Role of the conserved acidic residue Asp21 in the structure of phosphatidylinositol 3-kinase Src homology 3 domain: circular dichroism and nuclear magnetic resonance studies

To elucidate a role of the Src homology 3 (SH3)-conserved acidic residue Asp21 of the phosphatidylinositol 3-kinase (PI3K) SH3 domain, structural changes induced by the D21N mutation (Asp21 --> Asn) were examined by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. In the previous study, we demonstrated that environmental alterations occurred at the side chains of Trp55 and some Tyr residues from the comparison of the near-UV CD spectra of the PI3K SH3 domain with or without a D21N mutation [Okishio, N., et al. (2000) Biopolymers 57, 208-217]. In this work, the affected Tyr residues were identified as Tyr14 and Tyr73 by the CD analysis of a series of mutants, in which every single Tyr residue was replaced by a Phe residue with or without a D21N mutation. The (1)H and (15)N resonance assignments of the PI3K SH3 domain and its D21N mutant revealed that significant chemical shift changes occurred to the aromatic side-chain protons of Trp55 and Tyr14 upon the D21N mutation. All these aromatic residues are implicated in ligand recognition. In addition, the NMR analysis showed that the backbone conformations of Lys15-Asp23, Gly54-Trp55, Asn57-Gly58, and Gly67-Pro70 were affected by the D21N mutation. Furthermore, the (15)N[(1)H] nuclear Overhauser effect values of Tyr14, Glu19, and Glu20 were remarkably changed by the mutation. These results show that the D21N mutation causes structural deformation of more than half of the ligand binding cleft of the domain and provide evidence that Asp21 plays an important role in forming a well-ordered ligand binding cleft in cooperation with the RT loop (Lys15-Glu20).     

Three-dimensional model of the extracellular domain of the type 4a metabotropic glutamate receptor: new insights into the activation process

Metabotropic glutamate receptors (mGluRs) belong to the family 3 of G-protein-coupled receptors. On these proteins, agonist binding on the extracellular domain leads to conformational changes in the 7-transmembrane domains required for G-protein activation. To elucidate the structural features that might be responsible for such an activation mechanism, we have generated models of the amino terminal domain (ATD) of type 4 mGluR (mGlu4R). The fold recognition search allowed the identification of three hits with a low sequence identity, but with high secondary structure conservation: leucine isoleucine valine-binding protein (LIVBP) and leucine-binding protein (LBP) as already known, and acetamide-binding protein (AmiC). These proteins are characterized by a bilobate structure in an open state for LIVBP/LBP and a closed state for AmiC, with ligand binding in the cleft. Models for both open and closed forms of mGlu4R ATD have been generated. ACPT-I (1-aminocyclopentane 1,3,4-tricarboxylic acid), a selective agonist, has been docked in the two models. In the open form, ACPT-I is only bound to lobe I through interactions with Lys74, Arg78, Ser159, and Thr182. In the closed form, ACPT-I is trapped between both lobes with additional binding to Tyr230, Asp312, Ser313, and Lys317 from lobe II. These results support the hypothesis that mGluR agonists bind a closed form of the ATDs, suggesting that such a conformation of the binding domain corresponds to the active conformation.     

Holo- and apo-bound structures of bacterial periplasmic heme-binding proteins

An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an "in" position where it can coordinate the heme iron to an "out" orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg(228) in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg(228), and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B(12)-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B(12), compared with ligands for FhuD, a peptide siderophore.     

The structure of the ferric siderophore binding protein FhuD complexed with gallichrome

Siderophore binding proteins play a key role in the uptake of iron in many gram-positive and gram-negative bacteria. FhuD is a soluble periplasmic binding protein that transports ferrichrome and other hydroxamate siderophores. The crystal structure of FhuD from Escherichia coli in complex with the ferrichrome homolog gallichrome has been determined at 1.9 ? resolution, the first structure of a periplasmic binding protein involved in the uptake of siderophores. Gallichrome is held in a shallow pocket lined with aromatic groups; Arg and Tyr side chains interact directly with the hydroxamate moieties of the siderophore. FhuD possesses a novel fold, suggesting that its mechanisms of ligand binding and release are different from other structurally characterized periplasmic ligand binding proteins.     

