Identification by mutagenesis of conserved arginine and glutamate residues in the C-terminal domain of rat liver carnitine palmitoyltransferase I that are important for catalytic activity and malonyl-CoA sensitivity

Carnitine palmitoyltransferase I (CPTI) catalyzes the conversion of long chain fatty acyl-CoAs to acylcarnitines in the presence of l-carnitine. To determine the role of the conserved glutamate residue, Glu-603, on catalysis and malonyl-CoA sensitivity, we separately changed the residue to alanine, histidine, glutamine, and aspartate. Substitution of Glu-603 with alanine or histidine resulted in complete loss of L-CPTI activity. A change of Glu-603 to glutamine caused a significant decrease in catalytic activity and malonyl-CoA sensitivity. Substitution of Glu-603 with aspartate, a negatively charged amino acid with only one methyl group less than the glutamate residue in the wild type enzyme, resulted in partial loss in CPTI activity and a 15-fold decrease in malonyl-CoA sensitivity. The mutant L-CPTI with a replacement of the conserved Arg-601 or Arg-606 with alanine also showed over 40-fold decrease in malonyl-CoA sensitivity, suggesting that these two conserved residues may be important for substrate and inhibitor binding. Since a conservative substitution of Glu-603 to aspartate or glutamine resulted in partial loss of activity and malonyl-CoA sensitivity, it further suggests that the negative charge and the longer side chain of glutamate are essential for catalysis and malonyl-CoA sensitivity. We predict that this region of L-CPTI spanning these conserved C-terminal residues may be the region of the protein involved in binding the CoA moiety of palmitoyl-CoA and malonyl-CoA and/or the putative low affinity acyl-CoA/malonyl-CoA binding site.     

The extreme C terminus of rat liver carnitine palmitoyltransferase I is not involved in malonyl-CoA sensitivity but in initial protein folding

We previously reported that the N-terminal domain (1-147 residues) of rat liver carnitine palmitoyltransferase I (L-CPTI) was essential for import into the outer mitochondrial membrane and for maintenance of a malonyl-CoA-sensitive conformation. Malonyl-CoA binding experiments using mitochondria of Saccharomyces cerevisiae strains expressing wild-type L-CPTI or previously constructed chimeric CPTs (Cohen, I., Kohl, C., McGarry, J.D., Girard, J., and Prip-Buus, C. (1998) J. Biol. Chem. 273, 29896-29904) indicated that the N-terminal domain was unable, independently of the C-terminal domain, to bind malonyl-CoA with a high affinity, suggesting that the modulation of malonyl-CoA sensitivity occurred through N/C intramolecular interactions. To assess the role of the C terminus in malonyl-CoA sensitivity, a series of C-terminal deletion mutants was generated. The kinetic properties of Delta772-773 and Delta767-773 deletion mutants were similar to those of L-CPTI, indicating that the last two highly conserved Lys residues in all known L-CPTI species were not functionally essential. By contrast, Delta743-773 deletion mutant was totally inactive and unfolded, as shown by its sensitivity to trypsin proteolysis. Because the C terminus of the native folded L-CPTI could be cleaved by trypsin without inducing protein unfolding, we concluded that the last 31 C-terminal residues constitute a secondary structural determinant essential for the initial protein folding of L-CPTI.     

The first 28 N-terminal amino acid residues of human heart muscle carnitine palmitoyltransferase I are essential for malonyl CoA sensitivity and high-affinity binding

