Chemical modification of cationic residues in toxin a from king cobra (Ophiophagus hannah) venom

The cationic groups of arginine and lysine residues inα-neurotoxin, Toxin a, isolated from king cobra (Ophiophagus hannah) venom were subjected to modification with trinitrobenzene sulfonate (TNBS) andp-hydroxyphenylglyoxal (HPG), respectively. The trinitrophenylated (TNP) derivatives of Toxin a at Lys-10, 56, or 71 showed approximately 25% residual lethality, and modifications on Lys-10 and 56 or Lys-10 and 50 resulted in a decrease of lethality by 84% and 86%, respectively. Modifications on Arg-34, 37, and 70 and Arg-34, 37, and 72 in Toxin a caused a decrease in lethality by 92% and 93%, respectively, and it almost completely lost its lethality and binding activity to nicotinic acetylcholine receptor (nAChR) when all four arginine residues were modified. These results indicate that in addition to the cationic residues on loop II (Arg-34, 37), loop III (Lys-50, 56), and the C-terminal tail (Arg-70, 72; Lys-71), Lys-10 on loop I is also related to the neurotoxicity of Toxin a.     

Predicted Folding of β-Structure in Myelin Basic Protein

Predictions of myelin basic protein secondary structure have not previously considered a major role for beta-structure in the organization of the native molecule because optical rotatory dispersion and circular dichroism studies have provided little, if any, evidence for beta-structure, and because a polycationic protein is generally considered to resist folding into a compact structure. However, the Chou-Fasman, Lim, and Robson algorithms identify a total of five beta-strands in the amino acid sequence. Four of these hydrophobic amino acid sequences (37-45, 87-95, 110-118, and 150-158) could form a hairpin intermediate that initiates folding of a Greek-key-type beta-structure. A second fold on the more hydrophobic side, with the addition of a strand from the N-terminus (residues 13-21), would complete the five-stranded antiparallel beta-sheet. A unique strand alignment can be predicted by phasing the hydrophobic residues. The unusual triproline sequence of myelin basic protein (100-102) is enclosed in the 14-residue hairpin loop. If these prolines are in the trans conformation, models show that a reverse turn could occur at residues 102-105 (Pro-Ser-Gln-Gly). Algorithms do not agree on the prediction of alpha-helices, but each of the two large loops could accommodate an alpha-helix. Myelin basic protein is known to be phosphorylated in vivo on as many as five Ser/Thr residues. Phosphorylation might alter the dynamics of folding if the nascent polypeptide were phosphorylated in the cytoplasm. In particular, phosphorylation of Thr-99 could neutralize cationic residues Lys-106 and Arg-108 within the hairpin loop. In addition, the methylation of Arg-108 might stabilize the hairpin loop structure through hydrophobic interaction with the side chain of Pro-97. The cationic side chains of arginine and lysine residues located on the faces of the beta-sheet (Arg-43, Arg-114, Lys-13, Lys-92, Lys-153, and Lys-156) could provide sites for interaction with phospholipids and other anionic structures on the surface of the myelin lipid bilayer.     

Analysis of structure and function of the B subunit of cholera toxin by the use of site-directed mutagenesis

Oligonucleotide-directed mutagenesis of ctxB was used to produce mutants of cholera toxin B subunit (CT-B) altered at residues Cys-9, Gly-33, Lys-34, Arg-35, Cys-86 and Trp-88. Mutants were identified phenotypically by radial passive immune haemolysis assays and genotypically by colony hybridization with specific oligonucleotide probes. Mutant CT-B polypeptides were characterized for immunoreactivity, binding to ganglioside GM1, ability to associate with the A subunit, ability to form holotoxin, and biological activity. Amino acid substitutions that caused decreased binding of mutant CT-B to ganglioside GM1 and abolished toxicity included negatively charged or large hydrophobic residues for Gly-33 and negatively or positively charged residues for Trp-88. Substitution of lysine or arginine for Gly-33 did not affect immunoreactivity or GM1-binding activity of CT-B but abolished or reduced toxicity of the mutant holotoxins, respectively. Substitutions of Glu or Asp for Arg-35 interfered with formation of holotoxin, but none of the observed substitutions for Lys-34 or Arg-35 affected binding of CT-B to GM1. The Cys-9, Cys-86 and Trp-88 residues were important for establishing or maintaining the native conformation of CT-B or protecting the CT-B polypeptide from rapid degradation in vivo.     

