ABSTRACT
Activation of the high-osmolarity glycerol (HOG) pathway for osmoregulation in the yeast Saccharomyces cerevisiae involves interaction of the adaptor Ste50p with the cytoplasmic tail of single-transmembrane protein Opy2p. We have determined the solution structure of the Ste50p-RA (Ras association) domain, and it shows an atypical RA fold lacking the beta1 and beta2 strands of the canonical motif. Although the core of the RA domain is fully functional in the pheromone response, an additional region is required for the HOG pathway activation. Two peptide motifs within the intrinsically disordered cytoplasmic tail of Opy2p defined by NMR spectroscopy physically interact with the Step50p-RA domain. These Opy2p-derived peptides bind overlapping regions of the Step50p-RA domain with similarly weak affinities, suggesting a multivalent interaction of these proteins as a crucial point of control of the HOG pathway. As well, overall selection of signaling pathways depends on functionally distinct regions of the Ste50p-RA domain, implicating this element in the control of global regulatory decisions.
Subject(s)
Glycerol/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Protein Folding , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Cytoplasm/chemistry , Molecular Sequence Data , Osmolar Concentration , Osmosis , Peptides/chemistry , Peptides/metabolism , Protein Binding , Protein Interaction Mapping , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Deletion , Signal Transduction , Stress, Physiological , Structure-Activity RelationshipSubject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/chemistry , Protein Folding , Amino Acid Sequence , Conserved Sequence , Escherichia coli Proteins/classification , Escherichia coli Proteins/genetics , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
Major vault protein (MVP) is the main constituent of vaults, large ribonucleoprotein particles implicated in resistance to cancer therapy and correlated with poor survival prognosis. Here, we report the structure of the main repeat element in human MVP. The approximately 55 amino acid residue MVP domain has a unique, novel fold that consists of a three-stranded antiparallel beta-sheet. The solution NMR structure of a two-domain fragment reveals the interdomain contacts and relative orientations of the two MVP domains. We use these results to model the assembly of 672 MVP domains from 96 MVP molecules into the ribs of the 13MDa vault structure. The unique features include a thin, skin-like structure with polar residues on both the cytoplasmic and internal surface, and a pole-to-pole arrangement of MVP molecules. These studies provide a starting point for understanding the self-assembly of MVP into vaults and their interactions with other proteins. Chemical shift perturbation studies identified the binding site of vault poly(ADP-ribose) polymerase, another component of vault particles, indicating that MVP domains form a new class of interaction-mediating modules.
