Development of enzymatic activity during protein folding. Detection of a spectroscopically silent native-like intermediate of muscle acylphosphatase.

The recovery of enzymatic activity during the folding of muscle acylphosphatase and two single residue mutants (proline 54 to alanine and proline 71 to alanine) from 7 M urea has been monitored and compared with the development of intrinsic fluorescence emission. Fluorescence measurements reveal the presence in the wild-type protein of a major rapid refolding phase followed by a second low amplitude slow phase. The slow phase is absent in the fluorescence trace acquired with the proline 54 to alanine mutant, suggesting the involvement of this proline residue in the fluorescence-detected slow phase of the wild-type protein. The major kinetic phase is associated with a considerable recovery of enzymatic activity, indicating that a large fraction of molecules refolds with effective two-state behavior. The use of time-resolved enzymatic activity as a probe to follow the folding process reveals, however, the presence of another exponential slow phase arising from proline 71. This slow phase is not observable by utilizing optical probes, indicating that, unlike proline 54, the cis to trans isomerization of proline 71 can take place in an intermediate possessing a native-like fold. We suggest that, although spectroscopically silent and structurally insignificant, the cis-trans interconversion of proline residues in native-like intermediates may be crucial for the generation of enzymatic activity of functional enzymes

Distinct ubiquitin binding modes exhibited by SH3 domains: Molecular determinants and functional implications

SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination. © 2013 Ortega Roldan et al

Accurate characterization of weak macromolecular interactions by titration of NMR residual dipolar couplings: application to the CD2AP SH3-C:ubiquitin complex

The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100 μM, are remarkably common, but remain beyond the reach of most of structural biology. NMR spectroscopy, and in particular, residual dipolar couplings (RDCs) provide crucial conformational constraints on intermolecular orientation in molecular complexes, but the combination of free and bound contributions to the measured RDC seriously complicates their exploitation for weakly interacting partners. We develop a robust approach for the determination of weak complexes based on: (i) differential isotopic labeling of the partner proteins facilitating RDC measurement in both partners; (ii) measurement of RDC changes upon titration into different equilibrium mixtures of partially aligned free and complex forms of the proteins; (iii) novel analytical approaches to determine the effective alignment in all equilibrium mixtures; and (iv) extraction of precise RDCs for bound forms of both partner proteins. The approach is demonstrated for the determination of the three-dimensional structure of the weakly interacting CD2AP SH3-C:Ubiquitin complex (Kd = 132 ± 13 μM) and is shown, using cross-validation, to be highly precise. We expect this methodology to extend the remarkable and unique ability of NMR to study weak protein–protein complexes

Structural and Biophysical Characterization of Staphylococcus Aureus SaMazF Shows Conservation of Functional Dynamics

The Staphylococcus aureus genome contains three toxin-antitoxin modules, including one mazEF module, SamazEF. Using an on-column separation protocol we are able to obtain large amounts of wild-type SaMazF toxin. The protein is well-folded and highly resistant against thermal unfolding but aggregates at elevated temperatures. Crystallographic and nuclear magnetic resonance (NMR) solution studies show a well-defined dimer. Differences in structure and dynamics between the X-ray and NMR structural ensembles are found in three loop regions, two of which undergo motions that are of functional relevance. The same segments also show functionally relevant dynamics in the distantly related CcdB family despite divergence of function. NMR chemical shift mapping and analysis of residue conservation in the MazF family suggests a conserved mode for the inhibition of MazF by MazE

Mapping the encounter state of a transient protein complex by PRE NMR spectroscopy

Many biomolecular interactions proceed via a short-lived encounter state, consisting of multiple, lowly-populated species invisible to most experimental techniques. Recent development of paramagnetic relaxation enhancement (PRE) nuclear magnetic resonance (NMR) spectroscopy has allowed to directly visualize such transient intermediates in a number of protein-protein and protein-DNA complexes. Here we present an analysis of the recently published PRE NMR data for a protein complex of yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP). First, we describe a simple, general method to map out the spatial and temporal distributions of binding geometries constituting the Cc-CcP encounter state. We show that the spatiotemporal mapping provides a reliable estimate of the experimental coverage and, at higher coverage levels, allows to delineate the conformational space sampled by the minor species. To further refine the encounter state, we performed PRE-based ensemble simulations. The generated solutions reproduce well the experimental data and lie within the allowed regions of the encounter maps, confirming the validity of the mapping approach. The refined encounter ensembles are distributed predominantly in a region encompassing the dominant form of the complex, providing experimental proof for the results of classical theoretical simulations

