Isolating the Epstein-Barr virus gp350/220 binding site on complement receptor type 2 (CR2/CD21)

Complement receptor type 2 (CR2/CD21) is essential for the attachment of Epstein-Barr virus (EBV) to the surface of B-lymphocytes in an interaction mediated by the viral envelope glycoprotein gp350. The heavily glycosylated structure of EBV gp350 has recently been elucidated by x-ray crystallography, and the CR2 binding site on this protein has been characterized. To identify the corresponding gp350 binding site on CR2, we have undertaken a site-directed mutagenesis study targeting regions of CR2 that have previously been implicated in the binding of CR2 to the C3d/C3dg fragments of complement component C3. Wild-type or mutant forms of CR2 were expressed on K562 cells, and the ability of these CR2-expressing cells to bind gp350 was measured using flow cytometry. Mutations directed toward the two N-terminal extracellular domains of CR2 (SCR1-2) reveal that a large contiguous surface of CR2 SCR1-2 is involved in gp350 binding, including a number of positively charged residues (Arg-13, (Arg-28, (Arg-36, Lys-41, Lys-57, Lys-67, and Arg-83). These data appear to complement the CR2 binding site on gp350, which is characterized by a preponderance of negative charge. In addition to identifying the importance of charge in the formation of a CR2-gp350 complex, we also provide evidence that both SCR1 and SCR2 make contact with gp350. Specifically, two anti-CR2 monoclonal antibodies, designated as monoclonal antibodies 171 and 1048 whose primary epitopes are located within SCR2, inhibit binding of wild-type CR2 to EBV gp350; with regard to SCR1, both K562 cells expressing an S15P mutation and recombinant S15P CR2 proteins exhibit diminished gp350 binding.     

A bipartite mechanism for ERK2 recognition by its cognate regulators and substrates

Mitogen-activated protein (MAP) kinases control gene expression in response to extracellular stimuli and exhibit exquisite specificity for their cognate regulators and substrates. We performed a structure-based mutational analysis of ERK2 to identify surface areas that are important for recognition of its interacting proteins. We show that binding and activation of MKP3 by ERK2 involve two distinct protein-protein interaction sites in ERK2. Thus, the common docking (CD) site composed of Glu-79, Tyr-126, Arg-133, Asp-160, Tyr-314, Asp-316, and Asp-319 are important for high affinity MKP3 binding but not essential for ERK2-induced MKP3 activation. MKP3 activation requires residues Tyr-111, Thr-116, Leu-119, Lys-149, Arg-189, Trp-190, Glu-218, Arg-223, Lys-229, and His-230 in the ERK2 substrate-binding region, located distal to the common docking site. Interestingly, many of the residues important for MKP3 recognition are also used for Elk1 binding and phosphorylation. In addition to the shared residues, there are also residues that are unique to each target recognition. There is evidence indicating that the CD site and the substrate-binding region defined here are also utilized for MEK1 recognition, and indeed, we demonstrate that the binding of MKP3, Elk1, and MEK1 to ERK2 is mutually exclusive. Taken together, our data suggest that the efficiency and fidelity of ERK2 signaling is achieved by a bipartite recognition process. In this model, one part of the ERK2-binding proteins (e.g. the kinase interaction motif sequence) docks to the CD site located on the back side of the ERK2 catalytic pocket for high affinity association, whereas the interaction of the substrate-binding region with another structural element (e.g. the FXFP motif in MKP3 and Elk1) may not only stabilize binding but also provide contacts crucial for modulating the activity and/or specificity of ERK2 target molecules.     