Heart/skeletal muscle carnitine palmitoyltransferase I (M-CPTI) is 30-100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). To determine the role of the N-terminal region of human heart M-CPTI on malonyl CoA sensitivity and binding, a series of deletion mutations were constructed ranging in size from 18 to 83 N-terminal residues. All of the deletions except Delta83 were active. Mitochondria from the yeast strains expressing Delta28 and Delta39 exhibited a 2.5-fold higher activity compared to the wild type, but were insensitive to malonyl CoA inhibition and had complete loss of high-affinity malonyl CoA binding. The high-affinity site (K(D1), B(max1)) for binding of malonyl CoA to M-CPTI was completely abolished in the Delta28, Delta39, Delta51, and Delta72 mutants, suggesting that the decrease in malonyl CoA sensitivity observed in these mutants was due to the loss of the high-affinity binding entity of the enzyme. Delta18 showed only a 4-fold loss in malonyl CoA sensitivity but had activity and high-affinity malonyl CoA binding similar to the wild type. Replacement of the N-terminal domain of L-CPTI with the N-terminal domain of M-CPTI does not change the malonyl CoA sensitivity of the chimeric L-CPTI, suggesting that the amino acid residues responsible for the differing sensitivity to malonyl CoA are not located in this N-terminal region. These results demonstrate that the N-terminal residues critical for activity and malonyl CoA sensitivity in M-CPTI are different from those of L-CPTI.     

Pig liver carnitine palmitoyltransferase. Chimera studies show that both the N- and C-terminal regions of the enzyme are important for the unusual high malonyl-CoA sensitivity.

Pig and rat liver carnitine palmitoyltransferase I (L-CPTI) share common K(m) values for palmitoyl-CoA and carnitine. However, they differ widely in their sensitivity to malonyl-CoA inhibition. Thus, pig l-CPTI has an IC(50) for malonyl-CoA of 141 nm, while that of rat L-CPTI is 2 microm. Using chimeras between rat L-CPTI and pig L-CPTI, we show that the entire C-terminal region behaves as a single domain, which dictates the overall malonyl-CoA sensitivity of this enzyme. The degree of malonyl-CoA sensitivity is determined by the structure adopted by this domain. Using deletion mutation analysis, we show that malonyl-CoA sensitivity also depends on the interaction of this single domain with the first 18 N-terminal amino acid residues. We conclude that pig and rat L-CPTI have different malonyl-CoA sensitivity, because the first 18 N-terminal amino acid residues interact differently with the C-terminal domain. This is the first study that describes how interactions between the C- and N-terminal regions can determine the malonyl-CoA sensitivity of L-CPTI enzymes using active C-terminal chimeras.     

Substitution of glutamate-3, valine-19, leucine-23, and serine-24 with alanine in the N-terminal region of human heart muscle carnitine palmitoyltransferase I abolishes malonyl CoA inhibition and binding

The muscle isoform of carnitine palmitoyltransferase I (M-CPTI) is 30- to 100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). We have previously shown that deletion of the first 28 N-terminal amino acid residues in M-CPTI abolished malonyl CoA inhibition and high-affinity binding [Biochemistry 39 (2000) 712-717]. To determine the role of specific residues within the first 28 N-terminal amino acids of human heart M-CPTI on malonyl CoA sensitivity and binding, we constructed a series of substitution mutations and a mutant M-CPTI composed of deletion 18 combined with substitution mutations V19A, L23A, and S24A. All mutants had CPT activity similar to that of the wild type. A change of Glu3 to Ala resulted in a 60-fold decrease in malonyl CoA sensitivity and loss of high-affinity malonyl CoA binding. A change of His5 to Ala in M-CPTI resulted in only a 2-fold decrease in malonyl CoA sensitivity and a significant loss in the low- but not high-affinity malonyl CoA binding. Deletion of the first 18 N-terminal residues combined with substitution mutations V19A, L23A, and S24A resulted in a mutant M-CPTI with an over 140-fold decrease in malonyl CoA sensitivity and a significant loss in both high- and low-affinity malonyl CoA binding. This was further confirmed by a combined four-residue substitution of Glu3, Val19, Leu23, and Ser24 with alanine. Our site-directed mutagenesis studies demonstrate that Glu3, Val19, Leu23, and Ser24 in M-CPTI are important for malonyl CoA inhibition and binding, but not for catalysis.     

