Charged and Hydrophobic Surfaces on the A Chain of Shiga-Like Toxin 1 Recognize the C-Terminal Domain of Ribosomal Stalk Proteins

Shiga-like toxins are ribosome-inactivating proteins (RIP) produced by pathogenic E. coli strains that are responsible for hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A1 chain of Shiga-like toxin 1 (SLT-1), a representative RIP, first docks onto a conserved peptide SD[D/E]DMGFGLFD located at the C-terminus of all three eukaryotic ribosomal stalk proteins and halts protein synthesis through the depurination of an adenine base in the sarcin-ricin loop of 28S rRNA. Here, we report that the A1 chain of SLT-1 rapidly binds to and dissociates from the C-terminal peptide with a monomeric dissociation constant of 13 µM. An alanine scan performed on the conserved peptide revealed that the SLT-1 A1 chain interacts with the anionic tripeptide DDD and the hydrophobic tetrapeptide motif FGLF within its sequence. Based on these 2 peptide motifs, SLT-1 A1 variants were generated that displayed decreased affinities for the stalk protein C-terminus and also correlated with reduced ribosome-inactivating activities in relation to the wild-type A1 chain. The toxin-peptide interaction and subsequent toxicity were shown to be mediated by cationic and hydrophobic docking surfaces on the SLT-1 catalytic domain. These docking surfaces are located on the opposite face of the catalytic cleft and suggest that the docking of the A1 chain to SDDDMGFGLFD may reorient its catalytic domain to face its RNA substrate. More importantly, both the delineated A1 chain ribosomal docking surfaces and the ribosomal peptide itself represent a target and a scaffold, respectively, for the design of generic inhibitors to block the action of RIPs

RNAstructure: software for RNA secondary structure prediction and analysis

<p>Abstract</p> <p>Background</p> <p>To understand an RNA sequence's mechanism of action, the structure must be known. Furthermore, target RNA structure is an important consideration in the design of small interfering RNAs and antisense DNA oligonucleotides. RNA secondary structure prediction, using thermodynamics, can be used to develop hypotheses about the structure of an RNA sequence.</p> <p>Results</p> <p>RNAstructure is a software package for RNA secondary structure prediction and analysis. It uses thermodynamics and utilizes the most recent set of nearest neighbor parameters from the Turner group. It includes methods for secondary structure prediction (using several algorithms), prediction of base pair probabilities, bimolecular structure prediction, and prediction of a structure common to two sequences. This contribution describes new extensions to the package, including a library of C++ classes for incorporation into other programs, a user-friendly graphical user interface written in JAVA, and new Unix-style text interfaces. The original graphical user interface for Microsoft Windows is still maintained.</p> <p>Conclusion</p> <p>The extensions to RNAstructure serve to make RNA secondary structure prediction user-friendly. The package is available for download from the Mathews lab homepage at <url>http://rna.urmc.rochester.edu/RNAstructure.html</url>.</p

A stable human p53 heterotetramer based on constructive charge interactions within the tetramerization domain

This article is hosted on a website external to the CBCRA Open Access Archive. Selecting “View/Open” below will launch the full-text article in another browser window.The human p53 tetramerization domain (called p53tet; residues 325-355) spontaneously forms a dimer of dimers in solution. Hydrophobic interactions play a major role in stabilizing the p53 tetramer. However, the distinctive arrangement of charged residues at the dimer-dimer interface suggests that they also contribute to tetramer stability. Charge-reversal mutations at positions 343, 346, and 351 within the dimer-dimer interface were thus introduced into p53tet constructs and shown to result in the selective formation of a stable heterotetramer composed of homodimers. More precisely, mutants p53tet-E343K/E346K and p53tet-K351E preferentially associated with each other, but not with wild-type p53tet, to form a heterodimeric tetramer with enhanced thermal stability relative to either of the two components in isolation. The p53tet-E343K/E346K mutant alone assembled into a weakly stable tetramer in solution, whereas p53tet-K351E existed only as a dimer. Moreover, these mutants did not form heterocomplexes with wild-type p53tet, illustrating the specificity of the ionic interactions that form the novel heterotetramer. This study demonstrates the dramatic importance of ionic interactions in altering the stability of the p53 tetramer and in selectively creating heterotetramers of this protein scaffold

The A₁ chain of SLT-1 harbors a cationic surface composed of a cluster of arginine residues that interact with the ribosomal stalk protein P2 and the conserved C-terminal peptide.