Amino acid replacement studies of human cytochrome c by a complementation system using CYC1 deficient yeast

Various in vitro mutated human cytochrome c genes which encode displaced amino acid residues at the 14th, 17th, 28th, 37th, 38th, 56th, and/or 84th residues were constructed, and their degrees of complementation of yeast CYC1 deficiency were examined. Invariant Cys-17 and Arg-38 could not be replaced by alanine and tryptophan, respectively, without function impairment. Cytochrome c containing Ala-14 instead of conserved Cys-14, Gly-38 or Lys-38 instead of Arg-38, and Ser-84 instead of invariant Gly-84 were partly functional. These results indicate that these invariant or conserved residues are important. Cytochromes c containing Cys-56 instead of native Gly-56 was partly functional. Cytochrome c containing Arg-37 and Gly-38 instead of Gly-37 and Arg-38 was slightly functional. Replacement of variable Thr-28 and Gly-37 by Ile-28 and Arg-37, respectively, produced no effects. Our results are as a whole consistent with the view that conserved residues are important and variable residues are less important for cytochrome c to function.     

Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136 and Lys-167.

Vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes relaxation of supercoiled DNA by cleaving and rejoining DNA strands through a DNA- (3'-phosphotyrosyl)-enzyme intermediate. We have performed a kinetic analysis of mutational effects at four essential amino acids: Arg-130, Gly-132, Tyr-136 and Lys-167. Arg-130, Gly-132 and Lys-167 are conserved in all members of the type IB topoisomerase family. Tyr-136 is conserved in all poxvirus topoisomerases. We show that Arg-130 and Lys-167 are required for transesterification chemistry. Arg-130 enhances the rates of both cleavage and religation by 10(5). Lys-167 enhances the cleavage and religation reactions by 10(3) and 10(4), respectively. An instructive distinction between these two essential residues is that Arg-130 cannot be replaced by lysine, whereas substituting Lys-167 by arginine resulted in partial restoration of function relative to the alanine mutant. We propose that both basic residues interact directly with the scissile phosphate at the topoisomerase active site. Mutations at positions Gly-132 and Tyr-136 reduced the rate of strand cleavage by more than two orders of magnitude, but elicited only mild effects on religation rate. Gly-132 and Tyr-136 are suggested to facilitate a pre-cleavage activation step. The results of comprehensive mutagenesis of the vaccinia topoisomerase illuminate mechanistic and structural similarities to site-specific recombinases.     

Glycosylation and processing of pro-B-type natriuretic peptide in cardiomyocytes

B-type natriuretic peptide (BNP) and its related peptides are biomarkers for the diagnosis of heart failure. Recent studies identified several O-glycosylation sites, including Thr-71, on human pro-BNP but the functional significance was unclear. In this study, we analyzed glycosylation and proteolytic processing of pro-BNP in cardiomyocytes. Human pro-BNP wild-type (WT) and mutants were expressed in HEK 293 cells and murine HL-1 cardiomyocytes. Pro-BNP and BNP were analyzed by immunoprecipitation and Western blotting. Glycosidases and glycosylation inhibitors were used to examine carbohydrates on pro-BNP. The effects of furin and corin expression on pro-BNP processing in cells also were examined. We found that in HEK 293 cells, recombinant pro-BNP contained significant amounts of O-glycans with terminal oligosialic acids. Mutation at Thr-71 reduced O-glycans on pro-BNP and increased pro-BNP processing. In HL-1 cardiomyocytes, residue Thr-71 contained little O-glycans, and pro-BNP WT and T71A mutant were processed similarly. In HEK 293 cells, pro-BNP was processed by furin. Mutations at Arg-73 and Arg-76, but not Lys-79, prevented pro-BNP processing. In HL-1 cardiomyocytes, which express furin and corin, single or double mutations at Arg-73, Arg-76 and Lys-79 did not prevent pro-BNP processing. Only when all these three residues were mutated, was pro-BNP processing completely blocked. Our data indicate that pro-BNP glycosylation in cardiomyocytes differed significantly from that in HEK 293 cells. In HEK 293 cells, furin cleaved pro-BNP at Arg-76 whereas in cardiomyocytes corin cleaved pro-BNP at multiple residues including Arg-73, Arg-76 and Lys-79.     

