Three-dimensional structure and function study on the active region in the extracellular ligand-binding domain of human IL-6 receptor

In this study the three-dimensional (3-D) model of the ligand-binding domain (V106-P322) of human interleukin-6 receptor (hIL-6 R) was constructed by computer-guided homology modeling technique using the crystal structure of the ligand-binding domain (K52-L251) of human growth hormone receptor (hGHR) as templet. Furthermore, the active binding region of the 3-D model of hIL-6R with the ligand (hIL-6) was predicted. In light of the structural characteristics of the active region, a hydrophobic pocket shielded by two hydrophilic residues (E115 and E505) of the region was identified by a combination of molecular modelling and the site-directed or double-site mutation of the twelve crucial residues in the ligand-binding domain of hIL-6R (V106-P322). We observed and analyzed the effects of these mutants on the spatial conformation of the pocket-like region of hIL-6 R. The results indicated that any site-directed mutation of the five Cys residues (four conservative Cys residues: Cys121, Cys132, Cys165, Cys176; near membrane Cys residue: Cys193) or each double-site mutation of the five residues in WSEWS motif of hIL-6R (V106-P322) makes the corresponding spatial conformation of the pocket region block the linkage between hIL-6 R and hIL-6. However, the influence of the site-directed mutation of Cys211 and Cys277 individually on the conformation of the pocket region benefits the interaction between hIL-6R and hIL-6. Our study suggests that the predicted hydrophobic pocket in the 3-D model of hIL-6R (V106-P322) is the critical molecular basis for the binding of hIL-6R with its ligand, and the active pocket may be used as a target for designing small hIL-6R-inhibiting molecules in our further study.     

Prediction on the binding domain between human interleukin-6 and its receptor

Based on the spatial conformations of human interleukin-6 (hlL-6) derived from nuclear magnetic resonance analysis and human interleukin-6 receptor (hlL-6R) modeled with homology modeling method using human growth hormone receptor as template, the interaction between hlL-6 and its receptor (hIL-6R) is studied with docking program according to the surface electrostatic potential analysis and spatial conformation complement. The stable region structure composed of hlL-6 and hlL-6R is obtained on the basis of molecular mechanism optimization and molecular dynamics simulation. The binding domain between hIL-6 and hIL-6R is predicted theoretically. Furthermore, the especial binding sites that influence the interaction between hlL-6 and hlL-6R are confirmed. The results lay a theoretical foundation for confirming the active regions of hlL-6 and designing novel antagonist with computer-guided techniques.     

Prediction on the binding domain between human interleukin-6 and its receptor

Based on the spatial conformations of human interleukin-6 (hIL-6) derived from nuclear magnetic resonance analysis and human interleukin-6 receptor (hIL-6R) modeled with homology modeling method using human growth hormone receptor as template, the interaction between hIL-6 and its receptor (hIL-6R) is studied with docking program according to the surface electrostatic potential analysis and spatial conformation complement. The stable region structure composed of hIL-6 and hIL-6R is obtained on the basis of molecular mechanism optimization and molecular dynamics simulation. The binding domain between hIL-6 and hIL-6R is predicted theoretically. Furthermore, the especial binding sites that influence the interaction between hIL-6 and hIL-6R are confirmed. The results lay a theoretical foundation for confirming the active regions of hIL-6 and designing novel antagonist with computer-guided techniques.     

Comparative model building of interleukin-7 using interleukin-4 as a template: a structural hypothesis that displays atypical surface chemistry in helix D important for receptor activation