<p>(A) A vector expressing a catalytically inactive variant of the SLT-1 A₁ domain (CIA₁) or one of the arginine-to-alanine point mutants as fusion partners with the GAL4 DNA-BD domain were co-transformed in the yeast strain AH109 with a vector expressing ribosomal protein P2 as a fusion construct to the GAL4-AD. The transformed yeast cells were plated on SD agar −Trp/−Leu. The resulting yeast colonies were grown overnight, and spotted (10 µl) as 10-fold serial dilutions onto SD medium lacking Trp and Leu to select for the presence of each plasmid followed by spotting on SD media lacking Trp, Leu, and His to select for interacting partners leading to colony growth. (B) SPR profiles illustrating the decrease in relative units for the arginine-to-alanine SLT-1 A₁ chain variants in relation to the wild-type A₁ chain, at a concentration of 15 µM, when presented to the immobilized peptide SDDDMGFGLFD. (C) Increasing salt concentrations led to a decrease or loss of binding of wild-type SLT-1 A₁ chain when exposed to the peptide SDDDMGFGLFD. SPR traces were plotted for the wild-type SLT-1 A₁ chain (15 µM) as a function of increasing salt concentrations.</p

Wielkość pochłaniania tlenu w czasie treningu Nordic walking u chorych rehabilitowanych po incydentach wieńcowych

Background: Nordic walking (NW) is an effective form of endurance training in cardiac rehabilitation (CR). The key parameter for the safety and effectiveness of the training is its intensity. Training intensity may be directly measured by the volume of oxygen consumption (VO2), and indirectly by chronotropic cardiac response to exercise. No data have been published on the rates of VO2 during NW in field conditions among patients rehabilitated after coronary events. Aim: To assess the intensity of NW training in field conditions by measuring VO2, energy expenditure (EE), and heart rate (HR) in comparison with a treadmill cardiopulmonary exercise test (CPET) in a group of patients rehabilitated after coronary events. Methods: Thirteen men after percutaneous coronary intervention due to an acute coronary syndrome (STEMI, NSTEMI, or UA), aged 53.2 ± 8.2 years, were evaluated and recruited for comprehensive CR at 30.3 ± 15.7 days after the incident. Left ventricular ejection fraction was evaluated and treadmill exercise test (ExT) using an individualised ramp protocol was performed during initial functional assessment. Following risk stratification, patients began training at 50% of HR reserve (HRR). Participants at low and moderate risk qualified for field NW training in the second week of CR. Treadmill CPET using a ramp protocol was performed after the patients had mastered the technique of walking with poles. Next day, HR, parameters of ventilation, and respiratory gas concentrations were measured during NW using a portable spiroergometry system. Results: Exercise tolerance estimated during initial ExT was 9.1 ± 2.5 MET. Peak VO2 was 27.5 ± 5.4 mL/min/kg during CPET vs. 26.2 ± 7.7 mL/min/kg during NW (p &lt; 0.447). Mean VO2 during NW was 17.5 ± 4.5 mL/min/kg, which amounted to 59.4 ± 18.6% of VO2 reserve in CPET. Mean HR during NW was 104.8 ± 9.8 bpm, amounting to 63.7 ± 28.7% of HRR, and peak HR was 128.4 ± 13.7 bpm vs. 131.1 ± 18.0 bpm during CPET (p &lt; 0.628). EE during 24.7 ± 9.7 min of NW was 210.7 ± 149.0 kcal (8.1 ± 2.7 kcal/min). Conclusions: The intensity of NW training in field conditions in patients after coronary events was 59% of VO2 reserve, and its peak instantaneous intensity reached values obtained during CPET on a treadmill. EE during NW in the study group was 8.1 kcal/min. Chronotropic response during NW was 64% of HRR, and its instantaneous increase reached the maximum HR obtained during CPET.Wstęp: Nordic walking (NW) jest efektywną formą treningu wytrzymałościowego w rehabilitacji kardiologicznej (CR). Kluczowym parametrem dla bezpieczeństwa i efektywności treningu jest jego intensywność. Bezpośrednią jej miarę stanowi wielkość pochłaniania tlenu (VO2), a pośrednią — odpowiedź chronotropowa serca na wysiłek. Dane na temat wielkości VO2 w czasie treningu NW w terenie u chorych rehabilitowanych po incydentach wieńcowych nie były dotychczas publikowane. Cel: Celem pracy była ocena intensywności treningu NW w terenie za pomocą pomiaru VO2 i wydatku energetycznego oraz częstotliwości rytmu serca (HR) w porównaniu z wynikiem testu spiroergometrycznego (CPET) na bieżni mechanicznej u rehabilitowanych chorych po incydentach wieńcowych. Metody: Przebadano 13 mężczyzn w wieku 53,2 ± 8,2 roku po przezskórnej interwencji wieńcowej w przebiegu zawału serca (STEMI: 8 osób, NSTEMI: 3 osoby) i niestabilnej dławicy (2 pacjentów), włączonych do kompleksowej CR po 30,3 ± 15,7 dnia od incydentu. We wstępnej ocenie czynnościowej oznaczono echograficznie frakcję wyrzutową lewej komory i wykonano submaksymalny elektrokardiograficzny test wysiłkowy (ExT) na bieżni mechanicznej wg zindywidualizowanego protokołu ramp. Po stratyfikacji ryzyka zdarzeń sercowych chorzy rozpoczęli trening z intensywnością 50% rezerwy HR (50% HRR). W drugim tygodniu usprawniania pacjentów z niskim i umiarkowanym ryzykiem kwalifikowano do treningu NW w terenie. Po opanowaniu przez nich techniki marszu z kijkami wykonywano CPET na bieżni ruchomej wg protokołu ramp. Kolejnego dnia przeprowadzano rejestrację HR oraz parametrów wentylacyjnych i stężeń gazów oddechowych w trakcie treningu NW za pomocą przenośnego systemu do spiroergometrii. Wyniki: Estymowana wydolność chorych we wstępnym ExT wynosiła 9,1 ± 2,5 MET. Szczytowe VO2 (p VO2) w CPET na bieżni wynosiło 27,5 ± 5,4 vs. 26,2 ± 7,7 ml/min/kg zarejestrowane podczas NW (p &lt; 0,447). Średnie VO2 (m VO2) w czasie NW wynosiło 17,5 ± 4,5 ml/min/kg, co odpowiadało intensywności wysiłku równej 59,4 ± 18,6% rezerwy VO2 (VO2R) uzyskanej w CPET. Średnia HR (mHR) w czasie NW 104,8 ± 9,8 bpm stanowiła 63,7 ± 28,7% HRR, przy wartości pHR równej 128,4 ± 13,7 vs. 131,1 ± 18,0 bpm w CPET (p &lt; 0,628). Wydatek energii w trakcie 24,7 ± 9,7-minutowego NW wynosił 210,7 ± 149,0 kcal, co w przeliczeniu na 1 min marszu stanowiło 8,1 ± 2,7 kcal/min. Wnioski: Intensywność treningu NW prowadzonego w terenie u chorych po incydentach wieńcowych wynosiła 59% rezerwy VO2, a jego wzrost chwilowy osiągał wartość szczytową uzyskaną w CPET na bieżni mechanicznej. Wydatkowana energia w czasie NW w badanej grupie chorych wynosiła 8,1 kcal w ciągu 1 min marszu z kijkami. Odpowiedź chronotropowa w NW utrzymywała się na poziomie 64% rezerwy tętna, a jej chwilowy wzrost osiągał maksymalną częstość uzyskaną w czasie testu spiroergometrycznego.

