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Allosteric control of regulated scaffolding in membrane-associated guanylate kinases.

ABSTRACT: Membrane-associated guanylate kinases (MAGUKs) organize protein complexes at specific cellular sites by regulating interactions with their COOH-terminal guanylate kinase-like domains (GKs). Negative regulation of MAGUK GKs by an adjacent Src homology 3 domain (SH3) is critical for function, yet the mechanism is poorly understood. To gain insight into this process, we investigated SH3 regulation of the Discs large (Dlg) GK. Mutational analysis revealed that the binding site of the SH3-inhibited GK ligand GukHolder (GukH) is opposite the SH3 interacting surface, indicating that the SH3 does not directly occlude GukH binding. We screened for constitutively active SH3GK variants using yeast two-hybrid and a cell polarity/mitotic spindle orientation assay. Residues in both the SH3 and GK are required to maintain SH3GK inhibition, including those distant from both the SH3-GK and GK-GukH interaction sites. Activating mutations do not alter the ability of the SH3 and GK to interact in trans. On the basis of these observations, we propose that the SH3 modulates GK allostery to control its function.

SUBMITTER: Marcette J

PROVIDER: S-EPMC3535437 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Allosteric control of regulated scaffolding in membrane-associated guanylate kinases.

Marcette Jana J Hood Iris V IV Johnston Christopher A CA Doe Chris Q CQ Prehoda Kenneth E KE

Biochemistry 20091001 42

Membrane-associated guanylate kinases (MAGUKs) organize protein complexes at specific cellular sites by regulating interactions with their COOH-terminal guanylate kinase-like domains (GKs). Negative regulation of MAGUK GKs by an adjacent Src homology 3 domain (SH3) is critical for function, yet the mechanism is poorly understood. To gain insight into this process, we investigated SH3 regulation of the Discs large (Dlg) GK. Mutational analysis revealed that the binding site of the SH3-inhibited G ...[more]

PMID: 19769408

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Project description:Since the introduction of the induced-fit theory by D. E. Koshland Jr., it has been established that conformational motion invariably accompanies the execution of protein function. The catalytic activity of kinases, specifically, is associated with large conformational changes ( approximately 1 nm amplitude). In the case of guanylate kinase, upon substrate binding, the LID and nucleotide-monophosphate-binding domains are brought together and toward the CORE with large concerted movements about the alpha3 (helix 3) axis. However, whether the change in conformation mostly affects the catalytic rate or mostly increases binding affinities for one or the other substrate is unclear. We investigate this question using a nanotechnology approach based on mechanical stress. Using an "allosteric spring probe", we bias conformational states in favor of the "open" (substrate-free) conformation of the enzyme; the result is that the binding constant for the substrate guanosine monophosphate (GMP) is reduced by up to a factor of 10, whereas the binding constant for adenosine triphosphate (ATP) and the catalytic rate are essentially unaffected. The results show that the GMP-induced conformational change, which promotes catalysis, does not promote ATP binding, consistent with previous mutagenesis studies. Furthermore, they show that this conformational change is of the induced-fit type with respect to GMP binding (but not ATP binding). We elaborate on this point by proposing a quantitative criterion for the classification of conformational changes with respect to the induced-fit theory. More generally, these results show that the allosteric spring probe can be used to affect enzymatic activity in a continuously controlled manner, and also to affect specific steps of the reaction mechanism while leaving others unaffected. It is presumed that this will enable informative comparisons with the results of future molecular dynamics or statistical mechanics computations.

Dataset Information

Allosteric control of regulated scaffolding in membrane-associated guanylate kinases.

Publications

Allosteric control of regulated scaffolding in membrane-associated guanylate kinases.

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