Using a combination of theoretical sequence structure recognition predictions and experimental disulfide bond assignments, a three-dimensional (3D) model of human interleukin-7 (hIL-7) was constructed that predicts atypical surface chemistry in helix D that is important for receptor activation. A 3D model of hIL-7 was built using the X-ray crystal structure of interleukin-4 (IL-4) as a template (Walter MR et al., 1992, J Mol Biol. 224:1075-1085; Walter MR et al., 1992, J Biol Chem 267:20371-20376). Core secondary structures were constructed from sequences of hIL-7 predicted to form helices. The model was constructed by superimposing IL-7 helices onto the IL-4 template and connecting them together in an up-up down-down topology. The model was finished by incorporating the disulfide bond assignments (Cys3, Cys142), (Cys35, Cys130), and (Cys48, Cys93), which were determined by MALDI mass spectroscopy and site-directed mutagenesis (Cosenza L, Sweeney E, Murphy JR, 1997, J Biol Chem 272:32995-33000). Quality analysis of the hIL-7 model identified poor structural features in the carboxyl terminus that, when further studied using hydrophobic moment analysis, detected an atypical structural property in helix D, which contains Cys 130 and Cys142. This analysis demonstrated that helix D had a hydrophobic surface exposed to bulk solvent that accounted for the poor quality of the model, but was suggestive of a region in IL-7 that maybe important for protein interactions. Alanine (Ala) substitution scanning mutagenesis was performed to test if the predicted atypical surface chemistry of helix D in the hIL-7 model is important for receptor activation. This analysis resulted in the construction, purification, and characterization of four hIL-7 variants, hIL-7(K121A), hIL-7(L136A), hIL-7(K140A), and hIL-7(W143A), that displayed reduced or abrogated ability to stimulate a murine IL-7 dependent pre-B cell proliferation. The mutant hIL-7(W143A), which is biologically inactive and displaces [125I]-hIL-7, is the first reported IL-7R system antagonist.     

The effects of mutations in the carboxyl-terminal region on the catalytic activity of Escherichia coli signal peptidase I

Escherichia coli signal peptidase I (SPase I) is a membrane-bound serine endopeptidase that catalyses the cleavage of signal peptides from the pre-forms of membrane or secretory proteins. Our previous studies using chemical modification and site-directed mutagenesis suggested that Trp(300) and Arg(77), Arg(222), Arg(315) and Arg(318) are important for the proper and stable conformation of the active site of SPase I. Interestingly, many of these residues reside in the C-terminal region of the enzyme. As a continuation of these studies, we investigated in the present study the effects of mutations in the C-terminal region including amino acid residues at positions from 319 to 323 by deletions and site-directed mutagenesis. As a result, the deletion of the C-terminal His(323) was shown to scarcely affect the enzyme activity of SPase I, whereas the deletion of Gly(321)-His(323) or Ile(319)-His(323) as well as the point mutation of Ile(322) to alanine was shown to decrease significantly both the activity in vitro and in vivo without a big gross conformational change in the enzyme. These results suggest a significant contribution of Ile(322) to the construction and maintenance of the proper and critical local conformation backing up the active site of SPase I.     

Involvement of cysteine 306 and alanine 63 in the thermostability and oligomeric organization of glucose isomerase from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. SK

The implication of the original alanine 63 (Ala63) and the unique cysteine 306 (Cys306) residues in the thermostability of the Streptomyces sp. SK glucose isomerase (SKGI) were investigated by site-directed mutagenesis and homology modelling. The Cys306 to Ala mutation within SKGI dramatically affected its thermal stability by decreasing the half-life from 80 to 15 min at 90°C while the Ala63 to Ser replacement shifted this half-life to 65 min. The electrophoretic analysis proves that the residue Cys306 participates in oligomerization of the SKGI. Its stabilizing role is materialized by hydrogen bonds established with arginines at positions 284 and 259, as deduced from the constructed three-dimensional model. We have also shown that the presence of an Ala63 instead of Ser63 seems to be more suitable for enzyme thermostability by maintaining hydrophobic pocket that contributes to the protection of the enzyme active site.     

