Role of leucine residues in the C-terminal region of human interleukin-6 in the biological activity.

Site-directed mutagenesis of two sets of three periodic leucine residues which appear at every seventh position in the C-terminal region of human interleukin-6 (IL-6) was performed. Both receptor-binding and immunoglobulin (Ig)-induction activities of a triple mutant Leu168,175,182-->Val were only 1% compared with those of wild-type IL-6. However, the mutant Leu152,159,166-->Val had 13% receptor-binding and 2% Ig-induction activities of those of wild-type IL-6. In order to obtain more direct information on the receptor-binding region, we prepared two synthetic peptides. A significant binding activity was observed for the peptide Leu168-Met185, but not for the peptide Leu152-Arg169. These results indicate that leucine residues in the C-terminal region, especially Leu168, Leu175, and Leu182, play an important role in the receptor-binding and Ig-induction activities.     

Cyclic peptide interleukin 5 antagonists mimic CD turn recognition epitope for receptor alpha

The cyclic peptide AF17121 (Ac-VDECWRIIASHTWFCAEE) that inhibits interleukin 5 (IL-5) function and IL-5 receptor alpha-chain (IL-5Ralpha) binding has been derived from recombinant random peptide library screening and follow-up synthetic variation. To better understand the structural basis of its antagonist activity, AF17121 and a series of analogs of the parent peptide were prepared by solid phase peptide synthesis. Sequence variation was focused on the charged residues Asp(2), Glu(3), Arg(6), Glu(17), and Glu(18). Two of those residues, Glu(3) and Arg(6), form an EXXR motif that was found to be common among library-derived IL-5 antagonists. The E and R in the EXXR motif have a proximity similar to charged residues in a previously identified receptor alpha binding region, the beta-strand between the C- and D-helices of human IL-5. Optical biosensor interaction kinetics and cell proliferation assays were used to evaluate the antagonist activities of the purified synthetic peptides, by measuring competition with the highly active single chain IL-5. Analogs in which acidic residues (Asp(2), Glu(3), Glu(17), and Glu(18)) were replaced individually by Ala retained substantial competition activity, with multiple replacements in these residues leading to fractional loss of potency at most. In contrast, R6A analogs had strongly reduced competition activity. The results reveal that the arginine residue is crucial for the IL-5Ralpha binding of AF17121, while the acidic residues are not essential though likely complex-stabilizing particularly in the Asp(2)-Glu(3) region. By CD, AF17121 exhibited mostly disordered structure with evidence for a small beta-sheet content, and replacement of the arginine had no influence on the observed secondary structure of the peptides. The dominance of Arg(6) in AF17121 activity corresponds to previous findings of dominance of the positive charge balance in the antiparallel beta-sheet of IL-5 composed of (88)EERRR(92) in one strand of the CD turn region of IL-5 and with Arg(32) in the neighboring beta-strand. These results argue that AF17121 and related library-derived peptides function by mimicking the CD turn receptor alpha recognition epitope in IL-5 and open the way to small molecule antagonist design.     

Crystallographic structure of a rheumatoid arthritis MHC susceptibility allele, HLA-DR1 (DRB1*0101), complexed with the immunodominant determinant of human type II collagen

The expression of HLA-DR1 (DRB1*0101) is associated with an enhanced risk for developing rheumatoid arthritis (RA). To study its function, we have solved the three-dimensional structure of HLA-DR1 complexed with a candidate RA autoantigen, the human type II collagen peptide CII (259-273). Based on these structural data, the CII peptide is anchored by Phe263 at the P1 position and Glu266 at P4. Surprisingly, the Lys at the P2 position appears to play a dual role by participating in peptide binding via interactions with DRB1-His81 and Asn82, and TCR interaction, based on functional assays. The CII peptide is also anchored by the P4 Glu266 residue through an ionic interaction with DRB1-Arg71 and Glu28. Participation of DRB1-Arg71 is significant because it is part of the shared epitope expressed by DR alleles associated with RA susceptibility. Potential anchor residues at P6 and P9 of the CII peptide are both Gly, and the lack of side chains at these positions appears to result in both a narrower binding groove with the peptide protruding out of the groove at this end of the DR1 molecule. From the TCR perspective, the P2-Lys264, P5-Arg267, and P8-Lys270 residues are all oriented away from the binding groove and collectively represent a positive charged interface for CII-specific TCR binding. Comparison of the DR1-CII structure to a DR1-hemagglutinin peptide structure revealed that the binding of these two peptides generates significantly different interfaces for the interaction with their respective Ag-specific TCRs.     