Differential scanning calorimetric studies of E. coli aspartate transcarbamylase. III. The denaturational thermodynamics of the holoenzyme with single-site mutations in the catalytic chain

Aspartate transcarbamylase (EC 2.1.3.2) from E. coli is a multimeric enzyme consisting of two catalytic subunits and three regulatory subunits whose activity is regulated by subunit interactions. Differential scanning calorimetric (DSC) scans of the wild-type enzyme consist of two peaks, each comprised of at least two components, corresponding to denaturation of the catalytic and regulatory subunits within the intact holoenzyme (Vickers et al., J. Biol. Chem. 253 (1978) 8493; Edge et al., Biochemistry 27 (1988) 8081). We have examined the effects of nine single-site mutations in the catalytic chains. Three of the mutations (Asp-100-Gly, Glu-86-Gln, and Arg-269-Gly) are at sites at the C1: C2 interface between c chains within the catalytic subunit. These mutations disrupt salt linkages present in both the T and R states of the molecule (Honzatko et al., J. Mol. Biol. 160 (1982) 219; Krause et al., J. Mol. Biol. 193 (1987) 527). The remainder (Lys-164-Ile, Tyr-165-Phe, Glu-239-Gln, Glu-239-Ala, Tyr-240-Phe and Asp-271-Ser) are at the C1: C4 interface between catalytic subunits and are involved in interactions which stabilize either the T or R state. DSC scans of all of the mutants except Asp-100-Gly and Arg-269-Gly consisted of two peaks. At intermediate concentrations, Asp-100-Gly and Arg-269-Gly had only a single peak near the Tm of the regulatory subunit transition in the holoenzyme, although their denaturational profiles were more complex at high and low protein concentrations. The catalytic subunits of Glu-86-Gln, Lys-164-Ile and Asp-271-Ser appear to be significantly destabilized relative to wild-type protein while Tyr-165-Phe and Tyr-240-Phe appear to be stabilized. Values of delta delta G degree cr, the difference between the subunit interaction energy of wild-type and mutant proteins, evaluated as suggested by Brandts et al. (Biochemistry 28 (1989) 8588) range from -3.7 kcal mol-1 for Glu-86-Gln to 2.4 kcal mol-1 for Tyr-165-Phe.     

Identification of functionally relevant residues of the rat ileal apical sodium-dependent bile acid cotransporter

The mechanisms underlying the transport of bile acids by apical sodium-dependent bile acid transporter (Asbt) are not well defined. To further identify the functionally relevant residues, thirteen conserved negatively (Asp and Glu) and positively (Lys and Arg) charged residues plus Cys-270 of rat Asbt were replaced with Ala or Gln by site-directed mutagenesis. Seven of the fourteen residues of rat Asbt were identified as functionally important by taurocholate transport studies, substrate inhibition assays, confocal microscopy, and electrophysiological methods. The results showed that Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues of rat Asbt are critical for transport function and may determine substrate specificity. Arg-64 may be located at a different binding site to assist in interaction with non-bile acid organic anions. For bile acid transport by Asbt, Na(+) ion movement is a voltage-dependent process that tightly companied with taurocholate movement. Asp-122 and Glu-261 play a critical role in the interaction of a Na(+) ion and ligand with Asbt. Cys-270 is not essential for the transport process. These studies provide new details about the amino acid residues of Asbt involved in binding and transport of bile acids and Na(+).     

Charged amino acids of the N-terminal domain are involved in coupling binding and gating in alpha7 nicotinic receptors

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of conformational changes resulting in channel opening. Previously, we have shown that the aspartate residue Asp-266 at the M2-M3 linker of the alpha7 nicotinic receptor is involved in connecting binding and gating. High resolution structural data suggest that this region could interact with the so-called loops 2 and 7 of the extracellular N-terminal region. In this case, certain charged amino acids present in these loops could integrate together with Asp-266 and other amino acids, a mechanism involved in channel activation. To test this hypothesis, all charged residues in these loops, Asp-42, Asp-44, Glu-45, Lys-46, Asp-128, Arg-130, and Asp-135, were substituted with other amino acids, and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Glu-45, Lys-46, and Asp-135 exhibited poor or null functional responses to different nicotinic agonists regardless of significant membrane expression, whereas D128A showed a gain of function effect. Because the double reverse charge mutant K46D/D266K did not restore receptor function, a gating mechanism controlled by the pairwise electrostatic interaction between these residues is not likely. Rather, a network of interactions formed by residues Lys-46, Asp-128, Asp-135, Asp-266, and possibly others appears to link agonist binding to channel gating.     