Crystal structure of a glutamate/aspartate binding protein complexed with a glutamate molecule: structural basis of ligand specificity at atomic resolution

The crystal structure of a periplasmic l-aspartate/l-glutamate binding protein (DEBP) from Shigella flexneri complexed with an l-glutamate molecule has been determined and refined to an atomic resolution of 1.0 Å. There are two DEBP molecules in the asymmetric unit. The refined model contains 4462 non-hydrogen protein atoms, 730 water molecules, 2 bound glutamate molecules, and 2 Tris molecules from the buffer used in crystallization. The final R_cryst and R_free factors are 13.61% and 16.89%, respectively. The structure has root-mean-square deviations of 0.016 Å from standard bond lengths and 2.35° from standard bond angles.The DEBP molecule is composed of two similarly folded domains separated by the ligand binding region. Both domains contain a central five-stranded β-sheet that is surrounded by several α-helices. The two domains are linked by two antiparallel β-strands. The overall shape of DEBP is that of an ellipsoid approximately 55 Å × 45 Å × 40 Å in size.The binding of ligand to DEBP is achieved mostly through hydrogen bonds between the glutamate and side-chain and main-chain groups of DEBP. Side chains of residues Arg24, Ser72, Arg75, Ser90, and His164 anchor the deprotonated γ-carboxylate group of the glutamate with six hydrogen bonds. Side chains of Arg75 and Arg90 form salt bridges with the deprotonated α-carboxylate group, while the main-chain amide groups of Thr92 and Thr140 form hydrogen bonds with the same group. The positively charged α-amino group of the l-glutamate forms salt bridge interaction with the side-chain carboxylate group of Asp182 and hydrogen bond interaction with main-chain carbonyl oxygen of Ser90. In addition to these hydrogen bond and electrostatic interactions, other interactions may also play important roles. For example, the two methylene groups from the glutamate form van der Waals interactions with hydrophobic side chains of DEBP.Comparisons with several other periplasmic amino acid binding proteins indicate that DEBP residues involved in the binding of α-amino and α-carboxylate groups of the ligand and the pattern of hydrogen bond formation between these groups are very well conserved, but the binding pocket around the ligand side chain is not, leading to the specificity of DEBP. We have identified structural features of DEBP that determine its ability of binding glutamate and aspartate, two molecules with different sizes, but discriminating against very similar glutamine and asparagine molecules.     

Role of hydrophobic residues in the C1b domain of protein kinase C delta on ligand and phospholipid interactions

The C1 domains of conventional and novel protein kinase C (PKC) isoforms bind diacylglycerol and phorbol esters with high affinity. Highly conserved hydrophobic residues at or near the rim of the binding cleft in the second cysteine-rich domain of PKC-delta (PKC-deltaC1b) were mutated to probe their roles in ligand recognition and lipid interaction. [(3)H]Phorbol 12,13-dibutyrate (PDBu) binding was carried out both in the presence and absence of phospholipids to determine the contribution of lipid association to the ligand affinity. Lipid dependence was determined as a function of lipid concentration and composition. The binding properties of a high affinity branched diacylglycerol with lipophilicity similar to PDBu were compared with those of PDBu to identify residues important for ligand selectivity. As expected, Leu-20 and Leu-24 strongly influenced binding. Substitution of either by aspartic acid abolished binding in either the presence or absence of phosphatidylserine. Mutation of Leu-20 to Arg or of Leu-24 to Lys caused a dramatic (340- and 250-fold, respectively) reduction in PDBu binding in the presence of lipid but only a modest reduction in the weaker binding of PDBu observed in the absence of lipid, suggesting that the main effect was on C1 domain -phospholipid interactions. Mutation of Leu-20 to Lys or of Trp-22 to Lys had modest (3-fold) effects and mutation of Phe-13 to Tyr or Lys was without effect. Binding of the branched diacylglycerol was less dependent on phospholipid and was more sensitive to mutation of Trp-22 to Tyr or Lys, especially in the presence of phospholipid, than was PDBu. In terms of specific PKC isoforms, our results suggest that the presence of Arg-20 in PKC-zeta may contribute to its lack of phorbol ester binding activity. More generally, the results emphasize the interplay between the C1 domain, ligand, and phospholipid in the ternary binding complex.     

