The effect of a proline residue on the rate of growth and the space group of alpha-spectrin SH3-domain crystals.
alpha-Spectrin SH3-domain (Spc-SH3) crystallization is characterized by very fast growth of the crystals in the presence of ammonium sulfate as a precipitant agent. The origin of this behaviour can be attributed to the presence of a proline residue that participates in a crystal contact mimicking the binding of proline-rich sequences to SH3 domains. This residue, Pro20, is located in the RT loop and is the main contact in one of the interfaces present in the orthorhombic Spc-SH3 crystal structures. In order to understand the molecular interactions that are responsible for the very fast crystal growth of the wild-type (WT) Spc-SH3 crystals, the crystal structure of a triple mutant in which the residues Ser19-Pro20-Arg21 in the RT loop have been replaced by Gly19-Asp20-Ser21 (GDS Spc-SH3 mutant) has been solved. The removal of the critical proline residue results in slower nucleation of the Spc-SH3 crystals and a different arrangement of the protein molecules in the unit cell, leading to a crystal that belongs to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 42.231, c = 93.655 A, and that diffracts to 1.45 A resolution. For both WT Spc-SH3 and the GDS mutant, light-scattering experiments showed that a dimer was formed in solution within a few minutes of the addition of 2 M ammonium sulfate at pH 6.5 and allowed the proposal of a mechanism for the nucleation and crystal growth of Spc-SH3 in which the Pro20 residue plays a key role in the rate of crystal growth.
Cámara-Artigas
Ana
A
Departamento Química-Física, Bioquímica y Química Inorgánica, Universidad de Almería, Carretera Sacramento, Almería 04120, Spain. acamara@ual.es
Andújar-Sánchez
Monserrat
M
Ortiz-Salmerón
Emilia
E
Cuadri
Celia
C
Casares
Salvador
S
D003460:Crystallization; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009154:Mutation; D011392:Proline / Q000737:chemistry*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D013049:Spectrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018909:src Homology Domains
D013049:Spectrin; D011392:Proline