Functional mapping of charged residues of the 82-116 sequence in factor Xa: evidence that lysine 96 is a factor Va independent recognition site for prothrombin in the prothrombinase complex

It has been hypothesized that two antiparallel structures comprised of residues 82-91 and 102-116 in factor Xa (fXa) may harbor a factor Va- (fVa-) dependent prothrombin recognition site in the prothrombinase complex. There are 11 charged residues in the 82-116 loop of human fXa (Glu-84, Glu-86, Lys-90, Arg-93, Lys-96, Glu-97, Asp-100, Asp-102, Arg-107, Lys-109, and Arg-115). With the exception of Glu-84, which did not express, and Asp-102, which is a catalytic residue, we expressed the Ala substitution mutants of all other residues and evaluated their proteolytic and amidolytic activities in both the absence and presence of fVa. K96A and K109A activated prothrombin with 5-10-fold impaired catalytic efficiency in the absence of fVa. All mutants, however, exhibited normal activity toward the substrate in the presence of fVa. K109A also exhibited impaired amidolytic activity and affinity for Na(+); however, both fVa and higher Na(+) restored the catalytic defect caused by the mutation. Analysis of the X-ray crystal structure of fXa indicated that Glu-84 may interact by a salt bridge with Lys-109, explaining the lack of expression of E84A and the lower activity of K109A in the absence of fVa. These results suggest that none of the residues under study is a fVa-dependent recognition site for prothrombin in the prothrombinase complex; however, Lys-96 is a recognition site for the substrate independent of the cofactor. Moreover, the 82-116 loop is energetically linked to fVa and Na(+) binding sites of the protease.     

Glutamate 83 is important for stabilization of domain-domain conformation of Thermus aquaticus glycerol kinase

The gene glpK, encoding glycerol kinase (GlpK) of Thermus aquaticus, has recently been identified. The protein encoded by glpK was found to have an unusually high identity of 81% with the sequence of GlpK from Bacillus subtilis. Three residues (Arg-82, Glu-83, and Asp-244) of T. aquaticus GlpK are conserved in all the known GlpK sequences, including those from various bacteria, yeast and human. The roles that these three residues play in the catalytic mechanism were investigated by using site-directed mutagenesis to produce three mutants: Arg-82-Ala, Glu-83-Ala, and Asp-244-Ala. Replacement of Asp-244 by Ala resulted in a complete loss of activity, thus suggesting that Asp-244 is important for catalysis. Taking k(cat)/K(m) as a simple measure of catalytic efficiency, the mutants Arg-82-Ala and Glu-83-Ala were judged to cause 190- and 37,000-fold decrease, respectively, when compared to the wild-type GlpK. Thus, these three residues play a critical role in the catalytic mechanism. However, only mutant Glu-83-Ala was cleaved by alpha-chymotrypsin, and proteolysis studies showed that the mutant Glu-83-Ala involves a change in the exposure of Tyr-331 at the alpha-chymotrypsin site. This indicates a large domain conformational change, since the residues corresponding to Glu-83 and Tyr-331 in the Escherichia coli GlpK sequence are located in domain IB and domain IIB, respectively. The apparent conformational change caused by replacement of Glu-83 leads us to propose that Glu-83 is an important residue for stabilization of domain conformation.     

Isolation of four novel tachykinins from frog (Rana catesbeiana) brain and intestine

In a survey for unknown bioactive peptides in frog (Rana catesbeiana) brain and intestine, we isolated four novel peptides that exhibit potent stimulant effects on smooth muscle preparation of guinea pig ileum. By microsequencing and synthesis, these peptides were identified as Lys- Pro- Ser- Pro- Asp- Arg- Phe- Tyr- Gly- Leu- Met- NH2 (ranatachykinin A), Tyr- Lys- Ser- Asp- Ser- Phe- Tyr- Gly- Leu- Met- NH2 (ranatachykinin B), His- Asn- Pro- Ala- Ser- Phe- Ile- Gly- Leu- Met- NH2 (ranatachykinin C) and Lys- Pro- Ans- Pro- Glu- Arg- Phe- Tyr- Ala- Pro- Met- NH2 (ranatachykinin D). Ranatachykinin (RTK) A, B and C conserve the C- terminal sequence, Phe- X- Gly- Leu- Met- NH2, which is common to known members of the tachykinin family. On the other hand, RTK-D has a striking feature in its C-terminal sequence, Phe- Tyr- Ala- Pro- Met- NH2, which has never been found in other known tachykinins, and may constitute a new subclass in the tachykinin family.     

RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2

Here we report that bacteriophage T4 RNA ligase 2 (Rnl2) is an efficient catalyst of RNA ligation at a 3'-OH/5'-PO(4) nick in a double-stranded RNA or an RNA.DNA hybrid. The critical role of the template strand in approximating the reactive 3'-OH and 5'-PO(4) termini is underscored by the drastic reductions in the RNA-sealing activity of Rnl2 when the duplex substrates contain gaps or flaps instead of nicks. RNA nick joining requires ATP and a divalent cation cofactor (either Mg or Mn). Neither dATP, GTP, CTP, nor UTP can substitute for ATP. We identify by alanine scanning seven functionally important amino acids (Tyr-5, Arg-33, Lys-54, Gln-106, Asp-135, Arg-155, and Ser-170) within the N-terminal nucleotidyl-transferase domain of Rnl2 and impute specific roles for these residues based on the crystal structure of the AMP-bound enzyme. Mutational analysis of 14 conserved residues in the C-terminal domain of Rnl2 identifies 3 amino acids (Arg-266, Asp-292, and Glu-296) as essential for ligase activity. Our findings consolidate the evolutionary connections between bacteriophage Rnl2 and the RNA-editing ligases of kinetoplastid protozoa.     

NMR docking of a substrate into the X-ray structure of staphylococcal nuclease.

The conformation of the staphylococcal nuclease-bound metal-dTdA complex, previously determined by NMR methods [Weber, D.J., Mullen, G.P., Mildvan, A.S. (1991) Biochemistry 30:7425-7437] was docked into the X-ray structure of the enzyme-Ca(2+)-3',5'-pdTp complex [Loll, P.J., Lattman, E.E. (1989) Proteins: Struct., Funct., Genet. 5:183-201] by superimposing the metal ions, taking into account intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of the leaving dA moiety of dTdA, and energy minimization to relieve small overlaps. The proton resonances of the Phe, Tyr, and Trp residues of the enzyme in the ternary enzyme-La(3+)-dTdA complex were sequence specifically assigned by 2D phase-sensitive NOESY, with and without deuteration of the aromatic protons of the Tyr residues, and by 2D heteronuclear multiple quantum correlation (HMQC) spectroscopy and 3D NOESY-HMQC spectroscopy with 15N labeling. While resonances of most Phe, Tyr and Trp residues were unshifted by the substrate dTdA from those found in the enzyme-La(3+)-3',5'-pdTp complex and the enzyme-Ca(2+)-3',5'-pdTp complex, proton resonances of Tyr-85, Tyr-113, Tyr-115, and Phe-34 were shifted by 0.08 to 0.33 ppm and the 15N resonance of Tyr-113 was shifted by 2.1 ppm by the presence of substrate. The optimized position of enzyme-bound dTdA shows the 5'-dA leaving group to partially overlap the inhibitor, 3',5'-pdTp (in the X-ray structure). The 3'-TMP moiety of dTdA points toward the solvent in a channel defined by Ile-18, Asp-19, Thr-22, Lys-45, and His-46. The phosphate of dTdA is coordinated by the metal, and an adjacent inner sphere water ligand is positioned to donate a hydrogen bond to the general base Glu-43 and to attack the phosphorus with inversion. Arg-35 and Arg-87 donate monodentate hydrogen bonds to different phosphate oxygens of dTdA, with Arg-87 positioned to protonate the leaving 5'-oxygen of dA, thus clarifying the mechanism of hydrolysis. Model building of an additional 5'-dGMP onto the 3'-oxygen of dA placed this third nucleotide onto a surface cleft near residues Glu-80, Asp-83, Lys-84, and Tyr-115 with its 3'-OH group accessible to the solvent, thus defining the size of the substrate binding site as accommodating a trinucleotide.     

