Mutagenesis Of Charged Residues Research Articles

The Protease Activated Receptor 2 (PAR2) Polymorphic Variant F240S Constitutively Activates PAR2 Receptors and Potentiates Responses to Small-Molecule PAR2 Agonists

AC-55541 [N-[[1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl]-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl]-benzamide] and AC-264613 [2-oxo-4-phenylpyrrolidine-3-carboxylic acid [1-(3-bromo-phenyl)-(E/Z)-ethylidene]-hydrazide] are the first two small-molecule agonists described for the G protein-coupled receptor protease-activated receptor 2 (PAR2), but whether they activate PAR2 through a similar mechanism as its tethered peptide ligand or soluble peptide mimetics of its tethered peptide ligand is unclear. Extracellular loop 2 (ECL2) has been shown to play a critical role in the activation mechanism of PAR2. Therefore, we constructed a series of PAR2 receptors mutated in ECL2, including a previously described polymorphic variant of PAR2 (F240S), and compared AC-55541 and AC-264613 to SLIGRL and a potent analog of SLIGRL called 2-furoyl LIGRLO in a series of functional assays, including cellular proliferation, phosphatidylinositol hydrolysis, and β-arrestin recruitment assays. Surprisingly, receptors with the F240S mutation were constitutively active in all functional assays tested. Furthermore, AC-55541 and AC-264613 were potentiated over 30-fold at the receptors with the F240S mutation, whereas SLIGRL and 2-furoyl LIGRLO were much less affected. In contrast, mutagenesis of charged residues in ECL2 confirmed their important role in the actions of peptide agonists of PAR2, whereas these mutations did not significantly affect activation of PAR2 by AC-55541 or AC-264613. These results suggest that F240S PAR2 receptors may be useful in screens to detect novel small-molecule PAR2 modulators and that further work on the biological importance of the F240S PAR2 variant is warranted.

Sulphonylurea Receptors Regulate Kir6.2 Subunits Allosterically via a Salt Bridge in Cardiac K ATP Channels

ATP sensitive potassium (KATP) channels play important roles in many tissues, coupling metabolic status to membrane potential. In heart, they comprise subunit hetero-octamers of Kir6.24/SUR2A4.A minimal sequence (residues 1294-1358) in nucleotide binding domain-2 of SUR2A has been indentified previously by us to bind Kir6.2 (Rainbow et. al. Biochem. J. 2004;379:173-181). Co-immunoprecipitation of chimaeras of Kir6.2/Kir2.1 with maltose binding protein (MBP)-tagged SUR2A(1294-1358) fragments identified the cognate binding domain in Kir6.2 (residues 315-390). Mutagenesis of charged residues in Kir6.2, D323K and K338E, conserved in Kir6.1 but not Kir2.1, was alone sufficient to significantly reduce co-immunoprecipitation of MBP-SUR2A(1294-1358) by ∼50 % (P 0.5).Together, these data provide evidence for the transmission of allosteric information via a salt bridge between SUR2AE1318 and Kir6.2K338 in cardiac KATP channels, while ATP-sensitivity remains unaltered.

The cytotoxic A1 domain of shiga‐like toxin 1 interacts with two distinct sites on the ribosomal stalk

Ribosome‐inactivating proteins (RIPs) such as Shiga‐like toxin 1 (SLT‐1) halt protein synthesis in eukaryotic cells by depurinating a single adenine base in the sarcin‐ricin loop of 28S rRNA. The molecular details leading up to the site‐specific depurination are lacking, despite a general understanding of the biochemical basis of SLT‐1 toxicity. Here we present evidence that the mechanism by which the catalytic domain of RIPs cleaves its substrate involves initial docking interactions with the ribosomal stalk by virtue of a conserved C‐terminal domain common to all three stalk proteins P0, P1, and P2. The A1 chain of SLT‐1 transiently binds to this peptide with a modest binding constant and rapid on and off rates. Mutagenesis of charged residues within the A1 chain identified a cationic surface that interacts with the peptide motif. In addition, peptide phage‐display revealed a complementary charged surface and an additional hydrophobic motif which may be involved in the interaction. Moreover, deletion mutagenesis performed on the ribosomal protein P0 revealed that the A1 chain binds to an alternate site on P0 in proximity to the contact sites for P1/P2 heterodimers. These results demonstrate that the catalytic chain of RIPs such as SLT‐1 dock on ribosomes using two classes of binding sites located within the ribosomal stalk which may aid in orienting their catalytic domain in close proximity to the depurination site.