Identification by mutagenesis of conserved arginine and tryptophan residues in rat liver carnitine palmitoyltransferase I important for catalytic activity

Carnitine palmitoyltransferase I catalyzes the conversion of long-chain acyl-CoA to acylcarnitines in the presence of l-carnitine. To determine the role of the conserved arginine and tryptophan residues on catalytic activity in the liver isoform of carnitine palmitoyltransferase I (L-CPTI), we separately mutated five conserved arginines and two tryptophans to alanine. Substitution of arginine residues 388, 451, and 606 with alanine resulted in loss of 88, 82, and 93% of L-CPTI activity, respectively. Mutants R601A and R655A showed less than 2% of the wild type L-CPTI activity. A change of tryptophan 391 and 452 to alanine resulted in 50 and 93% loss in carnitine palmitoyltransferase activity, respectively. The mutations caused decreases in catalytic efficiency of 80-98%. The residual activity in the mutant L-CPTIs was sensitive to malonyl-CoA inhibition. Mutants R388A, R451A, R606A, W391A, and W452A had no effect on the K(m) values for carnitine or palmitoyl-CoA. However, these mutations decreased the V(max) values for both substrates by 10-40-fold, suggesting that the main effect of the mutations was to decrease the stability of the enzyme-substrate complex. We suggest that conserved arginine and tryptophan residues in L-CPTI contribute to the stabilization of the enzyme-substrate complex by charge neutralization and hydrophobic interactions. The predicted secondary structure of the 100-amino acid residue region of L-CPTI, containing arginines 388 and 451 and tryptophans 391 and 452, consists of four alpha-helices similar to the known three-dimensional structure of the acyl-CoA-binding protein. We predict that this 100-amino acid residue region constitutes the putative palmitoyl-CoA-binding site in L-CPTI.     

A single amino acid change (substitution of the conserved Glu-590 with alanine) in the C-terminal domain of rat liver carnitine palmitoyltransferase I increases its malonyl-CoA sensitivity close to that observed with the muscle isoform of the enzyme

Carnitine palmitoyltransferase I (CPTI) catalyzes the conversion of long-chain fatty acyl-CoAs to acylcarnitines in the presence of l-carnitine. To determine the role of the highly conserved C-terminal glutamate residue, Glu-590, on catalysis and malonyl-CoA sensitivity, we separately changed the residue to alanine, lysine, glutamine, and aspartate. Substitution of Glu-590 with aspartate, a negatively charged amino acid with only one methyl group less than the glutamate residue in the wild-type enzyme, resulted in complete loss in the activity of the liver isoform of CPTI (L-CPTI). A change of Glu-590 to alanine, glutamine, and lysine caused a significant 9- to 16-fold increase in malonyl-CoA sensitivity but only a partial decrease in catalytic activity. Substitution of Glu-590 with neutral uncharged residues (alanine and glutamine) and/or a basic positively charged residue (lysine) significantly increased L-CPTI malonyl-CoA sensitivity to the level observed with the muscle isoform of the enzyme, suggesting the importance of neutral and/or positive charges in the switch of the kinetic properties of L-CPTI to the muscle isoform of CPTI. Since a conservative substitution of Glu-590 to aspartate but not glutamine resulted in complete loss in activity, we suggest that the longer side chain of glutamate is essential for catalysis and malonyl-CoA sensitivity. This is the first demonstration whereby a single residue mutation in the C-terminal region of the liver isoform of CPTI resulted in a change of its kinetic properties close to that observed with the muscle isoform of the enzyme and provides the rationale for the high malonyl-CoA sensitivity of muscle CPTI compared with the liver isoform of the enzyme.     

