When a module is not a domain: the case of the REJ module and the redefinition of the architecture of polycystin-1

Abstract

Synopsis The extracellular region of a group of cell surface receptors known as the polycystic kidney disease 1 family, comprising amongst others polycystin-1, has been controversially described as containing four fibronectin type III (FNIII) domains or one REJ module in the same portion of polypeptide. Stimulated by recent atomic force microscopy work we re-examined the similarity of these four domains with a FNIII sequence profile showing the evolutionary relationship. Two of the predicted domains could be expressed in bacteria and refolded to give protein suitable for biophysical study and one of these expressed solubly. Circular dichroism spectroscopy showed that both domains contain a significant amount of β-sheet, in good agreement with theoretical predictions. Confirmation of independent folding as a domain is obtained from highly cooperative thermal and urea unfolding curves. Excellent dispersion of peaks in the high field region of one dimensional NMR spectra confirms the presence of a hydrophobic core. Analytical ultracentrifugation and analytical gel filtration agree very well with the narrow linewidths in the NMR spectra that at least one of the domains is monomeric. Based on this combined theoretical and experimental analysis we show that the extracellular portion of polycystin-1 does indeed contain β-sheet domains, very likely fibronectin type III, and that consequently the REJ module is not a single domain.

Analysis of the REJ Module of Polycystin-1 Using Molecular Modeling and Force-Spectroscopy Techniques

“…This is in agreement with a recent study that found that REJd1, -d2, -d3, and -d4 are very hard to express in E. coli [24]. In order to increase their solubility we flanked the REJ domains with maltose-binding protein (MBP) and titin I27 domains.…”

“…1 from PC1 (1b4r) and the human PKD domain from protein KIAA0319 (2e7m). The domain boundaries were based on Schröder et al sequence analysis [24] and our Clustal multiple sequence alignment. The overall identity between each of the REJ domains and the template structures was low (~10% overall identity, ~27% similarity).…”

“…More recently, Schröder et al used comprehensive sequence analysis together with CD spectroscopy and NMR techniques to analyze the first 425 amino acids of the REJ module [24]. They found that within this segment there are total of four predicted FNIII domain but only the first two domains could be expressed as soluble proteins, and only domain 2 was amenable for NMR analysis.…”

Altered trafficking and stability of polycystins underlie polycystic kidney disease

“…Between 65% and 75% of pathogenic mutations in PKD1 are either nonsense, splice site, or insertion/deletion changes that are predicted to result in premature termination of the PC1 pep like PKD repeats (34)(35)(36), a receptor egg jelly (REJ) domain (37) that includes fibronectin type III repeats (38) and is part of a recently identified structural GAIN domain (39). The REJ/GAIN domain is required for autoproteolytic cleavage of PC1 at a G protein-cou pled receptor proteolytic site (GPS) sequence, HL↓T 3049 (40), that yields an extracellular NH 2 terminal fragment (PC1-NTF) and an intramembranous COOHterminal fragment (PC1-CTF) (39,41,42).…”

Large-Scale Modelling of the Divergent Spectrin Repeats in Nesprins: Giant Modular Proteins

“…1D NMR spectra were recorded on a 700 MHz Bruker Avance spectrometer using watergate for water suppression [59] as described previously [60]. Diffusion coefficients were measured on a Bruker Avance 500 MHz spectrometer using a convection compensated double stimulated echo experiment [61].…”