The intrinsically disordered Tarp protein from chlamydia binds actin with a partially preformed helix

Tarp (translocated actin recruiting phosphoprotein) is an effector protein common to all chlamydial species that functions to remodel the host-actin cytoskeleton during the initial stage of infection. In C. trachomatis, direct binding to actin monomers has been broadly mapped to a 100-residue region (726-825) which is predicted to be predominantly disordered, with the exception of a ~10-residue α helical patch homologous to other WH2 actin-binding motifs. Biophysical investigations demonstrate that a Tarp726-825 construct behaves as a typical intrinsically disordered protein; within it, NMR relaxation measurements and chemical shift analysis identify the ten residue WH2-homologous region to exhibit partial α-helix formation. Isothermal titration calorimetry experiments on the same construct in the presence of monomeric G-actin show a well defined binding event with a 1:1 stoichiometry and Kd of 102 nM, whilst synchrotron radiation circular dichroism spectroscopy suggests the binding is concomitant with an increase in helical secondary structure. Furthermore, NMR experiments in the presence of G-actin indicate this interaction affects the proposed WH2-like α-helical region, supporting results from in silico docking calculations which suggest that, when folded, this α helix binds within the actin hydrophobic cleft as seen for other actin-associated proteins

Smad7 Binds Differently to Individual and Tandem WW3 and WW4 Domains of WWP2 Ubiquitin Ligase Isoforms

WWP2 is an E3 ubiquitin ligase that differentially regulates the contextual tumour suppressor/progressor TGFβ signalling pathway by alternate isoform expression. WWP2 isoforms select signal transducer Smad2/3 or inhibitor Smad7 substrates for degradation through different compositions of protein–protein interaction WW domains. The WW4 domain containing WWP2-C induces Smad7 turnover in vivo and positively regulates the metastatic epithelial–mesenchymal transition programme. This activity and the overexpression of these isoforms in human cancers make them candidates for therapeutic intervention. Here, we use NMR spectroscopy to solve the solution structure of the WWP2 WW4 domain and observe the binding characteristics of Smad7 substrate peptide. We also reveal that WW4 has an enhanced affinity for a Smad7 peptide phosphorylated at serine 206 adjacent to the PPxY motif. Using the same approach, we show that the WW3 domain also binds Smad7 and has significantly enhanced Smad7 binding affinity when expressed in tandem with the WW4 domain. Furthermore, and relevant to these biophysical findings, we present evidence for a novel WWP2 isoform (WWP2C-ΔHECT) comprising WW3–WW4 tandem domains and a truncated HECT domain that can inhibit TGFβ signalling pathway activity, providing a further layer of complexity and feedback to the WWP2 regulatory apparatus. Collectively, our data reveal a structural platform for Smad substrate selection by WWP2 isoform WW domains that may be significant in the context of WWP2 isoform switching linked to tumorigenesis

Mapping the Interacting Regions between Troponins T and C. Binding of TnT and TnI peptides to TnC and NMR mapping of the TnT-binding site on TnC

Muscular contraction is triggered by an increase in calcium concentration, which is transmitted to the contractile proteins by the troponin complex. The interactions among the components of the troponin complex (troponins T, C, and I) are essential to understanding the regulation of muscle contraction. While the structure of TnC is well known, and a model for the binary TnC·TnI complex has been recently published (Tung, C.-S., Wall, M. E., Gallagher, S. C., and Trewhella, J. (2000)Protein Sci. 9, 1312–1326), very little is known about TnT. Using non-denaturing gels and NMR spectroscopy, we have analyzed the interactions between TnC and five peptides from TnT as well as how three TnI peptides affect these interactions. Rabbit fast skeletal muscle peptide TnT-(160–193) binds to TnC with a dissociation constant of 30 ± 6 µm. This binding still occurs in the presence of TnI-(1–40) but is prevented by the presence of TnI-(56–115) or TnI-(96–139), both containing the primary inhibitory region of TnI. TnT-(228–260) also binds TnC. The binding site for TnT-(160–193) is located on the C-terminal domain of TnC and was mapped to the surface of TnC using NMR chemical shift mapping techniques. In the context of the model for the TnC·TnI complex, we discuss the interactions between TnT and the other troponin subunits