Acyl-CoA binding proteins interact with the acyl-CoA binding domain of mitochondrial carnitine palmitoyl transferase I

Although the rate limiting step in mitochondrial fatty acid oxidation, catalyzed by carnitine palmitoyl transferase I (CPTI), utilizes long-chain fatty acyl-CoAs (LCFA-CoA) as a substrate, how LCFA-CoA is transferred to CPTI remains elusive. Based on secondary structural predictions and conserved tryptophan residues, the cytoplasmic C-terminal domain was hypothesized to be the LCFA-CoA binding site and important for interaction with cytoplasmic LCFA-CoA binding/transport proteins to provide a potential route for LCFA-CoA transfer. To begin to address this question, the cytoplasmic C-terminal region of liver CPTI (L-CPTI) was recombinantly expressed and purified. Data herein showed for the first time that the L-CPTI C-terminal 89 residues were sufficient for high affinity binding of LCFA-CoA (K (d) = 2-10 nM) and direct interaction with several cytoplasmic LCFA-CoA binding proteins (K (d) < 10 nM), leading to enhanced CPTI activity. Furthermore, alanine substitutions for tryptophan in L-CPTI (W391A and W452A) altered secondary structure, decreased binding affinity for LCFA-CoA, and almost completely abolished L-CPTI activity, suggesting that these amino acids may be important for ligand stabilization necessary for L-CPTI activity. Moreover, while decreased activity of the W452A mutant could be explained by decreased binding of lipid binding proteins, W391 itself seems to be important for activity. These data suggest that both interactions with lipid binding proteins and the peptide itself are important for optimal enzyme activity.     

Identification of conserved amino acid residues in rat liver carnitine palmitoyltransferase I critical for malonyl-CoA inhibition. Mutation of methionine 593 abolishes malonyl-CoA inhibition

Carnitine palmitoyltransferase (CPT) I, which catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine facilitating its transport through the mitochondrial membranes, is inhibited by malonyl-CoA. By using the SequenceSpace algorithm program to identify amino acids that participate in malonyl-CoA inhibition in all carnitine acyltransferases, we found 5 conserved amino acids (Thr(314), Asn(464), Ala(478), Met(593), and Cys(608), rat liver CPT I coordinates) common to inhibitable malonyl-CoA acyltransferases (carnitine octanoyltransferase and CPT I), and absent in noninhibitable malonyl-CoA acyltransferases (CPT II, carnitine acetyltransferase (CAT) and choline acetyltransferase (ChAT)). To determine the role of these amino acid residues in malonyl-CoA inhibition, we prepared the quintuple mutant CPT I T314S/N464D/A478G/M593S/C608A as well as five single mutants CPT I T314S, N464D, A478G, M593S, and C608A. In each case the CPT I amino acid selected was mutated to that present in the same homologous position in CPT II, CAT, and ChAT. Because mutant M593S nearly abolished the sensitivity to malonyl-CoA, two other Met(593) mutants were prepared: M593A and M593E. The catalytic efficiency (V(max)/K(m)) of CPT I in mutants A478G and C608A and all Met(593) mutants toward carnitine as substrate was clearly increased. In those CPT I proteins in which Met(593) had been mutated, the malonyl-CoA sensitivity was nearly abolished. Mutations in Ala(478), Cys(608), and Thr(314) to their homologous amino acid residues in CPT II, CAT, and ChAT caused various decreases in malonyl-CoA sensitivity. Ala(478) is located in the structural model of CPT I near the catalytic site and participates in the binding of malonyl-CoA in the low affinity site (Morillas, M., Gómez-Puertas, P., Rubi, B., Clotet, J., Ari?o, J., Valencia, A., Hegardt, F. G., Serra, D., and Asins, G. (2002) J. Biol. Chem. 277, 11473-11480). Met(593) may participate in the interaction of malonyl-CoA in the second affinity site, whose location has not been reported.     

