ABSTRACT
The ability to sense and respond appropriately to environmental changes is a primary requirement of all living organisms. In response to phosphate limitation, Saccharomyces cerevisiae induces transcription of a set of genes involved in the regulation of phosphate acquisition from the ambient environment. A signal transduction pathway (the PHO pathway) mediates this response, with Pho85-Pho80 playing a vital role. Here we report the X-ray structure of Pho85-Pho80, a prototypic structure of a CDK-cyclin complex functioning in transcriptional regulation in response to environmental changes. The structure revealed a specific salt link between a Pho85 arginine and a Pho80 aspartate that makes phosphorylation of the Pho85 activation loop dispensable and that maintains a Pho80 loop conformation for possible substrate recognition. It further showed two sites on the Pho80 cyclin for high-affinity binding of the transcription factor substrate (Pho4) and the CDK inhibitor (Pho81) that are markedly distant to each other and the active site.
Subject(s)
Cyclin-Dependent Kinases/chemistry , Cyclins/chemistry , Phosphates/pharmacology , Repressor Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/enzymology , Signal Transduction/drug effects , Binding Sites , Crystallography, X-Ray , DNA-Binding Proteins/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors/metabolism , Models, Molecular , Mutant Proteins/chemistry , Phenylalanine , Phosphorylation/drug effects , Protein Binding/drug effects , Protein Structure, Secondary , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Structure-Activity Relationship , Substrate Specificity/drug effects , Transcription Factors/metabolismABSTRACT
The ABC transporter associated with antigen processing (TAP) shuttles cytosolic peptides into the endoplasmic reticulum for loading onto class I MHC molecules. Transport is fueled by ATP binding and hydrolysis at two distinct cytosolic ATPase sites. One site comprises consensus motifs shared among most ABC transporters, while the second has substituted, degenerate motifs. Biochemical and crystallography experiments with a TAP cytosolic domain demonstrate that the consensus ATPase site has high catalytic activity and facilitates ATP-dependent dimerization of the cytosolic domains, which is an important conformational change during transport. In contrast, the degenerate site is defective in dimerization and ATP hydrolysis. Full-length TAP mutagenesis demonstrates the necessity for at least one consensus site, supporting our conclusion that the consensus site is the principal facilitator of substrate transport. Since asymmetry of the ATPase site motifs is a feature of many mammalian homologs, our proposed model has broad implications for ABC transporters.