Interdomain salt bridges modulate ligand-induced domain motion of the sulfate receptor protein for active transport.

The refined crystal structure of the liganded form of the Salmonella typhimurium sulfate-binding protein, a periplasmic receptor of active transport, is made up of two globular domains bisected by a deep cleft wherein the dehydrated sulfate is completely engulfed and bound by hydrogen bonds and van der Waals' forces. Two salt bridges (between Glu15 and Arg174 and between Asp68 and Arg134) span the cleft opening. To elucidate the role of the inter-domain salt bridges in the ligand-induced domain motion, the acidic residues were changed (singly and together) to their corresponding amide side-chains by site-directed mutagenesis of the recombinant Escherichia coli sulfate-binding protein. Rapid kinetics and equilibrium measurements of sulfate binding to the purified mutant proteins demonstrate that these salt bridges stabilize the closed liganded form of the receptor and modulate the rate of cleft opening. Our results have new implications in understanding the dynamics of many other multidomain proteins that undergo similar large-scale domain motions.     

Mutational analysis of vaccinia virus topoisomerase identifies residues involved in DNA binding.

Vaccinia DNA topoisomerase catalyzes the cleavage and re-joining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(C/T)CCTT downward arrow. The 314 aa protein consists of three protease-resistant structural domains demarcated by protease-sensitive interdomain segments referred to as the bridge and the hinge. The bridge is defined by trypsin-accessible sites at Arg80, Lys83 and Arg84. Photocrosslinking and proteolytic footprinting experiments suggest that residues near the interdomain bridge interact with DNA. To assess the contributions of specific amino acids to DNA binding and transesterification chemistry, we introduced alanine substitutions at 16 positions within a 24 aa segment from residues 63 to 86(DSKGRRQYFYGKMHVQNRNAKRDR). Assays of the rates of DNA relaxation under conditions optimal for the wild-type topoisomerase revealed significant mutational effects at six positions; Arg67, Tyr70, Tyr72, Arg80, Arg84 and Asp85. The mutated proteins displayed normal or near-normal rates of single-turnover transesterification to DNA. The effects of amino acid substitutions on DNA binding were evinced by inhibition of covalent adduct formation in the presence of salt and magnesium. The mutant enzymes also displayed diminished affinity for a subset of cleavage sites in pUC19 DNA. Tyr70 and Tyr72 were subjected to further analysis by replacement with Phe, His, Gln and Arg. At both positions, the aromatic moiety was important for DNA binding.     

Amino acid mutagenesis of the ligand binding site and the dimer interface of the metabotropic glutamate receptor 1. Identification of crucial residues for setting the activated state