Electrostatic Contributions to the Stability of the GCN4 Leucine Zipper Structure

Ion pairs are ubiquitous in X-ray structures of coiled coils, and mutagenesis of charged residues can result in large stability losses. By contrast, p K a values determined by NMR in solution often predict only small contributions to stability from charge interactions. To help reconcile these results we used triple-resonance NMR to determine p K a values for all groups that ionize between pH 1 and 13 in the 33 residue leucine zipper fragment, GCN4p. In addition to the native state we also determined comprehensive p K a values for two models of the GCN4p denatured state: the protein in 6 M urea, and unfolded peptide fragments of the protein in water. Only residues that form ion pairs in multiple X-ray structures of GCN4p gave large p K a differences between the native and denatured states. Moreover, electrostatic contributions to stability were not equivalent for oppositely charged partners in ion pairs, suggesting that the interactions between a charge and its environment are as important as those within the ion pair. The pH dependence of protein stability calculated from NMR-derived p K a values agreed with the stability profile measured from equilibrium urea-unfolding experiments as a function of pH. The stability profile was also reproduced with structure-based continuum electrostatic calculations, although contributions to stability were overestimated at the extremes of pH. We consider potential sources of errors in the calculations, and how p K a predictions could be improved. Our results show that although hydrophobic packing and hydrogen bonding have dominant roles, electrostatic interactions also make significant contributions to the stability of the coiled coil.

Intracellular Domains of a Rat Brain GABA Transporter That Govern Transport

Plasma membrane neurotransmitter transporters determine in part the concentration, time course, and diffusion of extracellular transmitter. Much has been learned about how substrate translocation through the transporter occurs; however, the precise way in which transporter structure maps onto transporter function has not yet been fully elucidated. Here, biochemical and electrophysiological approaches were used to test the hypothesis that intracellular domains of the rat brain GABA transporter (GAT1) contribute to the transport process. Injection of a peptide corresponding to the presumed fourth intracellular loop of the transporter (IL4) into oocytes expressing GAT1 greatly reduced both forward and reverse transport and reduced the transport rate in a dose-dependent manner. Coinjection of the IL4 peptide with a peptide corresponding to the N-terminal cytoplasmic tail of GAT1 reversed the IL4-mediated inhibition; this reversal, and direct binding between these two domains, was prevented by mutagenesis of charged residues in either the IL4 or N-terminal domains. Furthermore, syntaxin 1A, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein that inhibits GAT1 transport rates via interactions with the N-terminal tail of GAT1 was unable to regulate the GAT1 IL4 mutant. Together, these data suggest a model in which the GAT1 IL4 domain serves as a barrier for transport, and this barrier can be regulated through intra-molecular and inter-molecular interactions.

The extracellular domain suppresses constitutive activity of the transmembrane domain of the human TSH receptor: implications for hormone-receptor interaction and antagonist design.