Deletion of N-terminal amino acids from human lecithin:cholesterol acyltransferase differentially affects enzyme activity toward alpha- and beta-substrate lipoproteins

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for generation of the majority of the cholesteryl esters (CE) in human plasma. Although most plasma cholesterol esterification occurs on high-density lipoprotein (HDL), via alpha-LCAT activity, esterification also occurs on low-density lipoprotein (LDL) via the beta-activity of the enzyme. Computer threading techniques have provided a three-dimensional model for use in the structure-function analysis of the core and catalytic site of the LCAT protein, but the model does not extend to the N-terminal region of the enzyme, which may mediate LCAT interaction with lipoprotein substrates. In the present study, we have examined the functional consequences of deletion of the highly conserved hydrophobic N-terminal amino acids (residues 1-5) of human LCAT. Western blot analysis showed that the mutant proteins (Delta 1-Delta 5) were synthesized and secreted from transfected COS-7 cells at levels approximately equivalent to those of wild-type hLCAT. The secreted proteins had apparent molecular weights of 67 kDa, indicating that they were correctly processed and glycosylated during cellular transit. However, deletion of the first residue of the mature LCAT protein (Delta 1 mutant) resulted in a dramatic loss of alpha-LCAT activity (5% of wild type using reconstituted HDL substrate, rHDL), although this mutant retained full beta-LCAT activity (108% of wild-type using human LDL substrate). Removal of residues 1 and 2 (Delta 2 mutant) abolished alpha-LCAT activity and reduced beta-LCAT activity to 12% of wild type. Nevertheless, LCAT Delta 1 and Delta 2 mutants retained their ability to bind to rHDL and LDL lipoprotein substrates. The dramatic loss of enzyme activity suggests that the N-terminal residues of LCAT may be involved in maintaining the conformation of the lid domain and influence activation by the alpha-LCAT cofactor apoA-I (in Delta 1) and/or loss of enzyme activity (in Delta 1-Delta 5). Since the Delta 1 and Delta 2 mutants retain their ability to bind substrate, other factor(s), such as decreased access to the substrate binding pocket, may be responsible for the loss of enzyme activity.     

A receptor-binding region in Escherichia coli alpha-haemolysin

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

Active-site residues of a plant membrane-bound fatty acid elongase β-ketoacyl-CoA synthase,FAE1 KCS

The fatty acid elongase-1 β-ketoacyl-CoA synthase, FAE1 KCS, a seed-specific elongase condensing enzyme from Arabidopsis, is involved in the production of eicosenoic (C20:1) and erucic (C22:1) acids. Alignment of the amino acid sequences of FAE1 KCS, KCS1, and five other putative elongase condensing enzymes (KCSs) revealed the presence of six conserved cysteine and four conserved histidine residues. Each of the conserved cysteine and histidine residues was individually converted by site-directed mutagenesis to both alanine and serine, and alanine and lysine respectively. After expression in yeast cells, the mutant enzymes were analyzed for their fatty acid elongase activity. Our results indicated that only cysteine 223 is an essential residue for enzyme activity, presumably for acyl chain transfer. All histidine substitutions resulted in complete loss of elongase activity. The loss of activity of these mutants was not due to their lower expression level since immunoblot analysis confirmed each was expressed to the same extent as the wild type FAE1 KCS.     

Mutational analysis of catalytic sites of the cell wall lytic N-acetylmuramoyl-L-alanine amidases CwlC and CwlV

The Bacillus subtilis CwlC and the Bacillus polymyxa var. colistinus CwlV are the cell wall lytic N-acetylmuramoyl-l-alanine amidases in the CwlB (LytC) family. Deletion in the CwlC amidase from the C terminus to residue 177 did not change the amidase activity. However, when the deletion was extended slightly toward the N terminus, the amidase activity was entirely lost. Further, the N-terminal deletion mutant without the first 19 amino acids did not have the amidase activity. These results indicate that the N-terminal half (residues 1-176) of the CwlC amidase, the region homologous to the truncated CwlV (CwlVt), is a catalytic domain. Site-directed mutagenesis was performed on 20 highly conserved amino acid residues within the catalytic domain of CwlC. The amidase activity was lost completely on single amino acid substitutions at two residues (Glu-24 and Glu-141). Similarly, the substitution of the two glutamic acid residues (E26Q and E142Q) of the truncated CwlV (CwlV1), which corresponded to Glu-24 and Glu-141 of CwlC, was critical to the amidase activity. The EDTA-treated CwlV1 did not have amidase activity. The amidase activity of the EDTA-treated CwlV1 was restored by the addition of Zn2+, Mn2+, and Co2+ but not by the addition of Mg2+ and Ca2+. These results suggest that the amidases in the CwlB family are zinc amidases containing two glutamic acids as catalytic residues.     