Previously, we determined the crystal structures of the dimeric ligand binding region of the metabotropic glutamate receptor subtype 1. Each protomer binds l-glutamate within the crevice between the LB1 and LB2 domains. We proposed that the two different conformations of the dimer interface between the two LB1 domains define the activated and resting states of the receptor protein. In this study, the residues in the ligand-binding site and the dimer interface were mutated, and the effects were analyzed in the full-length and truncated soluble receptor forms. The variations in the ligand binding activities of the purified truncated receptors are comparable with those of the full-length form. The mutated full-length receptors were also analyzed by inositol phosphate production and Ca(2+) response. The magnitude of the ligand binding capacities and the amplitude of the intracellular signaling were almost correlated. Alanine substitutions of four residues, Thr(188), Asp(208), Tyr(236), and Asp(318), which interact with the alpha-amino group of glutamate in the crystal, abolished their responses both to glutamate and quisqualate. The mutations of the Tyr(74), Arg(78), and Gly(293) residues, which interact with the gamma-carboxyl group of glutamate, lost their responsiveness to glutamate but not to quisqualate. Furthermore, a mutant receptor containing alanine instead of isoleucine at position 120 located within an alpha helix constituting the dimer interface showed no intracellular response to ligand stimulation. The results demonstrate the crucial role of the dimer interface in receptor activation.     

Roles of conserved basic amino acid residues and activation mechanism of the hyperthermophilic aspartate racemase at high temperature

X-ray crystallography has revealed two similar alpha/beta domains of the aspartate racemase from the hyperthermophilic archaeon, Pyrococcus horikoshii OT3. The active site is located in the cleft between the two domains where two cysteine residues face each other. This arrangement allows the substrate to enter the cleft and enables the two cysteine residues to act synergistically. However, the distance between their thiolates was estimated to be 9.6 angstroms, which is beyond the distance for cooperative action of them. We examined the molecular mechanism for the racemization reaction of this hyperthermophilic aspartate racemase by mutational analyses and molecular dynamics simulations. The mutational analyses revealed that Arg48 and Lys164 were essential for catalysis in addition to the putative catalytic cysteine residues. The molecular dynamics simulations revealed that the distance between the two active gamma-sulfur atoms of cysteine residues oscillate to periodically become shorter than the predicted cooperative distance at high temperature. In addition, the conformation of Tyr160, which is located at the entrance of the cleft and inhibits the entry of a substrate, changes periodically to open the entrance at 375 K. The opening of the gate is likely to be induced by the motion of the adjacent amino acid, Lys164. The entrance of an aspartate molecule was observed by molecular dynamics (MD) simulations driven by the force of the electrostatic interaction with Arg48, Lys164, and also Asp47. These results provide insights into the roles of amino acid residues at the catalytic site and also the activation mechanism of a hyperthermophilic aspartate racemase at high temperature.     

A domain of the manganese-stabilizing protein from Synechococcus elongatus involved in functional binding to photosystem II.

Site-directed mutagenesis was performed to investigate whether the two protease-sensitive sequences Phe(156)-Gly(163) and Arg(184)-Ser(191), of the manganese-stabilizing protein (MSP) from a thermophilic cyanobacterium, Synechococcus elongatus (Motoki, A., Shimazu, T., Hirano, M., and Katoh, S. (1998) Biochim. Biophys. Acta 1365, 492-502), are involved in functional interaction with photosystem II (PSII). The ability of MSP to bind to its functional site on the PSII complex and to reactivate oxygen evolution was dramatically reduced by the substitution of Arg(152), Asp(158), Lys(160), or Arg(162) with uncharged residues, by insertion of a single residue between Phe(156) and Leu(157), or by deletion of Leu(157). Substitution of each of the four charged residues with an identically charged residue showed that the charges at Asp(158), and possibly Lys(160), are important for the electrostatic interaction with PSII. The reactivating ability was also strongly affected by the alteration of Phe(156) to Leu. Replacement of Lys(188), the only strictly conserved charged residue in the Arg(184)-Ser(191) sequence, by Gln had only a marginal effect on the function of MSP. High affinity binding of MSP to PSII was also affected significantly by mutation at Arg(152), which is located in a region (Val(148)-Arg(152)) strictly conserved among the 14 sequences so far reported. These results imply that the Val(148)-Gly(163) sequence, which is well conserved among MSPs from cyanobacteria to higher plants, is a domain of MSP for functional interaction with PSII.     