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.     

Demonstration of the functional role of conserved Glu-Arg residues in the Staphylococcus aureus ferrichrome transporter

The features that govern the interaction of ligand binding proteins with membrane permeases of cognate ABC transporters are largely unknown. Using sequence alignments and structural modeling based on the structure of the Escherichia coli BtuCD vitamin B₁₂ transporter, we identified six conserved basic residues in the permease, comprised of FhuB and FhuG proteins, in the ferrichrome transporter of Staphylococcus aureus. Using alanine-scanning mutagenesis we demonstrate that two of these residues, FhuB Arg-71 and FhuG Arg-61, play a more dominant role in transporter function than FhuB Arg-74 and Arg-311, and FhuG Arg-64 and Lys-306. Moreover, we show that at positions 71 and 61 in FhuB and FhuG, respectively, arginine cannot be substituted for lysine without loss of transporter function. Previously, our laboratory demonstrated the importance of conserved acidic residues in the ferrichrome binding protein, FhuD2. Taken together, these results support the hypothesis that Glu-Arg salt bridges are critical for the interaction of the ligand binding protein with the transmembrane domains FhuB and FhuG. This hypothesis was further studied by “charge swapping” experiments whereby we constructed a S. aureus strain expressing FhuD2 with conserved residues Glu-97 and Glu-231 replaced by Arg and FhuB and FhuG with conserved basic residues Arg-71 and Arg-61, respectively, replaced by Glu. A strain containing this combination of substitutions restored partial function to the ferrichrome transporter. The results provide a direct demonstration of the functional importance of conserved basic residues on the extracellular surface of the ferrichrome permease in the Gram-positive bacterium S. aureus.     

Calcium-induced sensitization of the central helix of calmodulin to proteolysis.

The rate of proteolysis of trypsin-sensitive bonds was used to examine the nature of the structural changes accompanying Ca2+ and Mg2+ binding to calmodulin. In the Ca(2+)-free form, the rates of proteolysis at Arg-106 and Arg-37 are rapid (greater than 300 and 28 nmol min-1 mL-1, respectively), the bonds at Arg-74, Lys-75, and Lys-77, in the central helix, are cleaved more slowly (10 nmol min-1 mL-1), and a lag in the cleavage at the remaining bonds (Lys-13, Lys-30, Arg-86, Arg-90, and Arg-126) suggests that they are not cleaved in the native protein. High concentrations of Ca2+, but not Mg2+, almost completely abolish proteolysis at Arg-106 and drastically reduce the rate of cleavage at Arg-37. Both Ca2+ and Mg2+ exert a moderate protective effect on the proteolysis of the central helix. These results suggest that the F-helix of domains III and, to a lesser extent, the F-helix of domain I are somewhat flexible in the Ca(2+)-free form and are stabilized by Ca2+. Whereas full occupancy of the four Ca(2+)-binding sites produces little change in the susceptibility of the central helix to proteolytic attack, binding of two Ca2+ produces a 10-fold enhancement of the rate of proteolysis in this part of the molecule. We propose that at intermediate Ca2+ levels the flexibility of the central helix of calmodulin is greatly increased, resulting in the transient formation of intermediates which have not been detected by spectroscopic techniques but are trapped by the irreversible action of trypsin.     

Structure-function relationships of curaremimetic neurotoxin loop 2 and of a structurally similar segment of rabies virus glycoprotein in their interaction with the nicotinic acetylcholine receptor

Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic peptides to the acetylcholine receptor of Torpedo electric organ membranes was assessed by measuring their ability to inhibit the binding of 125I-alpha-bungarotoxin to the receptor. The peptides showing the highest affinity for the receptor were a peptide corresponding to the sequence of loop 2 (residues 25-44) of Ophiophagus hannah (king cobra) toxin b (IC50 = 5.7 x 10(-6) M) and the structurally similar segment (residues 173-203) of CVS rabies virus glycoprotein (IC50 = 2.6 x 10(-6) M). These affinities were comparable to those of d-tubocurarine (IC50 = 3.4 x 10(-6) M) and suberyldicholine (IC50 = 2.5 x 10(-6) M). These results demonstrate the importance of loop 2 in the neurotoxin interaction with the receptor. N- and C-terminal deletions of the loop 2 peptides and substitution of residues invariant or highly conserved among neurotoxins were performed in order to determine the role of individual residues in binding. Residues 25-40 are the most crucial in the interaction with the acetylcholine receptor. Modifications involving Lys-27, Trp-29, Phe-33, Arg-37, and Gly-38 reduced affinity of binding. R37D and F33T modifications reduced the affinity of alpha-bungarotoxin residues 28-40 by an order of magnitude. Arg-37 may correspond to the positively charged quaternary ammonium group and Phe-33 to the hydrophobic acetyl methyl group of acetylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of exosite residues of factor Xa involved in recognition of PAR-2 on endothelial cells

Recent results have indicated that factor Xa (FXa) cleaves protease-activated receptor 2 (PAR-2) to elicit protective intracellular signaling responses in endothelial cells. In this study, we investigated the molecular determinants of the specificity of FXa interaction with PAR-2 by monitoring the cleavage of PAR-2 by FXa in endothelial cells transiently transfected with a PAR-2 cleavage reporter construct in which the extracellular domain of the receptor was fused to cDNA encoding for alkaline phosphatase. Comparison of the cleavage efficiency of PAR-2 by a series of FXa mutants containing mutations in different surface loops indicated that the acidic residues of 39-loop (Glu-36, Glu-37, and Glu-39) and the basic residues of 60-loop (Lys-62 and Arg-63), 148-loop (Arg-143, Arg-150, and Arg-154), and 162-helix (Arg-165 and Lys-169) contribute to the specificity of receptor recognition by FXa on endothelial cells. This was evidenced by significantly reduced activity of mutants toward PAR-2 expressed on transfected cells. The extent of loss in the PAR-2 cleavage activity of FXa mutants correlated with the extent of loss in their PAR-2-dependent intracellular signaling activity. Further characterization of FXa mutants indicated that, with the exception of basic residues of 162-helix, which play a role in the recognition specificity of the prothrombinase complex, none of the surface loop residues under study makes a significant contribution to the activity of FXa in the prothrombinase complex. These results provide new insight into mechanisms through which FXa specifically interacts with its macromolecular substrates in the clotting and signaling pathways.     

Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The role of surface loop basic residues in substrate binding to the fructose-2,6-bisphosphatase domain.

Lys-356 has been implicated as a critical residue for binding the C-6 phospho group of fructose 2,6-bisphosphate to the fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Li, L., Lin, K., Correia, J., and Pilkis, S. J. (1992) J. Biol. Chem. 267, 16669-16675). To ascertain whether the three other basic residues (Arg-352, Arg-358, and Arg-360), which are located in a surface loop (residues 331-362) which contains Lys-356, are important in substrate binding, these arginyl residues were mutated to Ala, and each arginyl mutant was expressed in Escherichia coli and purified to homogeneity. The far UV circular dichroism spectra of the mutants were identical to that of the wild-type enzyme. The kinetic parameters of 6-phosphofructo-2-kinase of the mutants revealed only small changes. However, the Km for fructose 2,6-bisphosphate, Ki for fructose 6-phosphate, and Ka for inorganic phosphate of fructose-2,6-bisphosphatase for Arg352Ala were, respectively, 2,800-, 4,500-, and 1,500-fold higher than those for the wild-type enzyme, whereas there was no change in the maximal velocity or the Ki for inorganic phosphate. The Km for fructose 2,6-bisphosphate and Ki for inorganic phosphate of Arg360Ala were 10- and 12-fold higher, respectively, than those of the wild-type enzyme, whereas the maximal velocity and Ki for fructose 6-phosphate were unchanged. In addition, substrate inhibition was not observed with Arg352Ala and greatly reduced with Arg360Ala. The properties of the Arg358Ala mutant were identical to those of the wild-type enzyme. The results demonstrate that in addition to Lys-356, Arg-352 is another critical residue in fructose-2,6-bisphosphatase for binding the C-6 phospho group of fructose 2,6-bisphosphate and that Arg-360 binds the C-2 phospho group of fructose 2,6-bisphosphate in the phosphoenzyme.fructose 2,6-bisphosphate complex. The results also provide support for Arg-352, Lys-356, and Arg-360 constituting a specificity pocket for fructose-2,6-bisphosphatase.     

