Crystallographic studies of the structured core domain of Knr4 fromSaccharomyces cerevisiae

Abstract

The potentially structured core domain of the intrinsically disordered protein Knr4 from Saccharomyces cerevisiae, comprising residues 80-340, was expressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. Selenomethionine-containing (SeMet) protein was also purified and crystallized. Crystals of both proteins belonged to space group P6 5 22, with unit-cell parameters a = b = 112.44, c = 265.21 Å for the native protein and a = b = 112.49, c = 262.21 Å for the SeMet protein, and diffracted to 3.50 and 3.60 Å resolution, respectively. There are two molecules in the asymmetric unit related by a twofold axis. The anomalous signal of selenium was recorded and yielded an electron-density map of sufficient quality to allow the identification of secondary-structure elements.

Knr4: a disordered hub protein at the heart of fungal cell wall signalling

“…The three‐dimensional structure of Knr4 is unknown to date, as well as those of its fungal homologs. However, the central core of Knr4 has been recently crystallized and secondary structure elements revealed, indicating that this domain is indeed able to adopt a stable conformation (Julien et al ., ). This central core holds the essential of the protein biological function as attested by its ability to complement most of the S .…”

The Conserved Yeast Protein Knr4 Involved in Cell Wall Integrity is a Multi-Domain Intrinsically Disordered Protein

“…The purification of the Knr4 DNDC , its crystallisation and preliminary crystallographic data were described before, 40 but the low resolution of the data did not allow to solve the corresponding structure. Here, the core domain including or not the disordered loop DL (Knr4 DNDC and Knr4 DNDLDC ), were both purified, crystallised and both structures were solved.…”

“…Plasmids pGEX-6P-3::Knr4 and pGEX-6P-3:: Knr4 DNDC have been described. 23,40 Plasmid pGEX-6P-3:: Knr4 DC was constructed by introducing two in frame stop codons at codons 346 and 347 of KNR4 in pGEX-6P-3::Knr4 by mutagenesis using self-complementary PCR primers (KNR4-Mut-Top and -Bot; Supplementary Table 3). Vector pGEX-6P-3::Knr4 DNDLDC was constructed by inverse PCR on pGEX-6P-3 using pGEX-6P-3:: Knr4 as matrix and primers allowing the precise deletion of DL (DL-Fwd and -Rev; Supplementary Table 3).…”

“…Previously determined crystallization conditions 40 repeatedly yielded crystals diffracting at best to 3.5 A resolution, which nevertheless allowed to obtain Se-SAD phases of poor quality allowing to build a partial model. The new construct described herein allowed improved purification and identification of new crystallisation conditions.…”