ABSTRACT
Pleckstrin homology (PH) domains are small modular domains that occur once, or occasionally several times, in a large variety of signalling proteins. In a number of instances, PH domains act to target their host protein to the cytosolic face of cellular membranes through an ability to associate with phosphoinositides. In this review, we discuss recent advances in our understanding of PH domain function. In particular we describe the structural aspects of how PH domains have evolved to bind various phosphoinositides, how PH domains regulate phosphoinositide-mediated association to plasma and internals membranes, and finally raise the issue of PH domains in protein:protein interactions and the allosteric regulation of their host protein.
Subject(s)
Blood Proteins/chemistry , Blood Proteins/metabolism , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Allosteric Regulation , Amino Acid Sequence , Animals , Binding Sites , Blood Proteins/genetics , Cell Membrane/metabolism , Chemotaxis/physiology , Humans , Intracellular Membranes/metabolism , Models, Molecular , Molecular Sequence Data , Phosphatidylinositols/metabolism , Phosphoproteins/genetics , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Signal TransductionABSTRACT
The membrane phospholipid phosphatidylinositol is the precursor of a family of lipid second-messengers, known as phosphoinositides, which differ in the phosphorylation status of their inositol group. A major advance in understanding phosphoinositide signalling has been the identification of a number of highly conserved modular protein domains whose function appears to be to bind various phosphoinositides. Such 'cut and paste' modules are found in a diverse array of multidomain proteins and recruit their host protein to specific regions in cells via interactions with phosphoinositides. Here, with particular reference to proteins involved in membrane traffic pathways, we discuss recent advances in our understanding of phosphoinositide-binding domains.
Subject(s)
Blood Proteins/chemistry , Conserved Sequence , Endocytosis/physiology , Phagocytosis/physiology , Phosphatidylinositols/metabolism , Phosphoproteins/chemistry , Second Messenger Systems , Amino Acid Sequence , Binding Sites , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositol Phosphates/metabolism , Protein Structure, TertiaryABSTRACT
The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P(3) production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4, 5)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P(4) rather than PtdIns(3,4,5)P(3).
Subject(s)
Cell Membrane/metabolism , Inositol Phosphates/metabolism , Phosphatidylinositol Phosphates/metabolism , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/metabolism , Amino Acid Substitution , Animals , Binding Sites , COS Cells , Cell Nucleus/metabolism , HeLa Cells , Humans , Liposomes , Mutagenesis, Site-Directed , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Subcellular Fractions/metabolism , Sucrose , TransfectionABSTRACT
GAP1(IP4BP) is a Ras GTPase-activating protein (GAP) that in vitro is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P(4)]. We have studied Ins(1,3,4,5)P(4) binding to GAP1(IP4BP), and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)P(4) binding to Bruton's tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)P(4) binding. The crystal structure of the Btk pleckstrin homology (PH) domain in complex with Ins(1,3,4,5)P(4) has shown that the binding site is located in a partially buried pocket between the beta 1/beta 2- and beta 3/beta 4-loops. Many of the residues involved in the binding are conserved in GAP1(IP4BP). Therefore we generated a model of the PH domain of GAP1(IP4BP) in complex with Ins(1,3,4,5)P(4) based on the Btk-Ins(1,3,4,5)P(4) complex crystal structure. This model had the typical PH domain fold, with the proposed binding site modelling well on the Btk structure. The model has been verified by site-directed mutagenesis of various residues in and around the proposed binding site. These mutations have markedly reduced affinity for Ins(1,3,4,5)P(4), indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding.
