Conversion of a human low-density lipoprotein receptor ligandbinding repeat to a virus receptor: identification of residues important for ligand specificity

The amino-terminal region of the low-density lipoprotein receptor (LDLR) contains seven imperfect repeats of a cysteine-rich, roughly 40-aa module (LDL-A module) that are critical for apolipoprotein binding. LDL-A modules are found in numerous cell-surface and secreted proteins and are believed to mediate extracellular protein-protein interactions. The cellular receptor for subgroup A Rous sarcoma virus (RSV) contains a single LDL-A module that binds the RSV envelope protein and allows viral infection. To define residues in an LDL-A module responsible for ligand recognition, we used a gain of function assay by using a chimeric protein in which the LDL-A module of Tva was replaced with a highly homologous module from human LDLR (LDL-A4) and determined whether this chimera or mutants produced in it could mediate RSV infection. LDL-A4 does not function as an RSV receptor; however, systematic replacement of the nonconserved residues of the LDL-A4 module in the chimeric protein with the corresponding residues from Tva identified three residues sufficient to alter ligand specificity and convert LDL-A4 to an efficient viral receptor. Mutations of the corresponding residues in the Tva LDL-A module decreased both envelope binding and viral receptor function, confirming the importance of these residues in ligand recognition by this module. Analysis of the hLDL-A5 structure demonstrates that these three residues are clustered at one end of the LDL-A module. These results demonstrate that using a single LDL-A module in a gain of function assay is a useful model to investigate ligand recognition by this module.     

Functional mapping of the surface residues of human thrombin

Utilizing site-directed mutagenesis, 77 charged and polar residues that are highly exposed on the surface of human thrombin were systematically substituted with alanine. Functional assays using thrombin mutants identified residues that were required for the recognition and cleavage of the procoagulant substrate fibrinogen (Lys21, Trp50, Lys52, Asn53 + Thr55, Lys65, His66, Arg68, Tyr71, Arg73, Lys77, Lys106 + Lys107, Asp193 + Lys196, Glu202, Glu229, Arg233, Asp234) and the anticoagulant substrate protein C (Lys21, Trp50, Lys65, His66, Arg68, Tyr71, Arg73, Lys77, Lys106 + Lys107, Glu229, Arg233), interactions with the cofactor thrombomodulin (Gln24, Arg70) and inhibition by the thrombin aptamer, an oligonucleotide-based thrombin inhibitor (Lys65, His66, Arg70, Tyr71, Arg73). Although there is considerable overlap between the functional epitopes, distinct and specific residues with unique functions were identified. When the functional residues were mapped on the surface of thrombin, they were located on a single hemisphere of thrombin that included both the active site cleft and the highly basic exosite 1. No functional residues were located on the opposite face of thrombin. Residues with procoagulant or anticoagulant functions were not spatially separated but interdigitated with residues of opposite or shared function. Thus thrombin utilizes the same general surface for substrate recognition regardless of substrate function although the critical contact residues may vary.     

Further evidence for the presence of mitochondrially encoded subunits in cytochrome c oxidase of the trypanosomatid Crithidia fasciculata

Trimethylaminuria is an autosomal recessive human disorder affecting a small part of the population as an inherited polymorphism. Individuals diagnosed with trimethylaminuria excrete relatively large amounts of trimethylamine in their urine, sweat, and breath, and this results in a fishy odor characteristic of trimethylamine. Activity of the human flavin-containing monooxygenase (FMO) has been proposed to be deficient in trimethylaminuria patients causing a decrease in the metabolism of trimethylamine that results in a fishy body odor. Cohorts of Australian, American, and British individuals suffering from trimethylaminuria have been identified. The human FMO3 cDNA was amplified from lymphocytes of affected patients. We report preliminary evidence of substitutions detected by screening of the cDNA and genomic DNA. The variant human FMO3 cDNA was constructed from wild type human FMO3 cDNA by site-directed mutagenesis as maltose-binding protein fusions. Five distinct human FMO3 mutants were expressed as fusion proteins in Escherichia coli and compared with wild type human FMO3 maltose-binding proteins (FMO3-MBP) for the N-oxygenation of 10-[(N,N-dimethylamino)pentyl]-2-(trifluoromethyl)phenothiazine, tyramine, and trimethylamine. Human Lys158 FMO3-MBP and, to a greater extent, human Glu158 FMO3-MBP efficiently N-oxygenated the three amine substrates. Human Lys158 Ile66 FMO3-MBP, Glu158 Ile66 FMO3-MBP, Lys158 Leu153 FMO3-MBP, and Glu158 Leu153 FMO3-MBP were all constructed as mutants identified as possible FMO3 variants responsible for trimethylaminuria and were found to be inactive as N-oxygenases. The results suggest that mutations at codons 66 and 153 of FMO3 can cause trimethylaminuria in humans. We observed a common polymorphism of Lys to Glu at codon 158 of FMO3 that segregated with almost equal allele frequencies in a number of control Australian and North American samples studied. The Lys158 to Glu158 human FMO3 polymorphism does not decrease trimethylamine N-oxygenation for the cDNA-expressed enzyme and thus does not appear to be causative of trimethyaminuria. The data show that the functional activity of human FMO3 can be significantly altered by amino acid changes that have been observed in individuals with clinically diagnosed trimethylaminuria.     