Identification of positive and negative determinants of malonyl-CoA sensitivity and carnitine affinity within the amino termini of rat liver- and muscle-type carnitine palmitoyltransferase I

The extreme amino terminus and, in particular, residue Glu-3 in rat liver (L) carnitine palmitoyltransferase I (CPT I) have previously been shown to be essential for the sensitivity of the enzyme to inhibition by malonyl-CoA. Using the Pichia pastoris expression system, we now observe that, although mutants E3A (Glu-3 --> Ala) or Delta(3-18) of L-CPT I have markedly lowered sensitivity to malonyl-CoA compared with the wild-type protein, the mutant Delta(1-82) generated an enzyme that had regained much of the sensitivity of wild-type CPT I. This suggests that a region antagonistic to malonyl-CoA sensitivity is present within residues 19-82 of the enzyme. This was confirmed in the construct Delta(19-30), which was found to be 50-fold more sensitive than wild-type L-CPT I. Indeed, this mutant was >4-fold more sensitive than even the native muscle (M)-CPT I isoform expressed and assayed under identical conditions. This behavior was dependent on the presence of Glu-3, with the mutant E3A-Delta(19-30) having kinetic characteristics similar to those of the E3A mutant. The increase in the sensitivity of the L-CPT I-Delta(19-30) mutant was not due to a change in the mechanism of inhibition with respect to palmitoyl-CoA, nor to any marked change of the K(0.5) for this substrate. Conversely, for M-CPT I, a decrease in malonyl-CoA sensitivity was invariably observed with increasing deletions from Delta(3-18) to Delta(1-80). However, deletion of residues 3-18 from M-CPT I affected the K(m) for carnitine of this isoform, but not of L-CPT I. These observations (i) provide the first evidence for negative determinants of malonyl-CoA sensitivity within the amino-terminal segment of L-CPT I and (ii) suggest a mechanism for the inverse relationship between affinity for malonyl-CoA and for carnitine of the two isoforms of the enzyme.     

The CD14 region spanning amino acids 57-64 is critical for interaction with the extracellular Toll-like receptor 2 domain

CD14 has been shown to enhance Toll-like receptor 2 (TLR2)-mediated signaling in response to peptidoglycan. Anti-CD14 monoclonal antibody MEM-18, whose epitope was located at the amino acid residues 57-64, blocked the binding of sCD14 to the recombinant soluble form of the extracellular TLR2 domain (sTLR2). The deletion mutant sCD14Delta57-64 lacking the amino acid residues 57-64 failed to bind to sTLR2. Cotransfection of wild type mCD14 but not mCD14Delta57-64 with TLR2 enhanced NF-kappaB activation in response to peptidoglycan. These results indicate that the CD14 region spanning amino acids 57-64 is critical for interacting with TLR2 and enhancing TLR2-mediated peptidoglycan signaling.     

Mutational Analysis of Residues in the Coiled-Coil Domain of Human Immunodeficiency Virus Type 1 Transmembrane Protein gp41

