The impact of oncogenic mutations of the viral Src kinase on the structure and stability of the SH3 domain.

Src kinase belongs to the family of Src-related nonreceptor tyrosine kinases. Because of its physiological role in cell growth and proliferation, its activity is strictly controlled by several mechanisms. Nevertheless, in viral Src kinase (v-Src) some of these mechanisms fail, and its uncontrolled activity is responsible for the occurrence of cancer. Here, the crystal structures of three SH3-domain mutants of v-Src were determined to unveil the effects of these oncogenic mutations in this regulatory domain. Mutations in the n-Src and distal loops have a low impact on the overall structure of the domain and its capacity to form intertwined dimers. However, mutations in the RT loop compromise the stability of the domain and make the protein very prone to aggregation. Additionally, these mutations prevent the formation of intertwined dimers. The results show a synergistic effect between mutations in the RT loop and those in the n-Src and distal loops. Analysis of the structures of the v-Src SH3-domain mutants and the closed inactive conformation of cellular Src kinase (c-Src) point to a loss of the interactions that are required to establish the compact inactive form of the kinase. Nevertheless, an analysis of structures of the c-Src SH3 domain complexed with class I and II peptides points to minor changes in the interactions between the v-Src SH3 domain and these peptides. In this way, the structures reported here indicate that mutations in the RT loop might impair the kinase regulation mechanism without affecting the recognition of short proline-rich motifs in the target proteins of the kinase, thus explaining the oncogenic behaviour of the protein.

Lysozyme crystals dyed with bromophenol blue: where has the dye gone?

Protein crystals can easily be coloured by adding dyes to their mother liquor, but most structures of these protein-dye complexes remain unsolved. Here, structures of lysozyme in complex with bromophenol blue obtained by soaking orthorhombic and tetragonal crystals in a saturated solution of the dye at different pH values from 5.0 to 7.5 are reported. Two different binding sites can be found in the lysozyme-bromophenol blue crystals: binding site I is located near the amino- and carboxyl-termini, while binding site II is located adjacent to helices α1 (residues 4-15) and α3 (residues 88-100). In the orthorhombic crystals soaked at pH 7.0, binding of the dye takes place in both sites without significant changes in the unit cell. However, soaking tetragonal crystals with bromophenol blue results in two different complexes. Crystals soaked at pH 5.5 (HEWL-T1) show a single dye molecule bound to site II, and the crystals belong to space group P43212 without significant changes in the unit cell (a = b = 78.50, c = 37.34 Å). On the other hand, crystals soaked at pH 6.5 in the presence of imidazole (HEWL-T2) show up to eight molecules of the dye bound to site II, and display changes in space group (P212121) and unit cell (a = 38.00, b = 76.65, c = 84.86 Å). In all of the structures, the dye molecules are placed at the surface of the protein near to positively charged residues accessible through the main solvent channels of the crystal. Differences in the arrangement of the dye molecules at the surface of the protein suggest that the binding is not specific and is mainly driven by electrostatic interactions.

The effect of an engineered ATCUN motif on the structure and biophysical properties of the SH3 domain of c-Src tyrosine kinase.

Metal binding to sites engineered in proteins can provide an increase in their stability and facilitate new functions. Besides the sites introduced in purpose, sometimes they are present accidentally as a consequence of the expression system used to produce the protein. This happens with the copper- and nickel-binding (ATCUN) motif generated by the amino-terminal residues Gly-Ser-His. This ATCUN motif is fortuitously present in many proteins, but how it affects the structural and biophysical characterization of the proteins has not been studied. In this work, we have compared the structure and biophysical properties of a small modular domain, the SH3 domain of the c-Src tyrosine kinase, cloned with and without an ATCUN motif at the N terminus. At pH 7.0, the SH3 domain with the ATCUN motif binds nickel with a binding constant Ka = 28.0 ± 3.0 mM-1. The formation of the nickel complex increases the thermal and chemical stability of the SH3 domain. A comparison of the crystal structures of the SH3 domain with and without the ATCUN motif shows that the binding of nickel does not affect the overall structure of the SH3 domain. In all crystal structures analyzed, residues Gly-Ser-His in complex with Ni2+ show a square planar geometry. The CD visible spectrum of the nickel complex shows that this geometry is also present in the solution. Therefore, our results not only show that the ATCUN motif might influence the biophysical properties of the protein, but also points to an advantageous stabilization of the protein with potential biotechnological applications.