Site-directed mutagenesis of charged amino acids of the human mitochondrial carnitine/acylcarnitine carrier: Insight into the molecular mechanism of transport

The structure/function relationships of charged residues of the human mitochondrial carnitine/acylcarnitine carrier, which are conserved in the carnitine/acylcarnitine carrier subfamily and exposed to the water-filled cavity of carnitine/acylcarnitine carrier in the c-state, have been investigated by site-directed mutagenesis. The mutants were expressed in Escherichia coli, purified and reconstituted in liposomes, and their transport activity was measured as ³H-carnitine/carnitine antiport. The mutants K35A, E132A, D179A and R275A were nearly inactive with transport activities between 5 and 10% of the wild-type carnitine/acylcarnitine carrier. R178A, K234A and D231A showed transport function of about 15% of the wild-type carnitine/acylcarnitine carrier. The substitutions of the other residues with alanine had little or no effect on the carnitine/acylcarnitine carrier activity. Marked changes in the kinetic parameters with three-fold higher Km and lower Vmax values with respect to the wild-type carnitine/acylcarnitine carrier were found when replacing Lys-35, Glu-132, Asp-179 and Arg-275 with alanine. Double mutants exhibited transport activities and kinetic parameters reflecting those of the single mutants; however, lack of D179A activity was partially rescued by the additional mutation R178A. The results provide evidence that Arg-275, Asp-179 and Arg-178, which protrude into the carrier's internal cavity at about the midpoint of the membrane, are the critical binding sites for carnitine. Furthermore, Lys-35 and Glu-132, which are very probably involved in the salt-bridge network located at the bottom of the cavity, play a major role in opening and closing the matrix gate.     

Construction of Peptides Mimicking a HIV-1 gp41 Epitope Recognized by Virus-Neutralizing Antibodies 2F5

A phage peptide library was screened with virus-neutralizing monoclonal antibodies (MAb) 2F5 which recognize a conserved epitope of HIV-1 gp41. Phages that expose peptides specifically binding with MAb 2F5 were selected by ELISA. Amino acid sequence analysis revealed homology to region 662–671 of HIV-1 HB10 gp160 for most peptides. The major role in recognition was ascribed to Asp-664, Lys-665, and Trp-666. The epitope-mimicking peptides were tested for immunogenicity. Antibodies to gp41 were detected in the serum of immunized rabbits.     

Crystal structure of Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in complex with trypsin. Implications for KD1 specificity of inhibition

Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 inhibits trypsin, plasmin, and factor VIIa (FVIIa)/tissue factor with Ki values of 13, 3, and 1640 nM, respectively. To investigate the molecular specificity of KD1, crystals of the complex of KD1 with bovine beta-trypsin were obtained that diffracted to 1.8 A. The P1 residue Arg-15 (bovine pancreatic trypsin inhibitor numbering) in KD1 interacts with Asp-189 (chymotrypsin numbering) and with the carbonyl oxygens of Gly-219 and Ogamma of Ser-190. Leu-17, Leu-18, Leu-19, and Leu-34 in KD1 make van der Waals contacts with Tyr-39, Phe-41, and Tyr-151 in trypsin, forming a hydrophobic interface. Molecular modeling indicates that this complementary hydrophobic patch is composed of Phe-37, Met-39, and Phe-41 in plasmin, whereas in FVIIa/tissue factor, it is essentially absent. Arg-20, Tyr-46, and Glu-39 in KD1 interact with trypsin through ordered water molecules. In contrast, insertions in the 60-loop in plasmin and FVIIa allow Arg-20 of KD1 to directly interact with Glu-60 in plasmin and Asp-60 in FVIIa. Moreover, Tyr-46 in KD1 electrostatically interacts with Lys-60A and Arg-60D in plasmin and Lys-60A in FVIIa. Glu-39 in KD1 interacts directly with Arg-175 of the basic patch in plasmin, whereas in FVIIa, such interactions are not possible. Thus, the specificity of KD1 for plasmin is attributable to hydrophobic and direct electrostatic interactions. For trypsin, hydrophobic interactions are intact, and electrostatic interactions are weak, whereas for FVIIa, hydrophobic interactions are missing, and electrostatic interactions are partially intact. These findings provide insight into the protease selectivity of KD1.     