Near-complete backbone resonance assignments of acid-denatured human cytochrome c in dimethylsulfoxide: a prelude to studying interactions with phospholipids

Human cytochrome c plays a central role in the mitochondrial electron transfer chain and in the intrinsic apoptosis pathway. Through the interaction with the phospholipid cardiolipin, cytochrome c triggers release of pro-apoptotic factors, including itself, from the mitochondrion into the cytosol of cells undergoing apoptosis. The cytochrome c/cardiolipin complex has been extensively studied through various spectroscopies, most recently with high-field solution and solid-state NMR spectroscopies, but there is no agreement between the various studies on key structural features of cytochrome c in its complex with cardiolipin. In the present study, we report backbone 1H, 13C, 15N resonance assignments of acid-denatured human cytochrome c in the aprotic solvent dimethylsulfoxide. These have led to the assignment of a reference 2D 1H-15N HSQC spectrum in which out of the 99 non-proline residues 87% of the backbone amides are assigned. These assignments are being used in an interrupted H/D exchange strategy to map the binding site of cardiolipin on human cytochrome c

Atomic resolution insight into host cell recognition by Toxoplasma gondii.

Accepted versio

Study of divalent cations binding to EF-hand sites using smooth muscle myosin regulatory light chain

O objetivo deste trabalho é estudar a afinidade e especificidade de sítios EF-hand, e correlacionar estas propriedades com a estrutura primária do sítio, com as interações entre aminoácidos nas posições de coordenação, e com prováveis características da estrutura terciária da proteína. Os efeitos de três mutações no sítio EF-hand da cadeia leve regulatória de miosina (RLC) foram estudados: D5S, em que o aspartato presente na posição 5 do sítio foi substituído por uma serina; D9E, substituindo o aspartato da posição 9 por um glutamato, e D12E, substituindo o aspartato da posição 12 por um glutamato. Todas as combinações destas três mutações foram produzidas. Os mutantes simples D5S e D9E e o duplo mutante D5S/D9E têm baixa afinidade por cálcio. Todos os mutantes contendo a mutação D12E são específicos para cálcio, com afinidades maiores que RLC tipo selvagem. Todos os mutantes estudados possuem menor afinidade por magnésio que RLC tipo selvagem. As mudanças na energia livre de ligação e as energias de acoplamento sugerem que há interações inespecíficas entre todas as posições, e uma interação específica entre uma serina na posição 5 e um glutamato na posição 9. Esta interação ocorre somente na presença de magnésio, e quando há um aspartato na posição 12. O glutamato na posição 9 pode ser capaz de coordenar a ligação de magnésio diretamente no duplo mutante D5S/D9E. Embora um aminoácido ou um certo arranjo deles possa determinar características específicas do sítio EF-hand, o conjunto de propriedades depende da estrutura terciária, uma vez que sítios homólogos podem possuir afinidades e especificidades bastante diferentes.The aim of this thesis was to study affinity and specificity in EF-hand sites, and how these properties are related to the site primary structure, interactions between amino acids in coordinating positions, and probable tertiary structure properties. The effects of three mutations on the EF-hand Ca2+/Mg2+ binding site of smooth muscle myosin regulatory light chain (RLC) were studied: D5S, in which an aspartate is replaced by a serine in position 5 of the loop; D9E, in which an aspartate is replaced by a glutamate in position 9, and D12E, in which the aspartate in position 12 is replaced by a glutamate. All possible combinations of the three mutations were produced. The single mutants D5S and D9E and the double mutant D5S/D9E have low affinity for Ca2+. All the mutants containing mutation D12E are Ca2+-specific and have higher affinities than wild type, even when containing mutations D5S or D9E. All the mutants studied have lower affinity for Mg2+ than wild type RLC. Coupling energies and changes in binding free energy suggest that all positions interact in a non-specific way, and a specific interaction occurs between a serine in position 5 and a glutamate in position 9. This interaction can be seen only in the presence of magnesium, and with an apartate in position 12. Glutamate in position 9 may be able to coordinate Mg2+ directly in the double mutant D5S/D9E. Even though an amino acid or a few amino acids in certain positions can determine specific characteristics for an EF-hand site, the site properties depend on the tertiary structure, since homologue sites can have very different affinities and specificities