Expression and purification of recombinant neurotensin in Escherichia coli

An expression system has been designed for the rapid and economic expression of recombinant neurotensin for biophysical studies. A synthetic gene for neurotensin (Glu(1)-Leu(2)-Tyr(3)-Glu(4)-Asn(5)-Lys(6)-Pro(7)-Arg(8)-Arg(9)-Pro(1 0)-Tyr(11)-Ile(12)-Leu(13)) was cloned into the pGEX-5X-2 vector to allow expression of neurotensin as a glutathione S-transferase (GST) fusion protein. The inclusion of a methionine residue between the glutathione S-transferase and the neurotensin has facilitated the rapid cleavage of the neurotensin from its carrier protein. Purification of recombinant neurotensin was performed by reverse-phase HPLC. This method produced a relatively high yield of peptide and offers the potential for economic partial or uniform labeling of small peptides (<15 amino acids) with isotopes for NMR or other biophysical techniques.     

Epitope randomization redefines the functional role of glutamic acid 110 in interleukin-5 receptor activation

Sequence randomization through functional phage display of single chain human interleukin (IL)-5 was used to investigate the limits of replaceability of the Glu(110) residues that form a part of the receptor-binding epitope. Mutational analysis revealed unexpected affinity for IL-5 receptor alpha chain with variants containing E110W or E110Y. Escherichia coli-expressed Glu(110) variants containing E110W in the otherwise sequence-intact N-terminal half, including a variant with an E110A replacement in the sequence-disabled C-terminal half, were shown by their CD spectra to be folded into secondary structures similar to that of single chain human IL-5 (scIL-5). Biosensor kinetics analysis revealed that (E110W/A5)scIL-5 and (E110W/A6)scIL-5 had receptor alpha chain binding affinities similar to that of (wt/A5)scIL-5. However, (E110W/A6)scIL-5 had a significantly reduced bioactivity in TF-1 cell proliferation compared with both (wt/A5)scIL-5 and (E110W/A5)scIL-5, and this activity reduction was disproportionately greater than the much smaller effect of Glu(110) mutation on receptor binding affinity. The marked and disproportionate decrease in TF-1 proliferation observed with (E110W/A6)scIL-5 suggests a role for Glu(110) in the biological activity mediated by the signal transducing receptor betac subunit of the IL-5 receptor. This is also consistent with the lack of stimulation of JAK2 phosphorylation by the (E110W/A6)scIL-5 mutant in recombinant 293T cells, as compared with the concentration-dependent stimulation seen for scIL-5. The results reveal the dispensability of charge in the Glu(110) locus of IL-5 for receptor alpha chain binding and, in contrast, its heretofore underappreciated importance for receptor activation.     

G alpha i-G alpha s chimeras define the function of alpha chain domains in control of G protein activation and beta gamma subunit complex interactions

Gs and Gi2 are G proteins whose alpha subunits are 65% homologous. Within the 355 amino acid alpha i2 polypeptide, substitution of residues Ile213-Lys319 with the corresponding alpha s region (Ile235-Arg356) generated a chimera that activated adenylyl cyclase, indicating that the alpha s activation domain resides within this 122 amino acid alpha s sequence. Mutation within alpha s residues Glu15-Pro144 resulted in an alpha s polypeptide having an enhanced rate of GDP dissociation. Mutation within two regions of the N-terminus influenced the ability of pertussis toxin to ADP-ribosylate the alpha subunit polypeptide, a reaction controlled by the beta gamma subunit complex. The findings define the G protein alpha subunit N-terminus as a regulatory region controlling beta gamma subunit interactions and GDP dissociation independent of the GTPase and effector activation domains.     

