N-terminal truncation of the

The transmembrane (TM) and juxtamembrane (JM) regions of the ErbB family receptor tyrosine kinases connect the extracellular ligand-binding domain to the intracellular kinase domain. Evidence for the role of these regions in the mechanism of receptor dimerization and activation is provided by TM-JM peptides corresponding to the Neu (or rat ErbB2) receptor. Solid-state NMR and fluorescence spectroscopy show that there are tight interactions of the JM sequence with negatively charged lipids, including phosphatidylinositol 4,5-bisphosphate, in TM-JM peptides corresponding to the wildtype receptor sequence. We observe a release of the JM sequence from the negatively charged membrane surface using peptides containing an activating V664E mutation within the TM domain or in peptides engineered to form TM helix dimers with Val664 in the interface. These results provide the basis of a mechanism for coupling ligand binding to kinase activation in the full-length receptor.EGF receptor | HER2 receptor | PIP 2 T he receptor tyrosine kinases (RTKs) are a large family of membrane receptors that control cell growth, differentiation, and migration. These receptors have a three-domain architecture consisting of an extracellular ligand-binding domain, a single transmembrane helix, and an intracellular kinase domain. Receptor activation is triggered by ligand-mediated dimerization of receptor monomers or the structural rearrangement of inactive, preformed dimers (1, 2). Detailed information about the mechanisms of activation and regulation of RTKs has come largely from crystal structures of their extracellular and intracellular domains in inactive and active conformations. The activation mechanisms of the ErbB subfamily of RTKs are of particular interest because mutations and deletions that result in constitutive receptor activity have been identified in a number of human tumors (3, 4).In the epidermal growth factor receptor (EGFR), a member of the ErbB subfamily, the structures of the extracellular domain with (5) and without (6) bound EGF revealed that the unliganded structure has a tethered conformation that undergoes a dramatic rearrangement upon ligand binding. On the intracellular side of the EGFR, crystal structures show that the activation loop associated with the active site in the kinase domain is in an open, active conformation before ligand binding (7) or locked in a Src tyrosine kinase/cyclin-dependent kinase-like inactive conformation (8). Activation is produced not via phosphorylation of the activation loop, as in other RTKs, but through the association of the intracellular kinase domains as an asymmetric dimer (8). An open question has involved how structural changes induced by ligand binding lead to the formation of an active, asymmetric dimer on the intracellular side of the receptor.The single transmembrane (TM) helix and associated juxtamembrane (JM) sequences bridge the extracellular and intracellular domains and require a membrane environment to adopt their native structure. In general, ligand binding to the e...

The chemical synthesis of human interleukin-2 (IL-2) , having a core 1 sugar, by a ligation method is reported. Although IL-2 is a globular glycoprotein, its C-terminal region, in particular (99-133), is extremely insoluble when synthesized by solid-phase method. To overcome this problem, the side-chain carboxylic acid of the Glu residues was protected by a picolyl ester, thus reversing its polarity from negative to positive. This reverse polarity protection significantly increased the isoelectric point of the peptide segment and made it positive under acidic conditions and facilitated the purification. An efficient method to prepare the prolyl peptide thioester required for the synthesis of the (28-65) segment was also developed. These efforts resulted in the total synthesis of the glycosylated IL-2 having full biological activity.

Activation of the protein tyrosine kinase receptors requires the coupling of ligand binding to a change in both the proximity and orientation of the single transmembrane (TM) helices of receptor monomers to allow transphosphorylation of the receptor kinase domain. We make use of peptides corresponding to the TM and juxtamembrane (JM) regions of the fibroblast growth factor receptor 3 to assess how mutations in the TM region (G380R and A391E), which lead to receptor activation, influence the orientation of the TM domain and interactions of the intracellular JM sequence with the membrane surface. On the basis of fluorescence and Fourier transform infrared spectroscopy, we find that both activating mutations change the TM helix tilt angle relative to the membrane normal and release the JM region from the membrane. These results suggest a general mechanism regarding how the TM–JM region functionally bridges the extracellular and intracellular regions for these receptors.

Protein engineering has been used to remodel pores for applications in biotechnology. For example, the heptameric α-hemolysin pore (αHL) has been engineered to form a nanoreactor to study covalent chemistry at the single-molecule level. Previous work has been confined largely to the chemistry of cysteine side chains or, in one instance, to an irreversible reaction of an unnatural amino acid side chain bearing a terminal alkyne. Here, we present four different αHL pores obtained by coupling either two or three fragments by native chemical ligation (NCL). The synthetic αHL monomers were folded and incorporated into heptameric pores. The functionality of the pores was validated by hemolysis assays and by single-channel current recording. By using NCL to introduce a ketone amino acid, the nanoreactor approach was extended to an investigation of reversible covalent chemistry on an unnatural side chain at the single-molecule level.

The chemical synthesis of human interleukin-2 (IL-2) ,h aving ac ore 1s ugar,b yaligation method is reported. Although IL-2 is aglobular glycoprotein, its C-terminal region, in particular (99-133), is extremely insoluble when synthesized by solid-phase method. To overcome this problem, the sidechain carboxylic acid of the Glu residues was protected by ap icolyl ester,t hus reversing its polarity from negative to positive.This reverse polarity protection significantly increased the isoelectric point of the peptide segment and made it positive under acidic conditions and facilitated the purification. An efficient method to prepare the prolyl peptide thioester required for the synthesis of the (28-65) segment was also developed. These efforts resulted in the total synthesis of the glycosylated IL-2 having full biological activity.

Ethanolamine ammonia-lyase (EAL) catalyzes the adenosylcobalamin-dependent conversion of ethanolamine to acetaldehyde and ammonia. The wild-type enzyme shows a very low solubility. N-terminal truncation of the Escherichia coli EAL beta-subunit dramatically increases the solubility of the enzyme without altering its catalytic properties. Two deletion mutants of the enzyme [EAL(betaDelta4-30) and EAL(betaDelta4-43)] have been overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method. Crystals of EAL(betaDelta4-30) and EAL(betaDelta4-43) diffracted to approximately 8.0 and 2.1 A resolution, respectively.

HiLdn rO Keita Ainta (g) Kbichi Mbri e Jleisuo 7orava f2)-ontakL VZTywoku a] and loshiki Higuchi fll a Dept ofLije Science Gsaduate hchoelofLde Science IinzL of Flyego 2 Graduate5choolof ,Naniralheiencectnd lechnology OLayama enii) 2P-O13 BXF7;iN:as[lt67:-1itdiMvaN07t-JVFtafie Fold dependency of the amme acia oLcurrentL in thc bet i strand Kasuo Flu-ara Jkromi thdo ctnd ,urlrksamtchJ 1hegT{chi lpepartnlu:t of B;efnfoFmatscs