Nic1 Inactivation Enables Stable Isotope Labeling with 13C615N4-Arginine in Schizosaccharomyces pombe

Stable Isotope Labeling by Amino Acids (SILAC) is a commonly used method in quantitative proteomics. Because of compatibility with trypsin digestion, arginine and lysine are the most widely used amino acids for SILAC labeling. We observed that Schizosaccharomyces pombe (fission yeast) cannot be labeled with a specific form of arginine, ¹³C₆¹⁵N₄-arginine (Arg-10), which limits the exploitation of SILAC technology in this model organism. We hypothesized that in the fission yeast the guanidinium group of ¹³C₆¹⁵N₄-arginine is catabolized by arginase and urease activity to ¹⁵N₁-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni²⁺-dependent urease activity, through deletion of the sole Ni²⁺ transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable ¹³C₆¹⁵N₄-arginine labeling for SILAC strategies in S. pombe. Finally, we employed Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M, and G2 cell cycle phases in S. pombe.Stable Isotope Labeling by Amino acids in Cell culture (SILAC)¹ is one of the most widely used methods in quantitative proteomics (1). It involves in vivo metabolic labeling of cell cultures (or small organisms) with different versions of stable isotope-labeled amino acids (2). To maximize the number of peptides that can be quantified after proteome digestion with trypsin, proteins are usually differentially labeled with different forms of lysine and arginine (3): l-lysine (Lys-0) and l-arginine (Arg-0); ²H₄-lysine(Lys-4) and ¹³C₆-arginine (Arg-6); or ¹³C₆-¹⁵N₂-lysine (Lys-8) and ¹³C₆-¹⁵N₄-arginine (Arg-10). The availability of multiple forms of labeled lysine and arginine support the application of SILAC in duplex (comparison of two states) or triplex (comparison of three states) formats. Efficient anabolic pathways mean that lysine and arginine are not essential for growth of wild type yeast cells. Auxotrophic mutants that are defective in these pathways can be used to switch yeast to an absolute dependence upon the provision of these amino acids in the external medium. Consequently, mutations in arginine and lysine biosynthesis pathways can be used to drive the complete labeling of all tryptic peptides with specific forms of these amino acids (4, 5). SILAC has been used in quantitative proteomics in several yeast species, but most widely in Saccharomyces cerevisiae (6) (budding yeast) and Schizosaccharomyces pombe (fission yeast). S. pombe is extensively exploited to study cell cycle control (7), heterochromatin (8), and differentiation (9) and is increasingly the subject of large-scale quantitative proteomic studies (10, 11).A major challenge that is faced when using SILAC in fission yeast, is metabolic conversion of arginine to other amino acids such as proline, glutamine, and lysine (5). This partial labeling of additional amino acids after the conversion event produces spectra with complex isotope clusters that makes the downstream analysis challenging and error-prone. Inactivation of the “arginine conversion pathway” by removal of the orthinine transferase, Car2, effectively overcomes this problem to support the use of arginine labeling in SILAC-based experiments (5). Although this exploitation of the car2.Δ mutation now enables SILAC technology in fission yeast, the choice of amino acids that can be employed remains limited. Only one form of heavy arginine (R6) is currently used alongside three forms of heavy lysine (Lys-4, Lys-6, and Lys-8) (5, 12). Surprisingly, we could not find any studies that use arginine (Arg-10) in fission yeast, even though this is a widely exploited reagent for labeling other cell types (1).Here, we show that labeling of fission yeast with Arg-10 leads to a general misincorporation of the stable isotope label that prevents the identification of labeled peptides. We hypothesize that successive arginase and urease activities catabolize the guanidinium group of Arg-10 to ¹⁵N₁-labeled ammonia. This labeled ammonium is then used as a general precursor for amino acid biosynthesis. Disruption of Ni²⁺-dependent urease activity through deletion of Ni²⁺ transporter Nic1 in ammonium-supplemented medium, blocked this recycling to support ¹³C₆¹⁵N₄-arginine labeling SILAC strategies. As a proof of principle we employ Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M, and G2 cell cycle phases in S. pombe.     