The crystal structure of staphylococcal nuclease refined at 1.7 A resolution

The crystal structure of staphylococcal nuclease has been determined to 1.7 A resolution with a final R-factor of 16.2% using stereochemically restrained Hendrickson-Konnert least-squares refinement. The structure reveals a number of conformational changes relative to the structure of the ternary complex of staphylococcal nuclease 1,2 bound with deoxythymidine-3',5'-diphosphate and Ca2+. Tyr-113 and Tyr-115, which pack against the nucleotide base in the nuclease complex, are rotated outward creating a more open binding pocket in the absence of nucleotide. The side chains of Ca2+ ligands Asp-21 and Asp-40 shift as does Glu-43, the proposed general base in the hydrolysis of the 5'-phosphodiester bond. The significance of some changes in the catalytic site is uncertain due to the intrusion of a symmetry related Lys-70 side chain which hydrogen bonds to both Asp-21 and Glu-43. The position of a flexible loop centered around residue 50 is altered, most likely due to conformational changes propagated from the Ca2+ site. The side chains of Arg-35, Lys-84, Tyr-85, and Arg-87, which hydrogen bond to the 3'- and 5'-phosphates of the nucleotide in the nuclease complex, are unchanged in conformation, with packing interactions with adjacent protein side chains sufficient to fix the geometry in the absence of ligand. The nuclease structure presented here, in combination with the stereochemically restrained refinement of the nuclease complex structure at 1.65 A, provides a wealth of structural information for the increasing number of studies using staphylococcal nuclease as a model system of protein structure and function.     

Investigation of the catalytic site within the ATP-grasp domain of Clostridium symbiosum pyruvate phosphate dikinase

Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of ATP, P(i), and pyruvate with AMP, PP(i), and phosphoenolpyruvate (PEP) in three partial reactions as follows: 1) E-His + ATP --> E-His-PP.AMP; 2) E-His-PP.AMP + P(i) --> E-His-P.AMP.PP(i); and 3) E-His-P + pyruvate --> E.PEP using His-455 as the carrier of the transferred phosphoryl groups. The crystal structure of the Clostridium symbiosum PPDK (in the unbound state) reveals a three-domain structure consisting of consecutive N-terminal, central His-455, and C-terminal domains. The N-terminal and central His-455 domains catalyze partial reactions 1 and 2, whereas the C-terminal and central His-455 domains catalyze partial reaction 3. Attempts to obtain a crystal structure of the enzyme with substrate ligands bound at the nucleotide binding domain have been unsuccessful. The object of the present study is to demonstrate Mg(II) activation of catalysis at the ATP/P(i) active site, to identify the residues at the ATP/P(i) active site that contribute to catalysis, and to identify roles for these residues based on their positions within the active site scaffold. First, Mg(II) activation studies of catalysis of E + ATP + P(i) --> E-P + AMP + PP(i) partial reaction were carried out using a truncation mutant (Tem533) in which the C-terminal domain is absent. The kinetics show that a minimum of 2 Mg(II) per active site is required for the reaction. The active site residues used for substrate/cofactor binding/activation were identified by site-directed mutagenesis. Lys-22, Arg-92, Asp-321, Glu-323, and Gln-335 mutants were found to be inactive; Arg-337, Glu-279, Asp-280, and Arg-135 mutants were partially active; and Thr-253 and Gln-240 mutants were almost fully active. The participation of the nucleotide ribose 2'-OH and alpha-P in enzyme binding is indicated by the loss of productive binding seen with substrate analogs modified at these positions. The ATP, P(i), and Mg(II) ions were docked into the PPDK N-terminal domain crevice, in an orientation consistent with substrate/cofactor binding modes observed for other members of the ATP-Grasp fold enzyme superfamily and consistent with the structure-function data. On the basis of this docking model, the ATP polyphosphate moiety is oriented/activated for pyrophosphoryl transfer through interaction with Lys-22 (gamma-P), Arg-92 (alpha-P), and the Gly-101 to Met-103 loop (gamma-P) as well as with the Mg(II) cofactors. The P(i) is oriented/activated for partial reaction 2 through interaction with Arg-337 and a Mg(II) cofactor. The Mg(II) ions are bound through interaction with Asp-321, Glu-323, and Gln-335 and substrate. Residues Glu-279, Asp-280, and Arg-135 are suggested to function in the closure of an active site loop, over the nucleotide ribose-binding site.     