A receptor-binding region in Escherichia coli alpha-haemolysin

Escherichia coli alpha-hemolysin (HlyA) is a 107-kDa protein toxin with a wide range of mammalian target cells. Previous work has shown that glycophorin is a specific receptor for HlyA in red blood cells (Cortajarena, A. L., Go?i, F. M., and Ostolaza, H. (2001) J. Biol. Chem. 276, 12513-12519). The present study was aimed at identifying the glycophorin-binding region in the toxin. Data in the literature pointed to a short amino acid sequence near the C terminus as a putative receptor-binding domain. Previous sequence analyses of several homologous toxins that belong, like HlyA, to the so-called RTX toxin family revealed a conserved region that corresponded to residues 914-936 of HlyA. We therefore prepared a deletion mutant lacking these residues (HlyA Delta 914-936) and found that its hemolytic activity was decreased by 10,000-fold with respect to the wild type. This deletion mutant was virtually unable to bind human and horse red blood cells or to bind pure glycophorin in an affinity column. The peptide Trp914-Arg936 had no lytic activity of its own, but it could bind glycophorin reconstituted in lipid vesicles. Moreover, the peptide Trp914-Arg936 protected red blood cells from hemolysis induced by wild type HlyA. It was concluded that amino acid residues 914-936 constitute a major receptor-binding region in alpha-hemolysin.     

Molecular analysis of dibasic endoproteolytic cleavage signals.

Biologically active peptide hormones are synthesized from larger precursor proteins by a variety of post-translational processing reactions. To characterize these processing reactions further we have expressed preprogastrin in two endocrine cell lines and examined the molecular determinants involved in endoproteolysis at dibasic cleavage sites. The Gly93-Arg94-Arg95 carboxyl-terminal processing site of progastrin must be processed sequentially by an endoprotease, a carboxypeptidase, and an amidating enzyme to produce bioactive gastrin. For these studies the dibasic Arg94-Arg95 residues that serve as signals for the initiation of this processing cascade were mutated to Lys94-Arg95, Arg94-Lys95, and Lys94-Lys95. In the GH3 cells the Lys94-Arg95 mutation slightly diminished synthesis of carboxyl-terminally amidated gastrin, whereas in the MTC 6-23 cells this mutation had no effect on amidated gastrin synthesis. In contrast, both Arg94-Lys95 and Lys94-Lys95 mutations resulted in significantly diminished production of amidated gastrin in both cell lines. A specific hierarchy of preferred cleavage signals at this progastrin processing site was demonstrated in both cell lines, indicating that cellular dibasic endoproteases have stringent substrate specificities. Progastrins with the Lys94-Arg95 mutation in GH3 cells also demonstrated diminished processing at the Lys74-Lys75 dibasic site, thus single amino acid changes at one processing site may alter cleavage at distant sites. These studies provide insight into the post-translational processing and biological activation of not only gastrin but other peptide hormones as well.     

Structural characterization of lipopeptide agonists for the bradykinin B2 receptor

The conformational features of Pam-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PKD) and Pam-Gly(-1)-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PGKD), the Pam-Lys and Pam-Gly-Lys analogues of bradykinin, have been determined by high-resolution NMR in a zwitterionic lipoid environment. Radical-induced relaxation of the (1)H NMR signals was used to probe the topological orientation of the peptides with respect to the zwitterionic lipid interface. The radical-induced relaxation and molecular dynamics (MD) data indicated that the palmitic acid and N-terminal amino acid residues embed into the micelles, while the rest of the polypeptide chain is closely associated with the water-micelle interface. Throughout the entire nuclear Overhauser effect restrained MD simulation, a nonideal type I beta-turn was observed in the C-terminus of PKD between residues 6 and 9, and a gamma-turn was observed in the C-terminus of PGKD between residues 6 and 7. Therefore, the additional glycine has a dramatic effect on the structural preferences of the biologically important C-terminus, an effect brought about by the interaction with the lipid environment. These structural features are correlated to the biological activity at the bradykinin B2 receptor.     