Major conformational changes in the structure of lysozyme obtained from a crystal with a very low solvent content.

A new crystal form of lysozyme with a very low solvent content (26.35%) has been obtained in the orthorhombic space group P212121 (with unit-cell parameters a = 30.04, b = 51.68, c = 61.53 Å). The lysozyme structure obtained from these crystals does not show the typical overall fold. Instead, major conformational changes take place in some elements of the secondary structure and in the hydrophobic core of the protein. At the end of the central α-helix (α2), Glu35 is usually buried in the catalytic site and shows an abnormally high pKa value, which is key to the activity of the enzyme. The high pKa value of this glutamate residue is favoured by the hydrophobic environment, particularly by its neighbour Trp108, which is important for structural stability and saccharide binding. In this new structure, Trp108 shows a 90° rotation of its side chain, which results in the rearrangement of the hydrophobic core. Conformational changes also result in the exposure of Glu35 to the solvent, which impairs the catalytic site by increasing the distance between Glu35 and Asp52 and lowering the pKa value of the glutamate. Altogether, this new lysozyme structure reveals major conformational changes in the hydrophobic core and catalytic site that might play a role in the folding and bactericidal function of the protein.

Orthorhombic lysozyme crystallization at acidic pH values driven by phosphate binding.

The structure of orthorhombic lysozyme has been obtained at 298 K and pH 4.5 using sodium chloride as the precipitant and in the presence of sodium phosphate at a concentration as low as 5 mM. Crystals belonging to space group P212121 (unit-cell parameters a = 30, b = 56, c = 73 Å, α = ß = γ = 90.00°) diffracted to a resolution higher than 1 Å, and the high quality of these crystals permitted the identification of a phosphate ion bound to Arg14 and His15. The binding of this ion produces long-range conformational changes affecting the loop containing Ser60-Asn74. The negatively charged phosphate ion shields the electrostatic repulsion of the positively charged arginine and histidine residues, resulting in higher stability of the phosphate-bound lysozyme. Additionally, a low-humidity orthorhombic variant was obtained at pH 4.5, and comparison with those previously obtained at pH 6.5 and 9.5 shows a 1.5 Šdisplacement of the fifth α-helix towards the active-site cavity, which might be relevant to protein function. Since lysozyme is broadly used as a model protein in studies related to protein crystallization and amyloid formation, these results indicate that the interaction of some anions must be considered when analysing experiments performed at acidic pH values.

New crystal form of human ubiquitin in the presence of magnesium.

Ubiquitin is a small globular protein that has a considerable number of lysine residues on its surface. This results in a high surface entropy that precludes the formation of crystal-packing interactions. To date, only a few structures of the native form of ubiquitin have been solved, and most of the crystals that led to these structures were obtained in the presence of different divalent metal cations. In this work, a new crystallographic structure of human ubiquitin solved from crystals grown in the presence of magnesium is presented. The crystals belonged to a triclinic space group, with unit-cell parameters a = 29.96, b = 30.18, c = 41.41 Å, α = 88.52, ß = 79.12, γ = 67.37°. The crystal lattice is composed of stacked layers of human ubiquitin molecules with a large hydrophobic interface and a smaller polar interface in which the magnesium ion lies at the junction between adjacent layers in the crystal. The metal ion appears in a hexa-aquo coordination, which is key to facilitating the crystallization of the protein.