Identification of determinants of ligand binding affinity and selectivity in the prostaglandin D2 receptor CRTH2

The chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a G protein-coupled receptor that mediates the pro-inflammatory effects of prostaglandin D(2) (PGD(2)) generated in allergic inflammation. The CRTH2 receptor shares greatest sequence similarity with chemoattractant receptors compared with prostanoid receptors. To investigate the structural determinants of CRTH2 ligand binding, we performed site-directed mutagenesis of putative mCRTH2 ligand-binding residues, and we evaluated mutant receptor ligand binding and functional properties. Substitution of alanine at each of three residues in the transmembrane (TM) helical domains (His-106, TM III; Lys-209, TM V; and Glu-268, TM VI) and one in extracellular loop II (Arg-178) decreased PGD(2) binding affinity, suggesting that these residues play a role in binding PGD(2). In contrast, the H106A and E268A mutants bound indomethacin, a nonsteroidal anti-inflammatory drug, with an affinity similar to the wild-type receptor. HEK293 cells expressing the H106A, K209A, and E268A mutants displayed reduced inhibition of intracellular cAMP and chemotaxis in response to PGD(2), whereas the H106A and E268A mutants had functional responses to indomethacin similar to the wild-type receptor. Binding of PGE(2) by the E268A mutant was enhanced compared with the wild-type receptor, suggesting that Glu-268 plays a role in determining prostanoid ligand selectivity. Replacement of Tyr-261 with phenylalanine did not affect PGD(2) binding but decreased the binding affinity for indomethacin. These results provided the first details of the ligand binding pocket of an eicosanoid-binding chemoattractant receptor.     

Identification of amino acid residues essential for the catalytic reaction of Bacillus kaustophilus leucine aminopeptidase

The functional significance of amino acid residues Lys-265, Asp-270, Lys-277, Asp-288, Asp-347, Glu-349, and Arg-351 of Bacillus kaustophilus leucine aminopeptidase was explored by site-directed mutagenesis. Variants with an apparent molecular mass of approximately 54 kDa were overexpressed in Escherichia coli and purified to homogeneity by nickel-chelate chromatography. The purified mutant enzymes had no LAP activity, implying that these residues are important for the catalytic reaction of the enzyme.     

Structure-function analysis of the reactive site in the first Kunitz-type domain of human tissue factor pathway inhibitor-2

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type proteinase inhibitor that regulates a variety of serine proteinases involved in coagulation and fibrinolysis through their non-productive interaction with a P(1) residue (Arg-24) in its first Kunitz-type domain (KD1). Previous kinetic studies revealed that TFPI-2 was a more effective inhibitor of plasmin than several other serine proteinases, but the molecular basis for this specificity was unclear. In this study, we employed molecular modeling and mutagenesis strategies to produce several variants of human TFPI-2 KD1 in an effort to identify interactive site residues other than the P(1) Arg that contribute significantly to its inhibitory activity and specificity. Molecular modeling of KD1 based on the crystal structure of bovine pancreatic trypsin inhibitor revealed that KD1 formed a more energetically favorable complex with plasmin versus trypsin and/or the factor VIIa-tissue factor complex primarily due to strong ionic interactions between Asp-19 (P(6)) and Arg residues in plasmin (Arg-644, Arg-719, and Arg-767), Arg-24 (P(1)) with Asp-735 in plasmin, and Arg-29 (P(5)') with Glu-606 in plasmin. In addition, Leu-26 through Leu-28 (P(2)'-P(4)') in KD1 formed strong van der Waals contact with a hydrophobic cluster in plasmin (Phe-583, Met-585, and Phe-587). Mutagenesis of Asp-19, Tyr-20, Arg-24, Arg-29, and Leu-26 in KD1 resulted in substantial reductions in plasmin inhibitory activity relative to wild-type KD1, but the Asp-19 and Tyr-20 mutations revealed the importance of these residues in the specific inhibition of plasmin. In addition to the reactive site residues in the P(6)-P(5)' region of KD1, mutation of a highly conserved Phe at the P(18)' position revealed the importance of this residue in the inhibition of serine proteinases by KD1. Thus, together with the P(1) residue, the nature of other residues flanking the P(1) residue, particularly at P(6) and P(5)', strongly influences the inhibitory activity and specificity of human TFPI-2.     

