Electrostatic Effects in the Folding of the SH3 Domain of the c-Src Tyrosine Kinase: pH-Dependence in 3D-Domain Swapping and Amyloid Formation

The SH3 domain of the c-Src tyrosine kinase (c-Src-SH3) aggregates to form intertwined dimers and amyloid fibrils at mild acid pHs. In this work, we show that a single mutation of residue Gln128 of this SH3 domain has a significant effect on: (i) its thermal stability; and (ii) its propensity to form amyloid fibrils. The Gln128Glu mutant forms amyloid fibrils at neutral pH but not at mild acid pH, while Gln128Lys and Gln128Arg mutants do not form these aggregates under any of the conditions assayed. We have also solved the crystallographic structures of the wild-type (WT) and Gln128Glu, Gln128Lys and Gln128Arg mutants from crystals obtained at different pHs. At pH 5.0, crystals belong to the hexagonal space group P6522 and the asymmetric unit is formed by one chain of the protomer of the c-Src-SH3 domain in an open conformation. At pH 7.0, crystals belong to the orthorhombic space group P212121, with two molecules at the asymmetric unit showing the characteristic fold of the SH3 domain. Analysis of these crystallographic structures shows that the residue at position 128 is connected to Glu106 at the diverging β-turn through a cluster of water molecules. Changes in this hydrogen-bond network lead to the displacement of the c-Src-SH3 distal loop, resulting also in conformational changes of Leu100 that might be related to the binding of proline rich motifs. Our findings show that electrostatic interactions and solvation of residues close to the folding nucleation site of the c-Src-SH3 domain might play an important role during the folding reaction and the amyloid fibril formation.This research was funded by the Spanish Ministry of Science and Innovation and Ministry of Economy and Competitiveness and FEDER (EU): BIO2009-13261-C02-01/02 (ACA); BIO2012-39922-C02-01/02 (ACA); CTQ2013-4493 (JLN) and CSD2008-00005 (JLN); Andalusian Regional Government (Spain) and FEDER (EU): P09-CVI-5063 (ACA); and Valentian Regional Government (Spain) and FEDER (EU): Prometeo 2013/018 (JLN). Data collection was supported by European Synchrotron Radiation Facility (ESRF), Grenoble, France: BAG proposals MX-1406 (ACA) and MX-1541 (ACA); and ALBA (Barcelona, Spain) proposals 2012010072 (ACA) and 2012100378 (ACA)

Capillary crystallization and molecular-replacement solution of haemoglobin II from the clam Lucina pectinata

The haemoglobin II from the clam L. pectinata has been crystallized using counter-diffusion in single capillary in the presence of agarose to improve crystal quality. Initial phases have been obtained by molecular replacement

The chromatin nuclear protein NUPR1L is intrinsically disordered and binds to the same proteins as its paralogue

NUPR1 is a protumoral multifunctional intrinsically disordered protein (IDP), which is activated during the acute phases of pancreatitis. It interacts with other IDPs such as prothymosin alpha, as well as with folded proteins such as the C-terminal region of RING1-B (C-RING1B) of the Polycomb complex; in all those interactions, residues around Ala33 and Thr68 (the `hot-spot' region) of NUPR1 intervene. Its paralogue, NUPR1L, is also expressed in response to DNA damage, it is p53-regulated, and its expression down-regulates that of the NUPR1 gene. In this work, we characterized the conformational preferences of isolated NUPR1L and its possible interactions with the same molecular partners of NUPR1. Our results show that NUPR1L was an oligomeric IDP from pH 2.0 to 12.0, as judged by steady-state fluorescence, circular dichroism (CD), dynamic light scattering, 1D H-1-NMR (nuclear magnetic resonance), and as indicated by structural modelling. However, in contrast with NUPR1, there was evidence of local helical- or turn-like structures; these structures were not rigid, as judged by the lack of sigmoidal behaviour in the chemical and thermal denaturation curves obtained by CD and fluorescence. Interestingly enough, NUPR1L interacted with prothymosin alpha and C-RING1B, and with a similar affinity to that of NUPR1 (in the low micromolar range). Moreover, NUPR1L hetero-associated with NUPR1 with an affinity of 0.4 mu M and interacted with the `hot-spot' region of NUPR1. Thus, we suggest that the regulation of NUPR1 gene by NUPR1L does not only happen at the DNA level, but It could also Involve direct interactions with NUPR1 natural partners