Primary and tertiary structural comparisons between SLT-1 and SLT-2 highlighting the conservation of important ribosomal stalk peptide contact sites.

<p>(A) <i>Left Panel</i> - Surface rendering of the SLT-1 A₁ chain (PDB# 1DM0) depicting the cationic (blue) and hydrophobic (yellow) residues essential for optimal binding to the conserved stalk peptide SDDDMGFGLFD as well as Arg-188 (light blue) which has a modest effect on peptide binding. <i>Right Panel</i> – Structure as shown in the left panel rotated by 140°, highlighting the catalytic residues in green. (B) Three-dimensional stick structures of SLT-1 (left panel), SLT-2 (PDB# 1R4P; middle panel), and the structural alignment of the two toxins (right panel). Cationic residues are labeled in blue and red, while hydrophobic residues are labeled in yellow and orange for SLT-1 and SLT-2 respectively. (C) Primary amino acid sequence alignment of SLT-1 and SLT-2 within residues 158 and 250. Catalytic residues are highlighted in green and cationic and hydrophobic residues in blue and yellow, respectively. Surface and stick renderings and alignments were performed using the The PyMOL Molecular Graphics System (Version 1.3, Schrödinger, LLC), whereas amino acid sequences were aligned using BioEdit software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031191#pone.0031191-Tchorzewski2" target="_blank">[59]</a>.</p