Secondary substrate-binding exosite in the serine protease domain of activated protein C important for cleavage at Arg-506 but not at Arg-306 in factor Va

Proteolytic inactivation of activated factor V (FVa) by activated protein C (APC) is a key reaction in the regulation of hemostasis. We now demonstrate the importance of a positive cluster in loop 37 of the serine protease (SP) domain of APC for the degradation of FVa. Lysine residues in APC at positions 37, 38, and 39 form a secondary binding site for FVa, which is important for cleavage of FVa at Arg-506 while having no effect on Arg-306 cleavage. In contrast, topological neighbors Lys-62, Lys-63, and Arg-74 in APC appear of minor importance in FVa degradation. This demonstrates that secondary binding exosites of APC specifically guide the proteolytic action of APC, resulting in a more favorable degradation of the 506-507 peptide bond as compared with the 306-307 bond.     

Functional categorization of the conserved basic amino acid residues in TrmH (tRNA (Gm18) methyltransferase) enzymes

Transfer RNA (Gm18) methyltransferase (TrmH) catalyzes the methyl transfer from S-adenosyl-L-methionine (AdoMet) to the 2'-OH group of the G18 ribose in tRNA. To identify amino acid residues responsible for the tRNA recognition, we have carried out the alanine substitution mutagenesis of the basic amino acid residues that are conserved only in TrmH enzymes and not in the other SpoU proteins. We analyzed the mutant proteins by S-adenosyl-L-homocysteine affinity column chromatography, gel mobility shift assay, and kinetic assay of the methyl transfer reaction. Based on these biochemical studies and the crystal structure of TrmH, we found that the conserved residues can be categorized according to their role (i) in the catalytic center (Arg-41), (ii) in the initial site of tRNA binding (Lys-90, Arg-166, Arg-168, and Arg-176), (iii) in the tRNA binding site required for continuation the catalytic cycle (Arg-8, Arg-19, and Lys-32), (iv) in the structural element involved in release of S-adenosyl-L-homocysteine (Arg-11-His-71-Met-147 interaction), (v) in the assisted phosphate binding site (His-34), or (vi) in an unknown function (Arg-109). Furthermore, the difference between the Kd and Km values for tRNA suggests that the affinity for tRNA is enhanced in the presence of AdoMet. To confirm this idea, we carried out the kinetic studies, a gel mobility shift assay with a mutant protein disrupted in the catalytic center, and the analytical gel-filtration chromatography. Our experimental results clearly show that the enzyme has a semi-ordered sequential mechanism in which AdoMet both enhances the affinity for tRNA and induces formation of the tetramer structure.     

Interaction of gamma-glutamyl transpeptidase with glutathione involves specific arginine and lysine residues of the heavy subunit.

Gamma-glutamyl transpeptidase, an enzyme of importance in glutathione metabolism, consists of two subunits, one of which (the light subunit, Mr 22,000; residues 380-568; rat kidney) contains residue Thr-523, which selectively interacts with the substrate analog acivicin to form an adduct that is apparently analogous to the gamma-glutamyl enzyme intermediate formed in the normal reaction (Stole, E., Seddon, A. P., Wellner, D., and Meister, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1706-1709). The present studies indicate that specific arginine and lysine residues of the heavy subunit (Mr 51,000; residues 31-379) participate in catalysis by binding the substrates. Selective labeling studies of the enzyme with [14C]phenylglyoxal showed that Lys-99 and Arg-111 were modified. This appears to be the first instance in which phenylglyoxal was found to react with an enzyme lysine residue. Incorporation of [14C]phenylglyoxal into Lys-99 was decreased in the presence of acceptor site selective compounds. Incorporation into both Lys-99 and Arg-111 was decreased in the presence of glutathione. The findings suggest that Lys-99 and Arg-111 interact, respectively, with the omega- and alpha-carboxyl groups of glutathione. That these putative electrostatic binding sites are on the heavy subunit indicates that both subunits contribute to the active center. Two additional heavy subunit arginine residues become accessible to modification by phenylglyoxal when acivicin is bound, suggesting that interaction with acivicin is associated with a conformational change.     