Comparative structural analysis of the Erbin PDZ domain and the first PDZ domain of ZO-1. Insights into determinants of PDZ domain specificity

We report a structural comparison of the first PDZ domain of ZO-1 (ZO1-PDZ1) and the PDZ domain of Erbin (Erbin-PDZ). Although the binding profile of Erbin-PDZ is extremely specific ([D/E][T/S]WV(COOH)), that of ZO1-PDZ1 is similar ([R/K/S/T][T/S][W/Y][V/I/L](COOH)) but broadened by increased promiscuity for three of the last four ligand residues. Consequently, the biological function of ZO-1 is also broadened, as it interacts with both tight and adherens junction proteins, whereas Erbin is restricted to adherens junctions. Structural analyses reveal that the differences in specificity can be accounted for by two key differences in primary sequence. A reduction in the size of the hydrophobic residue at the base of the site(0) pocket enables ZO1-PDZ1 to accommodate larger C-terminal residues. A single additional difference alters the specificity of both site(-1) and site(-3). In ZO1-PDZ1, an Asp residue makes favorable interactions with both Tyr(-1) and Lys/Arg(-3). In contrast, Erbin-PDZ contains an Arg at the equivalent position, and this side chain cannot accommodate either Tyr(-1) or Lys/Arg(-3) but, instead, interacts favorably with Glu/Asp(-3). We propose a model for ligand recognition that accounts for interactions extending across the entire binding site but that highlights several key specificity switches within the PDZ domain fold.     

Deduced amino-acid sequence and possible catalytic residues of a novel pectate lyase from an alkaliphilic strain of Bacillus.

The nucleotide sequence of the gene for a highly alkaline, low-molecular-mass pectate lyase (Pel-15) from an alkaliphilic Bacillus isolate was determined. It harbored an open reading frame of 672 bp encoding the mature enzyme of 197 amino acids with a predicted molecular mass of 20 924 Da. The deduced amino-acid sequence of the mature enzyme showed very low homology (< 20.4% identity) to those of known pectinolytic enzymes in the large pectate lyase superfamily (the polysaccharide lyase family 1). In an integrally conserved region designated the BF domain, Pel-15 showed a high degree of identity (40.5% to 79.4%) with pectate lyases in the polysaccharide lyase family 3, such as PelA, PelB, PelC, and PelD from Fusarium solani f. sp. pisi, PelB from Erwinia carotovora ssp. carotovora, PelI from E. chrysanthemi, and PelA from a Bacillus strain. By site-directed mutagenesis of the Pel-15 gene, we replaced Lys20 in the N-terminal region, Glu38, Lys41, Glu47, Asp63, His66, Trp78, Asp80, Glu83, Asp84, Lys89, Asp106, Lys107, Asp126, Lys129, and Arg132 in the BF domain, and Arg152, Tyr174, Lys182, and Lys185 in the C-terminal region of the enzyme individually with Ala and/or other amino acids. Consequently, some carboxylate and basic residues selected from Glu38, Asp63, Glu83, Asp106, Lys107, Lys129, and Arg132 were suggested to be involved in catalysis and/or calcium binding. We constructed a chimeric enzyme composed of Ala1 to Tyr105 of Pel-15 in the N-terminal regions, Asp133 to Arg159 of FsPelB in the internal regions, and Gln133 to Tyr197 of Pel-15 in the C-terminal regions. The substituted PelB segment could also express beta-elimination activity in the chimeric molecule, confirming that Pel-15 and PelB share a similar active-site topology.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

The investigation of interactions of kappa-Hefutoxin1 with the voltage-gated potassium channels: a computational simulation