Non-active site residues Cys69 and Asp150 affected the enzymatic properties of glutathione S-transferase AdGSTD3-3

To elucidate how non-active site residues support the catalytic function, five selected residues of AdGSTD3-3 isoenzyme were changed to AdGSTD1-1 residues by means of site-directed mutagenesis. Analysis of the kinetic parameters indicated that Cys69Gln and Asp150Ser showed marked differences in Vmax and Km compared with the wild type enzyme. Both residues were characterized further by replacement with several amino acids. Both the Cys69 and Asp150 mutants showed differences with several GST substrates and inhibitors including affecting the interactions with pyrethroid insecticides. Cys69 and Asp150 mutants possessed a decreased half-life relative to the wild type enzyme. The Asp150 mutation appears to affect neighboring residues that support two important structural motifs, the N-capping box and the hydrophobic staple motif. The Cys69 mutants appeared to have subtle conformational changes near the active site residues resulting in different conformations and also directly affecting the active site region. The results show the importance of the cumulative effects of residues remote from the active site and demonstrate that minute changes in tertiary structure play a role in modulating enzyme activity.     

Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila melanogaster

The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acid-binding residue within the ligand-binding pocket, 1323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein.     

A Charge-inverting Mutation in the “Linker” Region of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Alters Agonist Binding and Gating Kinetics Independently of Allosteric Modulators

AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728–4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.     

Molecular modeling, affinity labeling, and site-directed mutagenesis define the key points of interaction between the ligand-binding domain of the vitamin D nuclear receptor and 1 alpha,25-dihydroxyvitamin D3

We have combined molecular modeling and classical structure-function techniques to define the interactions between the ligand-binding domain (LBD) of the vitamin D nuclear receptor (VDR) and its natural ligand, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)]. The affinity analogue 1alpha,25-(OH)(2)D(3)-3-bromoacetate exclusively labeled Cys-288 in the VDR-LBD. Mutation of C288 to glycine abolished this affinity labeling, whereas the VDR-LBD mutants C337G and C369G (other conserved cysteines in the VDR-LBD) were labeled similarly to the wild-type protein. These results revealed that the A-ring 3-OH group docks next to C288 in the binding pocket. We further mutated M284 and W286 (separately creating M284A, M284S, W286A, and W286F) and caused severe loss of ligand binding, indicating the crucial role played by the contiguous segment between M284 and C288. Alignment of the VDR-LBD sequence with the sequences of nuclear receptor LBDs of known 3-D structure positioned M284 and W286 in the presumed beta-hairpin of the molecule, thereby identifying it as the region contacting the A-ring of 1alpha, 25-(OH)(2)D(3). From the multiple sequence alignment, we developed a homologous extension model of the VDR-LBD. The model has a canonical nuclear receptor fold with helices H1-H12 and a single beta hairpin but lacks the long insert (residues 161-221) between H2 and H3. We docked the alpha-conformation of the A-ring into the binding pocket first so as to incorporate the above-noted interacting residues. The model predicts hydrogen bonding contacts between ligand and protein at S237 and D299 as well as at the site of the natural mutation R274L. Mutation of S237 or D299 to alanine largely abolished ligand binding, whereas changing K302, a nonligand-contacting residue, to alanine left binding unaffected. In the "activation" helix 12, the model places V418 closest to the ligand, and, consistent with this prediction, the mutation V418S abolished ligand binding. The studies together have enabled us to identify 1alpha,25-(OH)(2)D(3)-binding motifs in the ligand-binding pocket of VDR.     

Amphipathic property of free thiol group contributes to an increase in the catalytic efficiency of carboxypeptidase Y.

Cys341 of carboxypeptidase Y, which constitutes one side of the solvent-accessible surface of the S1 binding pocket, was replaced with Gly, Ser, Asp, Val, Phe or His by site-directed mutagenesis. Kinetic analysis, using Cbz-dipeptide substrates, revealed that polar amino acids at the 341 position increased K(m) whereas hydrophobic amino acids in this position tended to decrease K(m). This suggests the involvement of Cys341 in the formation of the Michaelis complex in which Cys341 favors the formation of hydrophobic interactions with the P1 side chain of the substrate as well as with residues comprising the surface of the S1 binding pocket. Furthermore, C341G and C341S mutants had significantly higher k(cat) values with substrates containing the hydrophobic P1 side chain than C341V or C341F. This indicates that the nonhydrophobic property conferred by Gly or Ser gives flexibility or instability to the S1 pocket, which contributes to the increased k(cat) values of C341G or C341S. The results suggest that Cys341 may interact with His397 during catalysis. Therefore, we propose a dual role for Cys341: (a) its hydrophobicity allows it to participate in the formation of the Michaelis complex with hydrophobic substrates, where it maintains an unfavorable steric constraint in the S1 subsite; (b) its interaction with the imidazole ring of His397 contributes to the rate enhancement by stabilizing the tetrahedral intermediate in the transition state.     