CXCR3 and heparin binding sites of the chemokine IP-10 (CXCL10)

The chemokine IP-10 (interferon-inducible protein of 10 kDa, CXCL10) binds the G protein-coupled receptor CXCR3, which is found mainly on activated T cells and NK cells, and plays an important role in Th1-type inflammatory diseases. IP-10 also binds to glycosaminoglycans (GAGs), an interaction thought to be important for its sequestration on endothelial and other cells. In this study, we performed an extensive mutational analysis to identify the CXCR3 and heparin binding sites of murine IP-10. The mutants were characterized for heparin binding, CXCR3 binding, and the ability to induce chemotaxis, Ca(2+) flux, and CXCR3 internalization. Double mutations neutralizing adjacent basic residues at the C terminus did not lead to a significant reduction in heparin binding, indicating that the main heparin binding site of IP-10 is not along the C-terminal alpha helix. Alanine exchange of Arg-22 had the largest effect on heparin binding, with residues Arg-20, Ile-24, Lys-26, Lys-46, and Lys-47 further contributing to heparin binding. A charge change mutation of Arg-22 resulted in further reduction in heparin binding. The N-terminal residue Arg-8, preceding the first cysteine, was critical for CXCR3 signaling. Mutations of charged and uncharged residues in the loop regions of residues 20-24 and 46-47, which caused reduced heparin binding, also resulted in reduced CXCR3 binding and signaling. CXCR3 expressing GAG-deficient Chinese hamster ovary cells revealed that GAG binding was not required for IP-10 binding and signaling through CXCR3, which suggests that the CXCR3 and heparin binding sites of IP-10 are partially overlapping.     

The role of positively charged amino acids in ATP recognition by human P2X(1) receptors

P2X receptors for ATP are a family of ligand-gated cation channels. There are 11 conserved positive charges in the extracellular loop of P2X receptors. We have generated point mutants of these conserved residues (either Lys --> Arg, Lys --> Ala, Arg --> Lys, or Arg --> Ala) in the human P2X(1) receptor to determine their contribution to the binding of negatively charged ATP. ATP evoked concentration-dependent (EC(50) approximately 0.8 microm) desensitizing responses at wild-type (WT) P2X(1) receptors expressed in Xenopus oocytes. Suramin produced a parallel rightward shift in the concentration response curve with an estimated pK(B) of 6.7. Substitution of amino acids at positions Lys-53, Lys-190, Lys-215, Lys-325, Arg-202, Arg-305, and Arg-314 either had no effect or only a small change in ATP potency, time course, and/or suramin sensitivity. Modest changes in ATP potency were observed for mutants at K70R and R292K/A (20- and 100-fold decrease, respectively). Mutations at residues K68A and K309A reduced the potency of ATP by >1400-fold and prolonged the time course of the P2X(1) receptor current but had no effect on suramin antagonism. Lys-68, Lys-70, Arg-292, and Lys-309 are close to the predicted transmembrane domains of the receptor and suggest that the ATP binding pocket may form close to the channel vestibule.     

The contribution of adjacent subunits to the active sites of D-3-phosphoglycerate dehydrogenase

D-3-Phosphoglycerate dehydrogenase (PGDH) from Escherichia coli is allosterically inhibited by L-serine, the end product of its metabolic pathway. Previous results have shown that inhibition by serine has a large effect on Vmax and only a small or negligible effect on Km. PGDH is thus classified as a V-type allosteric enzyme. In this study, the active site of PGDH has been studied by site-directed mutagenesis to assess the role of certain residues in substrate binding and catalysis. These consist of a group of cationic residues (Arg-240, Arg-60, Arg-62, Lys-39, and Lys-141') that potentially form an electrostatic environment for the binding of the negatively charged substrate, as well as the only tryptophan residue found in PGDH and which fits into a hydrophobic pocket immediately adjacent to the active site histidine residue. Interestingly, Trp-139' and Lys-141' are part of the polypeptide chain of the subunit that is adjacent to the active site. The results of mutating these residues show that Arg-240, Arg-60, Arg-62, and Lys-141' play distinct roles in the binding of the substrate to the active site. Mutants of Trp-139' show that this residue may play a role in stabilizing the catalytic center of the enzyme. Furthermore, these mutants appear to have a significant effect on the cooperativity of serine inhibition and suggest a possible role for Trp-139' in the cooperative interactions between subunits.