Calmodulin fragments can not activate target enzymes

Under conditions where nM level of calmodulin was able to show full activation of myosin light chain kinase and cyclic-nucleotide phosphodiesterase, the fragments of calmodulin at concentrations as high as 20 microM failed to activate these enzymes in the presence of Ca2+. The fragments tested were Ala1-Lys75 (F12), Ala1-Arg74 (F12'), Lys75-Lys148 (F34'), Met76-Lys148 (F34'), Asp78-Lys148 (F34), Ala1-Arg106 (F123), and His107-Lys148 (F4). Purification of the proteolytic fragments through HPLC was necessary to remove contaminant calmodulin. Among the fragments, that corresponding to the C-terminal half domain inhibited myosin light chain kinase activity with the inhibition constant of 13 microM. The integrated structure of calmodulin consisting of N-terminal half domain, C-terminal half domain, and the linker peptide was indispensable for the enzyme activation. We discuss the functions of the two structural domains (N-domain and C-domain) in the activation of various enzymes.     

Type I beta-turn conformation is important for biological activity of the melanocyte-stimulating hormone analogues.

In order to define which structure of alpha-melanocyte-stimulating hormone (MSH) analogues plays a critical role for ligand-receptor interaction and selectivity, we analysed receptor-binding and cAMP-generating activity in Chinese hamster ovary cell lines stably transfected with rMC3R and hMC4R, as well as the NMR structures of chemically synthesized alpha-MSH analogues. Compared with [Ahx4]alpha-MSH, the linear MTII designated as alpha-MSH-ND revealed a preference for the MC4R, whereas its IC50 and EC50 values were comparable to those of MTII reported previously. Truncation of Ahx4 and Asp5 of alpha-MSH-ND remarkably decreased the receptor-binding and cAMP-generating activity. Meanwhile, maximum cAMP-generating activity was observed at a higher concentration (10(-5) M) of alpha-MSH-ND(6-10), and MC4R preference was changed into MC3R preference. In contrast, [Gln6]alpha-MSH-ND(6-10) lost its cAMP-generating activity almost completely, even though it bound to both receptors. Whereas the solution conformation of alpha-MSH-ND revealed a stable type I beta-turn structure, [Gln6]alpha-MSH-ND(6-10) revealed a tight gamma-turn composed of Gln6-D-Phe7-Arg8. Replacement of the His6 residue of alpha-MSH-ND by Gln, Asn, Arg or Lys decreased not only the receptor binding, but also the cAMP-generating activity in both the MC3R and the MC4R. The structure of [Gln6]alpha-MSH-ND exhibited a stable type I' beta-turn comprising Asp5, Gln6, D-Phe7 and Arg8. [Lys6]alpha-MSH-ND showed a greatly reduced binding affinity and cAMP-generating activity with the loss of MC4R selectivity. In NMR studies, [Lys6]alpha-MSH-ND also demonstrated a gamma-turn conformation around Lys6-DPhe7-Arg8. From the above results, we conclude that a type I beta-turn conformation comprising the residues Asp5-His6-(D-Phe7)-Arg8 was important for receptor binding and activation, as well as the selectivity of MSH analogues.     

Toward understanding the role of insulin alpha-helix II in the growth-promoting activity of insulin.

For further understanding the contribution of the alpha-helix II (alphaII) in the growth-promoting activity of insulin, the residues A2Ile, A5Gln, and A8Thr located in alphaII were mutated to Leu, Glu, and Tyr, respectively. Three mutant insulins, [A2Leu]human insulin, [A5Glu]human insulin, and [A8Tyr]human insulin, were prepared by means of site-directed mutagenesis. The in vitro growth-promoting activities of the three mutant insulins, measured using GR2H6 cells, were 7.5%, 291%, and 250% of that of native insulin, respectively. Their receptor-binding activities to the insulin receptor were 2.3%, 46.7%, and 138.7%, respectively, compared with native insulin. Both the growth-promoting and receptor-binding activities of [A2Leu]human insulin and [A3Leu]insulin (Shi et al., 1997) were parallel and greatly decreased compared with native insulin. The results demonstrate that the residues A2Ile and A3Val in the alphaII are essential for the growth-promoting activity of insulin, and the growth-promoting function of insulin might be performed through, or mainly through, binding to the insulin receptor. The growth-promoting activities of [A5Glu]human insulin and [A8Tyr]human insulin were increased 6-fold and 2-fold, respectively, compared with native insulin, indicating that their growth-promoting activities might be expressed by, or mainly by, binding to the IGF-1 receptor.     