Clusters of transmembrane residues are critical for human prostacyclin receptor activation

Relaxation of vascular smooth muscle and prevention of blood coagulation are mediated by ligand-induced activation of the human prostacyclin (hIP) receptor, a seven-transmembrane-domain G-protein-coupled receptor (GPCR). In this study, we elucidate the molecular requirements for receptor activation within the region of the ligand-binding pocket, identifying transmembrane residues affecting potency. Eleven of 30 mutated residues in the region of the ligand-binding domain exhibited defective activation (decreased potency). These critical residues localized to four distinct clusters (analysis via a rhodopsin-based human prostacyclin receptor homology model). Residues Y75(2.65) (TMII), F95(3.28) (TMIII), and R279(7.40) (TMVII) comprised the immediate binding-pocket cluster and were shown to be essential for proper receptor activation, compared to equivalent expression levels of the wild-type hIP (WT EC(50) = 1.2 +/- 0.1 nM; Y75(2.65)A EC(50) = 347.3 +/- 62.8 nM, p < 0.001; F95(3.28)A EC(50) = 8.0 +/- 0.6 nM, p < 0.001; R279(7.40)A EC(50) = 130 +/- 63.0 nM, p < 0.001). Residues S20(1.39) (TMI), F24(1.43) (TMI), and F72(2.62) (TMII) were localized to a cluster involving P17(1.36), a critical residue thought to facilitate transmembrane movement during changes in activation conformation. A third cluster formed around amino acid D60(2.50) (TMII), containing the highly conserved (100% of prostanoid receptors) D288(7.49)/P289(7.50) motif located in TMVII. Last, a large hydrophobic cluster composed of aromatic residues F146(4.52) (TMIV), F150(4.56) (TMIV), F184(5.40) (TMV), and Y188(5.44) (TMV) was observed away from the ligand-binding pocket, but still necessary for hIP activation. These results assist in delineating the potential molecular requirements for agonist-induced signaling through the transmembrane domain. Such observations may be generally applicable, as many of these clusters are highly conserved among the prostanoid receptors as well as other class A GPCRs.     

Site-directed modification of the adenylation domain of the fusaricidin nonribosomal peptide synthetase for enhanced production of fusaricidin analogs

Fusaricidins produced by Paenibacillus polymyxa DBB1709 are lipopeptide antibiotics active against fungi and Gram-positive bacteria. The cyclic hexapeptide structures of fusaricidins are synthesized by fusaricidin synthetase, a non-ribosomal peptide synthetase. The adenylation domain of the third module (FusA-A3) can recruit L: -Tyr, L: -Val, L: -Ile, L: -allo-Ile, or L: -Phe, which diversifies the fusaricidin structures. Since the L: -Phe-incorporated fusaricidin analog (LI-F07) exhibits more potent antimicrobial activity than other analogs, we modified a specificity-conferring sequence in the substrate binding pocket of FusA-A3 to direct the enhanced production of LI-F07. Base on comparison to the adenylation domain of gramicidin S synthetase 1 and tyrocidine synthetase 1, both of which mainly activate L: -Phe, six mutant strains with altered FusA-A3 were generated using site-directed mutagenesis. M3 (I239W, I299V), M5 (I299V, G322A, V330I), and M6 (S239W, I299V, G322A, V330I) mutants produced significantly more LI-F07 than the wild-type strain.     

