)7CD3 or -(CD2)15CD3) is reported. Tethered bilayer lipid membrane (tBLM) formation of synthetic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine was initially probed on SAMs with controlled tether (d-alkyl tail) surface densities and lateral molecular packing using quartz crystal microbalance with dissipation monitoring (QCM-D). Long time-scale coarse-grained molecular dynamics (MD) simulations were then employed to elucidate the mechanisms behind the interaction between the SUVs and the different phases formed by the -(CD2)7CD3 and -(CD2)15CD3 tethers. Furthermore, a series of real time kinetics was recorded under different osmotic conditions using QCM-D to determine the accumulated lipid mass and for probing the fusion process. It is shown that the key factors driving the SUV fusion and tBLM formation on this type of surfaces involve tether insertion into the SUVs along with vesicle deformation. It is also evident that surface densities of the tethers as small as a few mol% are sufficient to obtain stable tBLMs with a high reproducibility. The described "sparsely tethered" tBLM system can be advantageous in studying different biophysical phenomena, such as membrane protein insertion, effects of receptor clustering, and raft formation.

Synthesis and structure-activity relationships of USP48 deubiquitinylase inhibitors.

Ubiquitin-specific proteases represent a family of enzymes that catalyze the cleavage of ubiquitin from specific substrate proteins to regulate their activity. USP48 is a rarely studied USP, which has recently been linked to inflammatory signaling via regulation of the transcription factor nuclear factor kappa B. Nonetheless, a crystal structure of USP48 has not yet been resolved and potent inhibitors are not known. We screened a set of 14 commercially available USP inhibitors for their activity against USP48 and identified the USP2 inhibitor "ML364" as a candidate for further optimization. Using a ligand-based approach, we derived and synthesized a series of ML364 analogs. The IC50 concentrations of the new compounds to inhibit USP48 were determined in a deubiquitinylase activity assay by measuring the fluorescence intensity using tetra-ubiquitin rhodamine110 as substrate. A compound containing a carboxylic acid functionalization (17e) inhibited USP48 activity toward tetra-ubiquitin rhodamine110 with an IC50 of 12.6 µM. Further structure-based refinements are required to improve the inhibition activity and specificity.

Structural Dynamics of Lys11-Selective Deubiquitinylase Cezanne-1 during the Catalytic Cycle.

Deubiquitinylating enzymes (DUBs) regulate the deubiquitinylation process of post-translationally modified proteins and thus control protein signaling in various cellular processes. The DUB Cezanne-1 catalyzes the cleavage of the iso-peptide bond of Lys11-linked polyubiquitin chains with high selectivity. Crystal structures of Cezanne-1 in different states provide important insight regarding the complex formation and global changes during the catalytic cycle but are lacking details of dynamics and control of activation. Activity-based probes are used to isolate intermediate states upon forming covalent bonds with the DUB active site. Those, however, may lead to structures that are non-native. Conformational changes of Cezanne-1, during its process of activation and proteolytic activity, are investigated using all-atom molecular dynamics (MD) simulations of the ubiquitin-free, diubiquitin-bound, and monoubiquitin-bound Cezanne-1 DUB for a total of ∼18 µs. Our results show that ubiquitin-free Cezanne-1 dynamically shuttles between catalytically competent and incompetent states which suggests that its activation is independent of substrate binding. The catalytically competent substrate-free Cezanne-1 promotes distal ubiquitin substrate access to the catalytic center. The subsequent binding of the proximal ubiquitin shifts the equilibrium toward the catalytically competent state of the dyad, thereby promoting proteolysis of the iso-peptide bond. After cleavage of the scissile bond, sequential dissociation of first the proximal ubiquitin induces the inactivation of Cezanne-1. The subsequent release of the distal ubiquitin fully reconstitutes the inactive substrate-free state of Cezanne-1. The process of activation and catalytic turnover of DUB Cezanne-1 is a multistage cycle with several critical dynamic transitions that cannot be characterized based on protein structures alone. Activity-based probes of cysteine proteases lead to non-native protein-protein contacts, which need to be resolved in order to be able to issue statements about physiological states and substrate binding.

Conformational Control of Fast Asparagine Deamidation in a Norovirus Capsid Protein.