The envelope glycoprotein (Env) of human immunodeficiency virus mediates virus entry into cells by undergoing conformational changes that lead to fusion between viral and cellular membranes. A six-helix bundle in gp41, consisting of an interior trimeric coiled-coil core with three exterior helices packed in the grooves (core structure), has been proposed to be part of a fusion-active structure of Env (D. C. Chan, D. Fass, J. M. Berger, and P. S. Kim, Cell 89:263–273, 1997; W. Weissenhorn, A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley, Nature 387:426–430, 1997; and K. Tan, J. Liu, J. Wang, S. Shen, and M. Lu, Proc. Natl. Acad. Sci. USA 94:12303, 1997). We analyzed the effects of amino acid substitutions of arginine or glutamic acid in residues in the coiled-coil (heptad repeat) domain that line the interface between the helices in the gp41 core structure. We found that mutations of leucine to arginine or glutamic acid in position 556 and of alanine to arginine in position 558 resulted in undetectable levels of Env expression. Seven other mutations in six positions completely abolished fusion activity despite incorporation of the mutant Env into virions and normal gp160 processing. Single-residue substitutions of glutamic acid at position 570 or 577 resulted in the only viable mutants among the 16 mutants studied, although both viable mutants exhibited impaired fusion activity compared to that of the wild type. The glutamic acid 577 mutant was more sensitive than the wild type to inhibition by a gp41 coiled-coil peptide (DP-107) but not to that by another peptide corresponding to the C helix in the gp41 core structure (DP-178). These results provide insight into the gp41 fusion mechanism and suggest that the DP-107 peptide may inhibit fusion by binding to the homologous region in gp41, probably by forming a peptide-gp41 coiled-coil structure.     

Differential determinants for peptide and non-peptidyl ligand binding to the motilin receptor. Critical role of second extracellular loop for peptide binding and action.

The predicted second extracellular loop domain of the motilin receptor is of particular interest because it is a region that is quite distinct from the analogous regions in other family members that are most closely related and because the initial report of the photoaffinity labeling of a domain of this receptor included this region (Coulie, B. J., Matsuura, B., Dong, M., Hadac, E. M., Pinon, D. I., Feighner, S. D., Howard, A. D., and Miller, L. J. (2001) J. Biol. Chem. 276, 35518-35522). In the current work, motilin receptor constructs were prepared that included sequential deletions ranging from single residues to twelve amino acid segments throughout this 67 amino acid domain. Each construct was expressed in COS cells and characterized for motilin radioligand binding and motilin-stimulated intracellular calcium responses. The only segments that had negative impact on motilin binding and biological activity included deletion constructs DeltaCys(235), Delta179-182, and Delta241-246. Cys(235) is likely involved in the highly conserved and functionally important disulfide bond linking the first and second loops of G protein-coupled receptors. Alanine replacements for each of the amino acid residues in the other two segments revealed that the perimembranous residues at both ends of this loop, Val(179) and Leu(245) and Arg(246), were responsible for the negative impact on motilin binding and biological activity. Of note, these mutants responded normally to the non-peptidyl agonist, erythromycin. These data support important functional roles for both amino-terminal and carboxyl-terminal perimembranous regions of the second loop for responses to the natural agonist peptide, while supporting independent determinants for action of a non-peptidyl agonist ligand.     

Identification of receptor binding and activation determinants in the N-terminal and N-loop regions of the CC chemokine eotaxin

Eotaxin is a CC chemokine that specifically activates the receptor CCR3 causing accumulation of eosinophils in allergic diseases and parasitic infections. Twelve amino acid residues in the N-terminal (residues 1-8) and N-loop (residues 11-20) regions of eotaxin have been individually mutated to alanine, and the ability of the mutants to bind and activate CCR3 has been determined in cell-based assays. The alanine mutants at positions Thr(7), Asn(12), Leu(13), and Leu(20) show near wild type binding affinity and activity. The mutants T8A, N15A, and K17A have near wild type binding affinity for CCR3 but reduced receptor activation. A third class of mutants, S4A, V5A, R16A, and I18A, display significantly perturbed binding affinity for CCR3 while retaining the ability to activate or partially activate the receptor. Finally, the mutant Phe(11) has little detectable activity and 20-fold reduced binding affinity relative to wild type eotaxin, the most dramatic effect observed in both assays but less dramatic than the effect of mutating the corresponding residue in some other chemokines. Taken together, the results indicate that residues contributing to receptor binding affinity and those required for triggering receptor activation are distributed throughout the N-terminal and N-loop regions. This conclusion is in contrast to the separation of binding and activation functions between N-loop and N-terminal regions, respectively, that has been observed previously for some other chemokines.