The free energy landscape of the oncogene protein E7 of human papillomavirus type 16 reveals a complex interplay between ordered and disordered regions.

When present, structural disorder makes it very challenging to characterise the conformational properties of proteins. This is particularly the case of proteins, such as the oncogene protein E7 of human papillomavirus type 16, which contain both ordered and disordered domains, and that can populate monomeric and oligomeric states under physiological conditions. Nuclear magnetic resonance (NMR) spectroscopy is emerging as a powerful method to study these complex systems, most notably in combination with molecular dynamics simulations. Here we use NMR chemical shifts and residual dipolar couplings as structural restraints in replica-averaged molecular dynamics simulations to determine the free energy landscape of E7. This landscape reveals a complex interplay between a folded but highly dynamical C-terminal domain and a disordered N-terminal domain that forms transient secondary and tertiary structures, as well as an equilibrium between a high-populated (98%) dimeric state and a low-populated (2%) monomeric state. These results provide compelling evidence of the complex conformational heterogeneity associated with the behaviour and interactions of this disordered protein associated with disease.University of Florence (Italy) “Science without borders” of the Brazilian Ministry of Science and Technology (CNPq

Structure and dynamics of the integrin LFA-1 I-domain in the inactive state underlie its inside-out/outside-in signaling and allosteric mechanisms.

Lymphocyte function-associated antigen 1 (LFA-1) is an integrin that transmits information in two directions across the plasma membrane of leukocytes, in so-called outside-in and inside-out signaling mechanisms. To investigate the structural basis of these mechanisms, we studied the conformational space of the apo I-domain using replica-averaged metadynamics simulations in combination with nuclear magnetic resonance chemical shifts. We thus obtained a free energy landscape that reveals the existence of three conformational substates of this domain. The three substates include conformations similar to existing crystallographic structures of the low-affinity I-domain, the inactive I-domain with an allosteric antagonist inhibitor bound underneath α helix 7, and an intermediate affinity state of the I-domain. The multiple substates were validated with residual dipolar coupling measurements. These results suggest that the presence of three substates in the apo I-domain enables the precise regulation of the binding process that is essential for the physiological function of LFA-1.This study was supported by the Wellcome Trust and the BBSRC.This is the final version of the article. It first appeared from Cell Press via http://dx.doi.org/10.1016/j.str.2014.12.02

Structural characterization of the interaction of α-synuclein nascent chains with the ribosomal surface and trigger factor

The ribosome is increasingly becoming recognized as a key hub for integrating quality control processes associated with protein biosynthesis and cotranslational folding (CTF). The molecular mechanisms by which these processes take place, however, remain largely unknown, in particular in the case of intrinsically disordered proteins (IDPs). To address this question, we studied at a residue-specific level the structure and dynamics of ribosome-nascent chain complexes (RNCs) of α-synuclein (αSyn), an IDP associated with Parkinson’s disease (PD). Using solution-state nuclear magnetic resonance (NMR) spectroscopy and coarse-grained molecular dynamics (MD) simulations, we find that, although the nascent chain (NC) has a highly disordered conformation, its N-terminal region shows resonance broadening consistent with interactions involving specific regions of the ribosome surface. We also investigated the effects of the ribosome-associated molecular chaperone trigger factor (TF) on αSyn structure and dynamics using resonance broadening to define a footprint of the TF–RNC interactions. We have used these data to construct structural models that suggest specific ways by which emerging NCs can interact with the biosynthesis and quality control machinery

The RNF168 paralog RNF169 defines a new class of ubiquitylated histone reader involved in the response to DNA damage.

Site-specific histone ubiquitylation plays a central role in orchestrating the response to DNA double-strand breaks (DSBs). DSBs elicit a cascade of events controlled by the ubiquitin ligase RNF168, which promotes the accumulation of repair factors such as 53BP1 and BRCA1 on the chromatin flanking the break site. RNF168 also promotes its own accumulation, and that of its paralog RNF169, but how they recognize ubiquitylated chromatin is unknown. Using methyl-TROSY solution NMR spectroscopy and molecular dynamics simulations, we present an atomic resolution model of human RNF169 binding to a ubiquitylated nucleosome, and validate it by electron cryomicroscopy. We establish that RNF169 binds to ubiquitylated H2A-Lys13/Lys15 in a manner that involves its canonical ubiquitin-binding helix and a pair of arginine-rich motifs that interact with the nucleosome acidic patch. This three-pronged interaction mechanism is distinct from that by which 53BP1 binds to ubiquitylated H2A-Lys15 highlighting the diversity in site-specific recognition of ubiquitylated nucleosomes

Implementing efficient concerted rotations using Mathematica and C code

In this article we demonstrate a general and efficient metaprogramming implementation of concerted rotations using Mathematica. Concerted rotations allow the movement of a fixed portion of a polymer backbone with fixed bending angles, like a protein, while maintaining the correct geometry of the backbone and the initial and final points of the portion fixed. Our implementation uses Mathematica to generate a C code which is then wrapped in a library by a Python script. The user can modify the Mathematica notebook to generate a set of concerted rotations suited for a particular backbone geometry, without having to write the C code himself. The resulting code is highly optimized, performing on the order of thousands of operations per second

Proteomic analysis of the cellular response to a potent sensitiser unveils the dynamics of haptenation in living cells

Haptenation of model nucleophiles, representing the key MIE in skin sensitisation, is routinely measured in chemico to provide data for skin allergy risk assessment. Better understanding of the dynamics of haptenation in human skin could provide the metrics required to improve determination of sensitiser potency for risk assessment of chemicals. We have previously demonstrated the applicability and sensitivity of the dual stable isotope labelling approach to detect low level haptenation in complex mixtures of proteins. In the present study, we investigated haptenation in a relevant living cell model over time at a subtoxic concentration. DNCB, an extremely potent sensitiser, caused minimal changes in overall protein differential expression in HaCaT cells and haptenated approximately 0.25 % of all available nucleophiles when applied at a subtoxic concentration (10μM) for 4 h. The data shows that the maximum level of haptenation occurs at 2 h and that DNCB, whilst being a promiscuous hapten, shows a preference for Cys residues, despite the considerably higher concentration of amine-based nucleophiles. Although a proportion of highly abundant proteins were haptenated, numerous haptenated sites were also detected on low abundant proteins. Certain proteins were modified at residues buried deep inside the protein structure which are less accessible to haptenation compared with surface exposed nucleophiles. The microenvironment of the buried residues may be a result of several factors influencing the reactivity of both the target nucleophile and the hapten.</p