Kappa-Hefutoxin1 is a K(+) channel-blocking toxin from the scorpion Heterometrus fluvipes. It is a 22-residue protein that adapts a novel fold of two parallel helices linked by two disulfide bridges without beta-sheets. Recognition of interactions of kappa-Hefutoxin1 with the voltage-gated potassium channels, Kv1.1, Kv1.2, and Kv1.3, was studied by 3D-Dock software package. All structures of kappa-Hefutoxin1 were considered during the simulations, which indicated that even small changes in the structure of kappa-Hefutoxin1 considerably affected both the recognition and the binding between kappa-Hefutoxin1 and the Kv1 channels. kappa-Hefutoxin1 is located around the extracellular part of the Kv1 channels, making contacts with its helices. Lys 19, Tyr 5, Arg 6, Trp 9, or Arg 10 in the toxin and residues Asp 402, His 404, Thr 407,Gly 401, and Asp 386 in each subunit of the Kv potassium channel are the key residues for the toxin-channel recognition. Moreover, the simulation result demonstrates that the hydrophobic interactions are important in interaction of negatively charged toxins with potassium channels. The results of our docking/molecular dynamics simulations indicate that our 3D model structure of the kappa-Hefutoxin1-complex is both reasonable and acceptable and could be helpful for smarter drug design and the blocking agents of Kv1 channels.     

Ligand binding specificity of the Escherichia coli periplasmic histidine binding protein,HisJ

The HisJ protein from Escherichia coli and related Gram negative bacteria is the periplasmic component of a bacterial ATP‐cassette (ABC) transporter system. Together these proteins form a transmembrane complex that can take up L‐histidine from the environment and translocate it into the cytosol. We have studied the specificity of HisJ for binding L‐His and many related naturally occurring compounds. Our data confirm that L‐His is the preferred ligand, but that 1‐methyl‐L‐His and 3‐methyl‐L‐His can also bind, while the dipeptide carnosine binds weakly and D‐histidine and the histidine degradation products, histamine, urocanic acid and imidazole do not bind. L‐Arg, homo‐L‐Arg, and post‐translationally modified methylated Arg‐analogs also bind with reasonable avidity, with the exception of symmetric dimethylated‐L‐Arg. In contrast, L‐Lys and L‐Orn have considerably weaker interactions with HisJ and methylated and acetylated Lys variants show relatively poor binding. It was also observed that the carboxylate group of these amino acids and their variants was very important for proper recognition of the ligand. Taken together our results are a key step towards designing HisJ as a specific protein‐based reagentless biosensor.     

A fluorescently labeled undecapeptide derived from a protein in royal jelly of the honeybee—royalisin—for specific detection of oxidized low‐density lipoprotein

The probes for detection of oxidized low‐density lipoprotein (ox‐LDL) in plasma and in atherosclerotic plaques are expected to facilitate the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that a heptapeptide (Lys‐Trp‐Tyr‐Lys‐Asp‐Gly‐Asp, KP6) coupled through the ε‐amino group of N‐terminal Lys to fluorescein isothiocyanate (FITC), (FITC)KP6, can be useful as a fluorescent probe for specific detection of ox‐LDL. In the present study, to develop a novel fluorescent peptide for specific detection of ox‐LDL, we investigated the interaction (with ox‐LDL) of an undecapeptide corresponding to positions 41 to 51 of a potent antimicrobial protein (royalisin, which consists of 51 residues; from royal jelly of honeybees), conjugated at the N‐terminus to FITC in the presence of 6‐amino‐n‐caproic acid (AC) linker, (FITC‐AC)‐royalisin P11, which contains both sequences, Phe‐Lys‐Asp and Asp‐Lys‐Tyr, similar to Tyr‐Lys‐Asp in (FITC)KP6. The (FITC‐AC)‐royalisin P11 bound with high specificity to ox‐LDL in a dose‐dependent manner, through the binding to major lipid components in ox‐LDL (lysophosphatidylcholine and oxidized phosphatidylcholine). In contrast, a (FITC‐AC)‐shuffled royalisin P11 peptide, in which sequences Phe‐Lys‐Asp and Asp‐Lys‐Tyr were modified to Lys‐Phe‐Asp and Asp‐Tyr‐Lys, respectively, hardly bound to LDL and ox‐LDL. These findings strongly suggest that (FITC‐AC)‐royalisin P11 may be an effective fluorescent probe for specific detection of ox‐LDL and that royalisin from the royal jelly of honeybees may play a role in the treatment of atherosclerosis through the specific binding of the region at positions 41 to 51 to ox‐LDL.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Roles of the P1, P2, and P3 residues in determining inhibitory specificity of kallistatin toward human tissue kallikrein