Among glycoprotein hormone receptors the TSH receptor (TSHR) is the most susceptible to constitutive activation by mutations in various regions of the molecule, including mutations in the extracellular domain (ECD) and extracellular loops of the transmembrane domain (TMD). To understand the role of the ECD in TSHR activation we have tested several TSHR constructs with major deletions of the ECD. Previous studies reported very low expression of such truncated glycoprotein hormone receptors, which prevented reliable assessment of their ligand-binding and basal constitutive activities. We have eliminated this problem using TSHR tagged at its N-terminus with a hemagglutinin tag (HA) recognized by the HA-specific monoclonal antibody. Based on such quantitation the TSHR deletion mutant missing 386 N-terminal amino acid residues, constituting 98% of the entire ECD, showed 4-7 fold higher normalized basal activity compared to activity of the corresponding wild-type (WT) TSHR construct. This increase in basal activity was significantly inhibited by linking the common alpha-subunit of glycoprotein hormones at the N-terminus of the truncated TSH receptor. The role of a hypothetical activating fragment (409-418) in TSHR activation was further studied using peptides and mutagenesis of charged residues. This study provides important evidence supporting the "two-state" model of TSHR activation and the potential role of proteolytic cleavage for receptor activation. Accordingly, the mechanism of hormone-induced receptor activation is dependent, at least in part, on the elimination of inhibitory interactions within the receptor. Such intra-molecular inhibition of TSHR may include electrostatic interactions between the ECD and extracellular loops of TMD. Moreover, the truncated, constitutively active receptors described herein provide new insights valuable in the design of TSHR antagonists.

The Extracellular Domain Suppresses Constitutive Activity of the Transmembrane Domain of the Human TSH Receptor: Implications for Hormone-Receptor Interaction and Antagonist Design

Among glycoprotein hormone receptors the TSH receptor (TSHR) in the most susceptible to constitutive by mutations in various regions of the molecule, including mutations in the extracellular domain (ECD) and extracellular loops of the transmembrane domain (TMD) to understand the role of hte ECD in TSHR activation we have tested several TSHR constructs with major deletions of the ECD. Previous studies reported very low expression of such truncated glycoprotein hormone receptors, which prevented reliable assessment of their ligand-binding and basal constitutive activities. We have eliminated this problem using TSHR at its N-terminus with a hemagglutinin tag (HA) recognized by the HA-specific monoclonal antibody. Based on such quantitation the TSHR deletion mutant missing 386 N-terminal amino acid residues, constituting 98% of the entire ECD, showed 4-7 fold higher normalized basal activity compared to activity of the corresponding wild-type (WT) TSHR construct. This increase in basal was significantly inhibited by linking the common α-subunit of glycoprotein hormones at the N-terninus of the truncated TSH receptor. Thr role of a hypothetical activating fragment (409-418) in TSHR activatoin was further studied using peptides and mutagenesis of charged residues. This study provides important evidence supporting the “two-state” model of TSHR activation and the potential role of proteolytic cleavage for recetor activation. Accordingly, the mechanism of hormone-induced receptor activation is dependent, at least in part, on the elimination of inhibitory within the receptor. Such intra-molecular inhibition of TSHR may include electrostatic interactions between the ECD and extracellular loops of TMD. Moreover, the truncated, constitutively active receptors described herein privide new insights valuable in the design of TSHR antagonists.

Mutagenesis of charged residues in a conserved sequence in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Mutagenesis of charged residues in a conserved sequence in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Mutagenesis of charged residues in a conserved sequence in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Arg-136, Glu-137, Arg-138 and Arg-139 are conserved in all sequences of the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Their role was studied by site-directed mutagenesis. All the mutations had little, if any, effect on fructose-2,6-bisphosphatase activity. Mutations of Arg-136 and Glu-137 into Ala caused only minor modifications of phosphofructo-2-kinase activity. In contrast, mutation of Arg138 into Ala increased 280-fold the Km for fructose 6-phosphate of phosphofructo-2-kinase. Mutation of Arg-139 into Ala resulted in decreases in phosphofructo-2-kinase Vmax/Km for MgATP and fructose 6-phosphate 600-fold and 5000-fold respectively. Mutation of Arg-139 into Lys and Gln increased the Km of phosphofructo-2-kinase for MgATP (20-fold and 25-fold respectively) and for fructose 6-phosphate (8-fold and 13-fold), and the IC50 for MgADP (30-fold and 50-fold) and for magnesium citrate (7-fold and 25-fold). However, these two mutations did not affect nucleotide binding, as measured by quenching of intrinsic fluorescence. The changes in kinetic properties induced by mutations could not be attributed to structural changes. It is proposed that Arg-138 is involved in fructose 6-phosphate binding and that Arg-139 is probably involved in the stabilization of the transition state and so participates in catalysis.