Electron Transfer Interactome of Cytochrome <em>c</em>

<div><p>Lying at the heart of many vital cellular processes such as photosynthesis and respiration, biological electron transfer (ET) is mediated by transient interactions among proteins that recognize multiple binding partners. Accurate description of the ET complexes – necessary for a comprehensive understanding of the cellular signaling and metabolism – is compounded by their short lifetimes and pronounced binding promiscuity. Here, we used a computational approach relying solely on the steric properties of the individual proteins to predict the ET properties of protein complexes constituting the functional interactome of the eukaryotic cytochrome <em>c</em> (Cc). Cc is a small, soluble, highly-conserved electron carrier protein that coordinates the electron flow among different redox partners. In eukaryotes, Cc is a key component of the mitochondrial respiratory chain, where it shuttles electrons between its reductase and oxidase, and an essential electron donor or acceptor in a number of other redox systems. Starting from the structures of individual proteins, we performed extensive conformational sampling of the ET-competent binding geometries, which allowed mapping out functional epitopes in the Cc complexes, estimating the upper limit of the ET rate in a given system, assessing ET properties of different binding stoichiometries, and gauging the effect of domain mobility on the intermolecular ET. The resulting picture of the Cc interactome 1) reveals that most ET-competent binding geometries are located in electrostatically favorable regions, 2) indicates that the ET can take place from more than one protein-protein orientation, and 3) suggests that protein dynamics within redox complexes, and not the electron tunneling event itself, is the rate-limiting step in the intermolecular ET. Further, we show that the functional epitope size correlates with the extent of dynamics in the Cc complexes and thus can be used as a diagnostic tool for protein mobility.</p> </div

(1)H, (13)C and (15)N backbone and side-chain chemical shift assignment of the Fyn SH2 domain and its complex with a phosphotyrosine peptide.

SH2 domains are interaction modules uniquely dedicated to recognize phosphotyrosine sites, playing a central role in for instance the activation of tyrosine kinases or phosphatases. Here we report the (1)H, (15)N and (13)C backbone and side-chain chemical shift assignments of the SH2 domain of the human protein tyrosine kinase Fyn, both in its free state and bound to a high-affinity phosphotyrosine peptide corresponding to a specific sequence in the hamster middle-T antigen. The BMRB accession numbers are 17,368 and 17,369, respectively.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe

Conformational sampling in the Cc-Cb₅ complex.

<p>(A) Distribution of the heme-heme distances in the simulation runs with the full vdW potential and side-chain optimization (black); full vdW potential without side-chain optimization (vdW cut-off values of 300 kcal/mol and 10 kcal/mol; red and orange, respectively); and reduced vdW term, set to zero for all atoms extending beyond C_β (vdW cut-off values 300 kcal/mol and 10 kcal/mol; blue and green, respectively). In these runs, only translational degrees of freedom of Cc were simulated. (B) Correlation plots of heme-heme distance occurrences in the side-chain optimization run (horizontal axis) and the other control runs (vertical axis), color-coded as in (A). Pearson correlation coefficients are indicated in the plots. (C) Distribution of the heme-heme distances in the runs with the reduced vdW term (vdW cut-off 10 kcal/mol, green dataset in A) and a given rotational increment (δχ = δψ = δξ, 0°≤χ, ψ<360°, 0°≤ξ<180°). For the composite run, δχ = δψ = 5°, δξ = 15°; −45°≤χ, ψ≤45°, 0°≤ξ<360°. (D) Number of rotamers for each Cc CM, the total number of structures sampled per run, and the total run time (for 1 CPU).</p

Cc binding interface and ET properties in redox complexes.

<p>(A) Combined binding interface of ferric yeast Cc (PDB entry 2YCC) <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002807#pcbi.1002807-Berghuis1" target="_blank">[73]</a> in complexes with CcP <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002807#pcbi.1002807-Worrall1" target="_blank">[74]</a>, Cb₅<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002807#pcbi.1002807-Volkov4" target="_blank">[56]</a> and CCO <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002807#pcbi.1002807-Sakamoto1" target="_blank">[67]</a> as determined by solution NMR spectroscopy. Residues experiencing binding effects in any of the three, two of the three, and all three complexes are colored light blue, green, and dark blue, respectively. The heme group is indicated by the white rectangle. Several lysine residues affected by the binding are labeled. (B) Absolute counts of Cc CMs with k_ET>10⁶ s⁻¹ in the ET complexes studied in this work.</p