Structural basis of the auto-inhibition mechanism of nonreceptor tyrosine kinase PTK6

Protein tyrosine kinase 6 (PTK6) is composed of SH3, SH2, and Kinase domains, with a linker region (Linker) between the SH2 and Kinase domains. Here, we report the structural basis of the SH3-Linker interaction that results in auto-inhibition of PTK6. The solution structures of the SH3 domain and SH3/Linker complex were determined by NMR spectroscopy. The structure of the SH3 domain forms a conventional β-barrel with two β-sheets comprised of five β-strands. However, the molecular topology and charge distribution of PTK6-SH3 slightly differs from that of the other SH3 domains. The structure of the N-terminal Linker within the complex showed that the proline-rich region (P175-P187) of the Linker forms a compact hairpin structure through hydrophobic interactions. The structure of the SH3/Linker complex revealed intra-molecular interaction between the amino acid pairs R22/E190, W44/W184, N65/P177, and Y66/P179. Mutations in PTK6 at R22, W44, N65, and Y66 residues in the SH3 domain increased catalytic activity compared with wild-type protein, implying that specific interactions between hydrophobic residues in the proline-rich linker region and hydrophobic residues in the SH3 domain are mainly responsible for down-regulating the catalytic activity of PTK6.     

Difference in nucleocytoplasmic shuttling sequences of rat and human constitutive active/androstane receptor

Fluorescence recovery after photobleaching (FRAP) in spontaneous multinuclear cells shows that both rat and human constitutive active/androstane receptors (CARs) are shuttling proteins with both nuclear localization signals (NLSs) and nuclear export signals (NESs). We previously identified two NLSs in rat CAR: NLS1 in the hinge region (residues 100-108) and NLS2 in the ligand-binding domain (residues 111-320). In the present study, we compared the intracellular localization signals between rat and human CARs. There was a marked difference in their intracellular localization in COS-7 cells because, unlike rat CAR, human CAR does not contain NLS1 due to an amino acid change at position 106. A CRM1-dependent leucine-rich NES, which is sensitive to an inhibitory effect of leptomycin B, was found in the cytoplasmic retention region previously identified within the ligand-binding domain of rat CAR (residues 220-258). We found that human CAR instead has a NES in the ligand-binding domain between residues 170 and 220. Also, we detected CRM1-independent C-terminal NESs between residues 317-358 of rat and human CARs. Removal of NLS1 by N-terminal truncation and mutation of xenochemical response signal caused rat CAR to localize in the cytoplasm of COS-7 cells, which we suspect is due to the masking of NLS2.     

The steady-state kinetics of the enzyme reaction tested by site-directed mutagenesis of hydrophobic residues (Val, Leu, and Cys) in the C-terminal alpha-helix of human adenylate kinase

To elucidate whether the C-terminal region in human adenylate kinase participates in the interaction with the substrate (MgATP(2-) and/or AMP(2-)), hydrophobic residues (Val182, Val186, Cys187, Leu190, and Leu193) were substituted by site-directed mutagenesis and the steady-state kinetics of fifteen mutants were analyzed. A change in the hydrophobic residues in the C-terminal domain affects the affinity for substrates (K(m)), that is, not only for MgATP(2-) but also for AMP(2-), and the catalytic efficiency (k(cat)). The results obtained have led to the following conclusions: (i) Val182 may interact with both MgATP(2-) and AMP(2-) substrates, but to a greater extent with MgATP(2-), and play a role in catalysis. (ii) Val186 appears to play a functional role in catalysis by interacting with both MgATP(2-) and AMP(2-) to nearly the same extent. (iii) Cys187 appears to play a functional role in catalysis. (iv) Leu190 appears to interact with both MgATP(2-) and AMP(2-) substrates but to a greater extent with AMP(2-). (v) Leu193 appears to interact with both MgATP(2-) and AMP(2-) but to a greater extent with AMP(2-). The activity of all mutants decreased due to the change in substrate-affinity. The closer the residue is located to the C-terminal end, the more its mutation affects not only MgATP(2-) but also AMP(2-) substrate binding. The hydrophobic alterations disrupt hydrophobic interactions with substrates and that might destabilize the conformation of the active site. The more C-terminal part of the alpha-helix appears to interact with AMP, as if it has swung out and rotated to cover the adenine moieties. The C-terminal alpha-helix of human adenylate kinase appears to be essential for the interaction with adenine substrates by swinging out during catalysis.