Monitoring the structural consequences of Phe12-->D-Phe and Leu15-->Aib substitution in human/rat corticotropin releasing hormone. Implications for design of CRH antagonists.

A new human/rat CRH analogue has been synthesized using the Fmoc/tBu solid-phase synthetic protocol. The sequence of the new peptide differs from the original in two positions, 12 and 15, at which the native amino acids l-phenylalanine 12 and l-leucine 15 have been replaced by the nonprotein amino acids d-phenylalanine and alpha-aminoisobutyric acid (Aib), respectively. The high resolution three-dimensional solution structure of [d-Phe12, Aib15]CRH has been determined by 688 distance constraints (656 meaningful NOE and 32 H-bonds distance limits) and 21 angle constraints. A family of 40 energy-minimized conformers was obtained with average rmsd of 0.39 +/- 0.16 A and 0.99 +/- 0.13 A for backbone and heavy atoms, respectively, and distance penalty functions of 0.42 +/- 0.03 A2. The NMR data acquired in a solvent system of water/trifluoroethanol (34%/66%, v/v) revealed that this 41-polypeptide adopts an almost linear helical structure in solution with helical content which reaches an 84% of the residues. Structural analysis confirmed the existence of two helical peptide fragments. The first was comprised of residues Ile6-Arg16 and the second of residues Glu20-Ile40, forming an angle of 34.2 degrees. The structural differences with respect to the native peptide have been identified in the region d-Phe12-Glu20 where double substitution at positions 12 and 15 seems to perturb the elements of the native 35-residue helix. These structural rearrangements promote non-native intramolecular interactions in the region of the molecule between either the hydrophobic side-chains of d-Phe12, Aib15 and Leu18, or the charged groups of the residue pairs Arg16-Glu20 and His13-Glu17 being responsible for changes in hormonal functionality. This CRH analogue currently exhibits lack of any activity.     

Novel topology in C-terminal region of the human plasma membrane anion exchanger,AE1

Human AE1 performs electroneutral exchange of Cl(-) for HCO(3)(-) across the erythrocyte membrane. We examined the topology of the AE1 C-terminal region using cysteine-scanning mutagenesis and sulfhydryl-specific chemistry. Eighty individual cysteine residues, introduced into an otherwise cysteine-less mutant between Phe(806) and Cys(885), were expressed by transient transfection of HEK293 cells. Topology of the region was determined by comparing cysteine labeling with the membrane-permeant cysteine-directed reagent biotin maleimide, with or without prior labeling with the membrane-impermeant reagents, bromotrimethylammoniumbimane bromide (qBBr) and lucifer yellow iodoacetamide (LYIA). Phe(806)-Leu(835), Ser(852)-Ala(855), and Ile(872)-Cys(885) were labeled by biotin maleimide, suggesting their location in an aqueous environment. In contrast, Phe(836)-Lys(851) and Ser(856)-Arg(871) were not labeled by biotin maleimide and therefore localize to the plane of the bilayer, as transmembrane segments (TM). Labeling by qBBr revealed that Pro(815)-Lys(829) and Ser(852)-Ala(855) are accessible to the extracellular medium. Pro(815)-Lys(829) mutants were also labeled with LYIA. Mutants Ile(872)-Cys(885) were inaccessible to the extracellular medium and thus localized to the intracellular surface of AE1. Functional assays revealed that one face of each of two AE1 TMs was sensitive to mutation. Based on these results, we propose a topology model for the C-terminal region of the membrane domain of human AE1.     

Structural changes in the C-terminus of Ca2+-bound rat S100B (beta beta) upon binding to a peptide derived from the C-terminal regulatory domain of p53.