Accelerated spontaneous deamidation of asparagine 373 and subsequent conversion into an isoaspartate has been shown to attenuate the binding of histo blood group antigens (HBGAs) to the protruding domain (P-domain) of the capsid protein of a prevalent norovirus strain (GII.4). Here, we link an unusual backbone conformation of asparagine 373 to its fast site-specific deamidation. NMR spectroscopy and ion exchange chromatography have been used to monitor the deamidation reaction of P-domains of two closely related GII.4 norovirus strains, specific point mutants, and control peptides. MD simulations over several microseconds have been instrumental to rationalize the experimental findings. While conventional descriptors such as available surface area, root-mean-square fluctuations, or nucleophilic attack distance fail as explanations, the population of a rare syn-backbone conformation distinguishes asparagine 373 from all other asparagine residues. We suggest that stabilization of this unusual conformation enhances the nucleophilicity of the backbone nitrogen of aspartate 374, in turn accelerating the deamidation of asparagine 373. This finding should be relevant to the development of reliable prediction algorithms for sites of rapid asparagine deamidation in proteins.

The COP9 signalosome: A versatile regulatory hub of Cullin-RING ligases.

The COP9 signalosome (CSN) is a universal regulator of Cullin-RING ubiquitin ligases (CRLs) - a family of modular enzymes that control various cellular processes via timely degradation of key signaling proteins. The CSN, with its eight-subunit architecture, employs multisite binding of CRLs and inactivates CRLs by removing a small ubiquitin-like modifier named neural precursor cell-expressed, developmentally downregulated 8 (Nedd8). Besides the active site of the catalytic subunit CSN5, two allosteric sites are present in the CSN, one of which recognizes the substrate recognition module and the presence of CRL substrates, and the other of which can 'glue' the CSN-CRL complex by recruitment of inositol hexakisphosphate. In this review, we present recent findings on the versatile regulation of CSN-CRL complexes.

Ovarian tumor domain proteases in pathogen infection.

With the aim of overcoming host immune responses, and to permit persistence, numerous bacterial and viral pathogens have evolved effective strategies to control the activity of ovarian tumor domain proteases (OTUs), a group of deubiquitinylases crucial for regulating ubiquitin-modified proteins. Due to the important role of eukaryotic OTUs in cellular physiology, it is not surprising that pathogens have evolutionarily developed effector proteins which mimic host OTUs. Here, we focus on recent findings that illustrate how pathogen-encoded OTUs modulate eukaryotic host proteins and how they are implicated in cellular dysregulation. Further, we discuss the biological effects of OTUs in the context of structural features and pharmacological targeting. We point out the potentiality of selective OTU inhibitors, which shield ubiquitin-binding sites, as pharmacologic targets to treat harmful infections.

Substrate-assisted activation and selectivity of the bacterial RavD effector deubiquitinylase.

Deubiquitinylases (DUBs) catalyze the peptide bond cleavage of specific ubiquitin linkages at distinct protein substrates. Pathogens from viruses and bacteria independently developed effector proteins with DUB activity to mimic host DUB functions and circumvent immune responses. The effector protein RavD from Legionella pneumophila cleaves linear ubiquitin chains with an exclusive methionine-1 selectivity. It thus performs as a functional analogue of the human DUB OTULIN, which achieves its selectivity only via a specialized proximal ubiquitin S1' binding site as well as a substrate-assisted activation of the catalytic triad. An analysis of the crystal structures of bacterial RavD in its free and di-ubiquitin-bound forms, in order to rationalize the structural basis for its selectivity and activation mechanism, is not fully conclusive. As these ambiguities might arise from the introduced double mutation of the di-ubiquitin substrate in the RavD-di-ubiquitin complex crystal structure, biomolecular modeling, and molecular dynamics sampling (1-2 µs for each system of RavD and OTULIN) were employed to reconstitute the physiological RavD-di-ubiquitin complex. The simulations show that the distal S1 ubiquitin binding sites of RavD and OTULIN are similar in terms of interface area, composition, and ubiquitin binding affinity. The proximal S1' site of RavD, in contrast, is significantly smaller and ubiquitin binding is weaker and more flexible than in OTULIN. Upon substrate access, the residues of the catalytic triad of RavD show a reduction of flexibility and a conformational transition toward a catalytically active state. Thus, the enzymatic activation of RavD is presumably also substrate-assisted and a clear rationale for the common M1-substrate selectivity.