Structural and Thermodynamic Analysis of HIV-1 Fusion Inhibition Using Small gp41 Mimetic Proteins

Development of effective inhibitors of the fusion between HIV-1 and the host cell membrane mediated by gp41 continues to be a grand challenge due to an incomplete understanding of the molecular and mechanistic details of the fusion process. We previously developed single-chain, chimeric proteins (named covNHR) that accurately mimic the N-heptad repeat (NHR) region of gp41 in a highly stable coiled-coil conformation. These molecules bind strongly to peptides derived from the gp41 C-heptad repeat (CHR) and are potent and broad HIV-1 inhibitors. Here, we investigated two covNHR variants differing in two mutations, V10E and Q123R (equivalent to V38E and Q40R in gp41 sequence) that reproduce the effect of HIV-1 mutations associated with resistance to fusion inhibitors, such as T20 (enfuvirtide). A detailed calorimetric analysis of the binding between the covNHR proteins and CHR peptides (C34 and T20) reveals drastic changes in affinity due to the mutations as a result of local changes in interactions at the site of T20 resistance. The crystallographic structure of the covNHR:C34 complex shows a virtually identical CHR-NHR binding interface to that of the post-fusion structure of gp41 and underlines an important role of buried interfacial water molecules in binding affinity and in development of resistance against CHR peptides. Despite the great difference in affinity, both covNHR variants demonstrate strong inhibitory activity for a wide variety of HIV-1 strains. These properties support the high potential of these covNHR proteins as new potent HIV-1 inhibitors. Our results may guide future inhibition approaches

Overall fold of the monomeric structure of the c-Src-SH3 domain.

<p>Overall fold of the monomeric species of the WT c-Src-SH3 domain (WT_M, PDB code 4JZ4). The AU is composed by two chains of the SH3 domain; both chains are represented as a cartoon (white). The n-Src loop residues in chains A and B are shown in red. In chain B, the poor electronic density in the difference maps does not allow to model residues 114-115. Both chains show a nickel-binding site at the N-terminal formed by the residues His83-Ser82-Gly81, with slight differences in the conformation and in the axial ligand (nickel ion is represented with a green sphere). All the figures were performed using the program Pymol 1.7 (distributed by Schrödinger).</p

Nucleation site of the WT c-Src SH3 domain.

<p>Hydrogen-bond interactions among the residues belonging to the diverging β-turn and those of the distal loop are shown in green dotted lines. WT_M (PDB code 4JZ4) chains A (panel A) and B (panel B) are shown in blue and cyan, respectively. (C) Intertwined dimer structure of the WT c-Src SH3 domain (PDB code 4JZ3), residues at chain A are shown in white sticks and those belonging to the symmetry related molecule (chain B) are in magenta sticks.</p

X-ray data collection and refinement statistics.

<p>Statistics for the highest-resolution shell are shown in parentheses.</p><p>X-ray data collection and refinement statistics.</p

Water network at the nucleation site of the intertwined structures of the c-Src-SH3 mutants.

<p>Representation of the superposition of the 2Fo-Fc electron density map of the distal loop and the diverging β-turn of the WT (blue) and (A) Gln128Glu (red); (B) Gln128Arg (magenta); and (C) Gln128Lys (yellow) mutants. W1, W2 and W3 present in each structure are shown in the same color as the corresponding coordinates.</p

Hydrogen-bond distances in the water network at the distal loop and diverging β-turn.

<p>Hydrogen-bond distances in the water network at the distal loop and diverging β-turn.</p