S100B(beta beta) is a dimeric Ca2+-binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C-terminal regulatory domain of p53 has been shown to interact with S100B(beta beta) in a Ca2+-dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca2+-loaded S100B(beta beta) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, 3J(NH-H alpha) coupling constants, and chemical shift index data show that, like apo- and Ca2+-bound S100B(beta beta), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2-Arg20; helix 2, Lys29-Asn38; helix 3, Gln50-Asp61; helix 4, Phe70-Phe87), four loops (loop 1, Glu21-His25; loop 2, Glu39-Glu49; loop 3, Glu62-Gly66; loop 4, Phe88-Glu91), and two beta-strands (beta-strand 1, Lys26-Lys28; beta-strand 2, Glu67-Asp69), which forms a short antiparallel beta-sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo- or Ca2+-bound S100B(beta beta). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca2+-bound S100B(beta beta). Together, these observations plus intermolecular NOE correlations between the p53 peptide and S100B(beta beta) support the notion that the p53 peptide binds in a region of S100B(beta beta), which includes residues in helix 2, helix 3, loop 2, and the C-terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.     

Interaction between the N-terminal and middle regions is essential for the in vivo function of HSP90 molecular chaperone

At the primary structure level, the 90-kDa heat shock protein (HSP90) is composed of three regions: the N-terminal (Met(1)-Arg(400)), middle (Glu(401)-Lys(615)), and C-terminal (Asp(621)-Asp(732)) regions. In the present study, we investigated potential subregion structures of these three regions and their roles. Limited proteolysis revealed that the N-terminal region could be split into two fragments carrying residues Met(1) to Lys(281) (or Lys(283)) and Glu(282) (or Tyr(284)) to Arg(400). The former is known to carry the ATP-binding domain. The fragments carrying the N-terminal two-thirds (Glu(401)-Lys(546)) and C-terminal one-third of the middle region were sufficient for the interactions with the N- and C-terminal regions, respectively. Yeast HSC82 that carried point mutations in the middle region causing deficient binding to the N-terminal region could not support the growth of HSP82-depleted cells at an elevated temperature. Taken together, our data show that the N-terminal and middle regions of the HSP90 family protein are structurally divided into two respective subregions. Moreover, the interaction between the N-terminal and middle regions is essential for the in vivo function of HSP90 in yeast.     

The importance of the N-terminal beta-turn in bradykinin antagonists

Three peptides, B-10148 (Lys-1-Lys0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6- DF5F7-Oic8; where Hyp is trans-4-hydroxyproline, Igl is alpha-(2-indanyl)glycine, F5F is 2,3,4,5,6-pentafluorophenylalanine and Oic is (3aS,7aS)-octahydroindole-2-carboxylic acid), B-10206 (DArg0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6-DF 5F7-Nc7G8-Arg9; where Nc7G is N-cycloheptylglycine) and B- 10284 (Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-DTic7-Oic8- NH2; where Tic is 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), were studied in detail by NMR spectroscopy in 60% CD3OH /40% H2O and modeled by a simulated annealing protocol to determine their solution structure. B-10148, an extremely potent BK B1 receptor antagonist with very high BK B2 receptor antagonist activity, despite lacking a C-terminal Arg, displayed an ideal type II beta-turn from Pro2 to Igl5, as well as a salt bridge between the guanidino group of Arg1 and the carboXylate group of Oic8. B-10206, the most potent B2 antagonist, also displayed an ideal type II beta-turn from Pro2 to Igl5 but secondary structure was not observed at the C-terminal end. The third peptide, B-10284, a des-Arg9 analog with a C-terminal amide and a very potent B2 antagonist, had no definite solution structure. The high activity of these peptides emphasizes the importance of the N-terminal beta-turn and the hydrophobic character at the C-terminus in determining the activity of bradykinin antagonists.     