Subject(s)
Blood Proteins/chemistry , Inositol Phosphates/chemistry , Phosphoproteins/chemistry , Receptors, Cytoplasmic and Nuclear/chemistry , Amino Acid Sequence , Binding Sites , Calcium/metabolism , Lysine/chemistry , Methionine/chemistry , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Protein Binding , Protein Folding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Tryptophan/chemistryABSTRACT
The requirements for substrate binding in the quinoprotein glucose dehydrogenase (GDH) in the membranes of Escherichia coli are described, together with the changes in activity in a site-directed mutant in which His262 has been altered to a tyrosine residue (H262Y-GDH). The differences in catalytic efficiency between substrates are mainly related to differences in their affinity for the enzyme. Remarkably, it appears that, if a hexose is able to bind in the active site, then it is also oxidized, whereas some pentoses are able to bind (and act as competitive inhibitors), but are not substrates. The activation energies for the oxidation of hexoses and pentoses are almost identical. In a previously published model of the enzyme, His262 is at the entrance to the active site and appears to be important in holding the prosthetic group pyrroloquinoline quinone (PQQ) in place, and it has been suggested that it might play a role in electron transfer from the reduced PQQ to the ubiquinone in the membrane. The H262Y-GDH has a greatly diminished catalytic efficiency for all substrates, which is mainly due to a marked decrease in their affinities for the enzyme, but the rate of electron transfer to oxygen is unaffected. During the processing of the PQQ into the apoenzyme to give active enzyme, its affinity is markedly dependent on the pH, four groups with pK values between pH7 and pH8 being involved. Identical results were obtained with H262Y-GDH, showing that His262 it is not directly involved in this process.
Subject(s)
Amino Acid Substitution , Cell Membrane/enzymology , Escherichia coli/enzymology , Glucose Dehydrogenases/metabolism , Histidine/genetics , Tyrosine/genetics , Apoenzymes/biosynthesis , Apoenzymes/chemistry , Apoenzymes/isolation & purification , Apoenzymes/metabolism , Binding Sites , Electron Transport , Escherichia coli/cytology , Escherichia coli/genetics , Glucose Dehydrogenases/chemistry , Glucose Dehydrogenases/genetics , Glucose Dehydrogenases/isolation & purification , Hexoses/chemistry , Hexoses/metabolism , Histidine/metabolism , Hydrogen-Ion Concentration , Kinetics , Magnesium/metabolism , Mutagenesis, Site-Directed , Oxidation-Reduction , Oxygen/metabolism , PQQ Cofactor , Pentoses/chemistry , Pentoses/metabolism , Quinolones/metabolism , Quinones/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity , Tyrosine/metabolismSubject(s)
Escherichia coli/enzymology , Glucose Dehydrogenases/metabolism , Histidine , Quinolones/chemistry , Quinolones/metabolism , Quinones/chemistry , Quinones/metabolism , Binding Sites , Coenzymes/metabolism , Glucose Dehydrogenases/chemistry , Models, Molecular , Molecular Conformation , Mutagenesis, Site-Directed , PQQ Cofactor , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
The structure of methanol dehydrogenase (MDH) at 0.194 nm (1.94 A) has been used to provide a model structure for part of a membrane quinoprotein glucose dehydrogenase (GDH). The basic superbarrel structure is retained, along with the tryptophan-docking motifs. The active-site regions are similar, but there are important differences, the most important being that GDH lacks the novel disulphide ring structure formed from adjacent cysteines in MDH; in GDH the equivalent region is occupied by His-262. Because of the overall similarities in the active-site region, the mechanism of action of GDH is likely to be similar to that of MDH. The differences in co-ordination to the cation and bonding to the pyrrolo-quinoline quinone (PQQ) in the active site may explain the relative ease of dissociation of the prosthetic group from the holo-GDH. There are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site, the configuration of which influences substrate specificity. The proposed model is consistent in many respects with previous proposals for the active-site structure based on the effects of chemical modification on binding of PQQ and enzymic activity.
Subject(s)
Alcohol Oxidoreductases/chemistry , Escherichia coli/enzymology , Glucose Dehydrogenases/chemistry , Gram-Negative Aerobic Bacteria/enzymology , Models, Molecular , Amino Acid Sequence , Binding Sites , Consensus Sequence , Molecular Sequence Data , PQQ Cofactor , Quinolones/metabolismABSTRACT
The 1.94 A structure of methanol dehydrogenase has been used to provide a model structure for part of a membrane quinohaemoprotein alcohol dehydrogenase. The basic superbarrel structure and the active-site region are retained, indicating essentially similar mechanisms of action, but there are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site.