Kallistatin is a serpin with a unique P1 Phe, which confers an excellent inhibitory specificity toward tissue kallikrein. In this study, we investigated the P3-P2-P1 residues (residues 386-388) of human kallistatin in determining inhibitory specificity toward human tissue kallikrein by site-directed mutagenesis and molecular modeling. Human kallistatin mutants with 19 different amino acid substitutions at each P1, P2, or P3 residue were created and purified to compare their kallikrein binding activity. Complex formation assay showed that P1 Arg, P1 Phe (wild type), P1 Lys, P1 Tyr, P1 Met, and P1 Leu display significant binding activity with tissue kallikrein among the P1 variants. Kinetic analysis showed the inhibitory activities of the P1 mutants toward tissue kallikrein in the order of P1 Arg > P1 Phe > P1 Lys >/= P1 Tyr > P1 Leu >/= P1 Met. P1 Phe displays a better selectivity for human tissue kallikrein than P1 Arg, since P1 Arg also inhibits several other serine proteinases. Heparin distinguishes the inhibitory specificity of kallistatin toward kallikrein versus chymotrypsin. For the P2 and P3 variants, the mutants with hydrophobic and bulky amino acids at P2 and basic amino acids at P3 display better binding activity with tissue kallikrein. The inhibitory activities of these mutants toward tissue kallikrein are in the order of P2 Phe (wild type) > P2 Leu > P2 Trp > P2 Met and P3 Arg > P3 Lys (wild type). Molecular modeling of the reactive center loop of kallistatin bound to the reactive crevice of tissue kallikrein indicated that the P2 residue required a long and bulky hydrophobic side chain to reach and fill the hydrophobic S2 cleft generated by Tyr(99) and Trp(219) of tissue kallikrein. Basic amino acids at P3 could stabilize complex formation by forming electrostatic interaction with Asp(98J) and hydrogen bond with Gln(174) of tissue kallikrein. Our results indicate that tissue kallikrein is a specific target proteinase for kallistatin.     

Topology and structure of the C1q-binding site on C-reactive protein

The host defense functions of human C-reactive protein (CRP) depend to a great extent on its ability to activate the classical complement pathway. The aim of this study was to define the topology and structure of the CRP site that binds C1q, the recognition protein of the classical pathway. We have previously reported that residue Asp(112) of CRP plays a major role in the formation of the C1q-binding site, while the neighboring Lys(114) hinders C1q binding. The three-dimensional structure of CRP shows the presence of a deep, extended cleft in each protomer on the face of the pentamer opposite that containing the phosphocholine-binding sites. Asp(112) is part of this marked cleft that is deep at its origin but becomes wider and shallower close to the inner edge of the protomer and the central pore of the pentamer. The shallow end of the pocket is bounded by the 112-114 loop, residues 86-92 (the inner loop), the C terminus of the protomer, and the C terminus of the pentraxin alpha-helix 169-176, particularly Tyr(175). Mutational analysis of residues participating in the formation of this pocket demonstrates that Asp(112) and Tyr(175) are important contact residues for C1q binding, that Glu(88) influences the conformational change in C1q necessary for complement activation, and that Asn(158) and His(38) probably contribute to the correct geometry of the binding site. Thus, it appears that the pocket at the open end of the cleft is the C1q-binding site of CRP.