Crystal structure and mutational analysis of heparan sulfate 3-O-sulfotransferase isoform 1

Heparan sulfate interacts with antithrombin, a protease inhibitor, to regulate blood coagulation. Heparan sulfate 3-O-sulfotransferase isoform 1 performs the crucial last step modification in the biosynthesis of anticoagulant heparan sulfate. This enzyme transfers the sulfuryl group (SO(3)) from 3'-phosphoadenosine 5'-phosphosulfate to the 3-OH position of a glucosamine residue to form the 3-O-sulfo glucosamine, a structural motif critical for binding of heparan sulfate to antithrombin. In this study, we report the crystal structure of 3-O-sulfotransferase isoform 1 at 2.5-A resolution in a binary complex with 3'-phosphoadenosine 5'-phosphate. This structure reveals residues critical for 3'-phosphoadenosine 5'-phosphosulfate binding and suggests residues required for the binding of heparan sulfate. In addition, site-directed mutagenesis analyses suggest that residues Arg-67, Lys-68, Arg-72, Glu-90, His-92, Asp-95, Lys-123, and Arg-276 are essential for enzymatic activity. Among these essential amino acid residues, we find that residues Arg-67, Arg-72, His-92, and Asp-95 are conserved in heparan sulfate 3-O-sulfotransferases but not in heparan N-deacetylase/N-sulfotransferase, suggesting a role for these residues in conferring substrate specificity. Results from this study provide information essential for understanding the biosynthesis of anticoagulant heparan sulfate and the general mechanism of action of heparan sulfate sulfotransferases.     

DC-SIGN binds to HIV-1 glycoprotein 120 in a distinct but overlapping fashion compared with ICAM-2 and ICAM-3

DC-SIGN is a C-type lectin that binds to endogenous adhesion molecules ICAM-2 and ICAM-3 as well as the viral envelope glycoprotein human immunodeficiency virus, type 1, glycoprotein (gp) 120. We wished to determine whether DC-SIGN binds differently to its endogenous ligands ICAM-2 and ICAM-3 versus HIV-1 gp120. We found that recombinant soluble DC-SIGN bound to gp120-Fc more than 100- and 50-fold better than ICAM-2-Fc and ICAM-3-Fc, respectively. This relative difference was maintained using DC-SIGN expressed on three different CD4-negative cell lines. Although the cell surface affinity for gp120 varied by up to 4-fold on the cell lines examined, the affinity for gp120 was not a correlate of the ability of the cell line to transfer virus. Monosaccharides with equatorial 4-OH groups competed as well as D-mannose for gp120 binding to DC-SIGN, regardless of how the other hydroxyl groups were positioned. Disaccharide competitors and glycan chip analysis showed that DC-SIGN has a preference for oligosaccharides linked in an alpha-anomeric configuration. Alanine-scanning mutagenesis of DC-SIGN revealed that highly conserved residues that coordinate calcium (Asp-366) and/or are involved in both calcium and specific carbohydrate interactions (Glu-347, Asn-349, Glu-354, and Asp-355) significantly compromised binding to all three ligands. Mutating non-conserved residues (Asn-311, Arg-345, Val-351, Gly-352, Glu-353, Ser-360, Gly-361, and Asn-362) minimally affected binding except for the Asp-367 mutant, which enhanced gp120 binding but diminished ICAM-2 and ICAM-3 binding. Conversely, mutating the moderately conserved residue (Gly-346) abrogated gp120 binding but enhanced ICAM-2 and ICAM-3 binding. Thus, DC-SIGN appears to bind in a distinct but overlapping manner to gp120 when compared with ICAM-2 and ICAM-3.