Proteosynthetic activity of immobilized Staphylococcus aureus V8 protease: application in the semisynthesis of molecular variants of alpha-globin

The proteosynthetic activity of Staphylococcus aureus V8 protease (endoproteinase Glu-C) immobilized onto cross-linked agarose beads by reductive alkylation procedure has been investigated. The overall substrate specificity of the enzyme, as judged by peptide mapping of performic acid oxidized RNase A, as well as the high propensity of the protease to slice selectively the alpha-chain of hemoglobin (Hb) A at the Glu(30)-Arg(31) peptide bond at pH 4.0 and 37 degrees C was essentially unperturbed by the immobilization process. This high susceptibility of Glu(30) of the alpha-chain for proteolysis appears to be a consequence of the conformational aspects of the polypeptide in this region. The proteolysis of two mutant forms of alpha-chain, namely, those of Hb I (K16E) and Hb Sealy (D47H) by immobilized V8 protease at the Glu(30)-Arg(31) peptide bond proceeds with the same selectivity. The immobilized protease also retained the proteosynthetic activity, i.e., the ability to ligate the unprotected alpha-globin fragments at the Glu(30)-Arg(31) peptide bond in the presence of 30% 1-propanol. The use of the insoluble enzyme simplifies the procedures for the construction of new semisynthetic, molecular variants of alpha-globin. The general applicability of the immobilized enzyme for protein semisynthesis has been demonstrated by the construction of a doubly mutated alpha-globin. The complementary fragments from two natural mutant forms of alpha-globin, viz., alpha 1-30 (K16E) from Hb I and alpha 31-141 (D47H) from Hb Sealy, are readily ligated to form the double mutant alpha 1-141 (K16E;D47H).     

Molecular modeling of human BAD and its interaction with PKAc or PP1c

To build up the structure of human BAD (Bcl-2 antagonist of cell death), subsequently combined with PKAc or PP1c (protein phosphatase 1), to investigate the interaction relationship between BAD and its kinase/PTPese at the molecular level. Additionally, it is concerned with the search for all optimal positions and orientations of a set of amino acid residues of BAD, while its binding sites include N-termini (Glu19, Ala27, and Ser34-Lys35), BH3-located helical domain (Arg98-Lys126), and C-termini (Trp154-Ser163 and Ser167-Gln168). The related sites of PKAc are mainly assembled in C-terminal α/β-domain of PKAc, which comprises the KTL motif (47-49), Glu203 residue, a helical region (Asp241-Arg256), and the span from 328 to 333; while the interaction sites with BAD converge at C-terminal β-domain of PP1c, which includes the DEK motif (166-168), the stretch from 179 to 197 including a helix (Glu184-Arg188), Glu230-Asp242 segment containing Val232-His237 helix, and Glu287-Leu289 loop. In conclusion, analysis of the complex between BAD and PKAc or PP1c provides a novel viewpoint on the structural origins of molecular recognition. And the complex models suggest that BH3 domain of BAD interact with PKAc or PP1c by electrostatic, van der Waals contacts, hydrogen bond and salt bridge. This is helpful for our development and research of some new drugs, especially mimetic BH3 peptides and inspires scientists with BAD complex and molecular mechanism of its integrating glycolysis and apoptosis.     

Toll-like receptor 4 region Glu24-Lys47 is a site for MD-2 binding: importance of CYS29 and CYS40

Toll-like receptor 4 (TLR4) is a signaling receptor for lipopolysaccharide (LPS), but its interaction with MD-2 is required for efficient responses to LPS. Previous studies with deletion mutants indicate a critical role of the amino-terminal TLR4 region in interaction with MD-2. However, it is uncertain which region in the TLR4 molecule directly binds to MD-2. The purpose of this study was to determine a critical stretch of primary sequence in the TLR4 region that directly binds MD-2 and is critical for LPS signaling. The synthetic TLR4 peptide corresponding to the TLR4 region Glu(24)-Lys(47) directly binds to recombinant soluble MD-2 (sMD-2). The TLR4 peptide inhibited the binding of a recombinant soluble form of the extracellular TLR4 domain (sTLR4) to sMD-2 and significantly attenuated LPS-induced NF-kappaB activation and IL-8 secretion in wild type TLR4-transfected cells. Reduction and S-carboxymethylation of sTLR4 abrogated its association with sMD-2. The TLR4 mutants, TLR4(C29A), TLR4(C40A), and TLR4(C29A,C40A), were neither co-precipitated with MD-2 nor expressed on the cell surface and failed to transmit LPS signaling. These results demonstrate that the TLR4 region Glu(24)-Lys(47) is a site for MD-2 binding and that Cys(29) and Cys(40) within this region are critical residues for MD-2 binding and LPS signaling.