Astressin analogues (corticotropin-releasing factor antagonists) with extended duration of action in the rat

In earlier reports we identified specific point substitutions (DPhe12,Nle21,38), cyclization strategies [in particular, introduction of lactam rings such as that of cyclo(Glu30,Lys33)], and deletions (residues 1-7) in the CRF molecule that led to agonists. We also noted that further deletions (residues 8-14) produced antagonists such as astressin ?cyclo(30-33)[DPhe12,Nle21,38, Glu30, Lys33]hCRF(12-41)? (1). We hypothesized that the lactam ring promoted conformational stability to yield analogues with increased potency both in vitro and in vivo as compared to that of their linear counterparts. Additionally, we reported that cyclo(30-33)[DPhe12,Nle21,38, Glu30,DHis32,Lys33]hCRF(12-41) (3) and dicyclo(26-36,30-33)[Ac-Asp9,DPhe12,Nle21,38, Cys26, Glu30,Lys33, Cys36]hCRF(9-41) were ca. twice and 1/100 as potent as astressin, respectively, suggesting a putative turn that encompasses residues 30-33 (previous paper: Koerber et al. J. Med. Chem. 1998, 41). To increase the potency of 1 and/or 3 in vivo, we extended their chain length by one (5-8), two (9, 10), and three (11, 12) residues at the N-terminus and acetylated (6, 8, 10, 12). Of the compounds tested for duration of action (1, 3-6, 8), we found 6 and 8 to be slightly longer-acting than astressin or [DHis32]astressin, while their potencies in vitro were not significantly different from that of 3. Additionally, we introduced CalphaMe-leucine residues in lieu of leucine at positions 14, 15, 19, 27, and 37 in [DHis32]astressin. The analogue [CalphaMe-Leu27,DHis32]astressin (16) was more potent (although not statistically in all cases) than the other four analogues in vitro. While acetylation of the N-terminus of 16 (i.e., 18) or of [CalphaMe-Leu27]astressin (i.e., 19) did not have a significant effect on in vitro potency, elongation of the N-terminus by one or three residues in addition to acetylation resulted in cyclo(30-33)[DPhe12,Nle21,CalphaMe-Leu27,Glu3 0,DHis32,Lys33, Nle38]Ac-hCRF(11-41) (21), cyclo(30-33)[DPhe12,Nle21,CalphaMe-Leu27, Glu30,Lys33,Nle38]Ac-hCRF(9-41) (22), and cyclo(30-33)[DPhe12, Nle21, CalphaMe-Leu27,Glu30,DHis32,Lys33,Nle38 ]Ac-hCRF(9-41) (23) that were longer-acting than 6 and 8 (ca. 2 h inhibition of ACTH secretion at 25 micrograms/adrenalectomized rat). Analogues 22 and 23 were also more potent than astressin at reversing intracisternal CRF- and abdominal surgery-induced delay of gastric emptying in conscious rats.     

Site-directed mutagenesis of recombinant human beta 2-glycoprotein I identifies a cluster of lysine residues that are critical for phospholipid binding and anti-cardiolipin antibody activity

beta2-Glycoprotein I (beta2GPI) is a phospholipid-binding serum protein with anticoagulant properties. It plays a vital role in the binding of anti-cardiolipin Abs purified from patients with autoimmune disease when assayed in a cardiolipin (CL) ELISA. Based on a three-dimensional model of beta2GPI, electrostatic calculations, and earlier peptide studies, a highly positively charged amino acid sequence, Lys282-Asn-Lys-Glu-Lys-Lys287, located in the fifth domain of beta2GPI, has been predicted to be the phospholipid binding site. We tested this hypothesis by site-directed mutagenesis of residues in the predicted phospholipid binding site and by assessing the mutants for phospholipid binding and anti-beta2GPI activity. A single amino acid change from Lys286 to Glu significantly decreased the binding of beta2GPI to CL. Double and triple mutants 2k (from Lys286, 287 to Glu286, 287), 2ka (from Lys284, 287 to Glu284, 287), and 3k (from Lys284, 286, 287 to Glu284, 286, 287) possessed no binding of Ab to beta2GPI in a CL ELISA, as well as no inhibitory activity on the binding of iodinated native beta2GPI to CL. These results indicate that the residues Lys284, Lys286, and Lys287 in the fifth domain of beta2GPI are critical for its binding to anionic phospholipids and its subsequent capture for binding of anti-beta2GPI Abs.     

Mutation of protease domain residues Lys37-39 in human protein C inhibits activation by the thrombomodulin-thrombin complex without affecting activation by free thrombin

Activated protein C (aPC) is an important feedback regulator of the clotting cascade. In vivo, the conversion of protein C (PC) from its zymogen to activated form is mediated primarily by thrombin bound to thrombomodulin (TM), an endothelial cell surface protein. Molecular modeling suggests residues Lys37-Lys38-Lys39 of protein C's serine protease domain reside in a surface-exposed loop (variable region 1) whose high concentration of positive charge might be involved in protein-protein interactions. In this study, we have examined the role of the conserved tribasic Lys37-39 charge center in human protein C activation. This sequence was changed to acidic by substitution with Asp37-Glu38-Asp39 (DED) and Glu37-Glu38-Glu39 (EEE), or to neutrality by substitution with Gly37-Gly38-Gly39 (GGG). These mutant PCs, expressed and purified from recombinant human 293 cells, appeared normal with regard to intracellular processing, ability to be secreted, and formation of a viable active site for tripeptidyl-p-nitroanilide substrate cleavage. For activation by free thrombin, wild-type (wt) and mutant PCs displayed equivalent activation rates, as well as identical calcium-dependent inhibition of such activation. Activation of wt-PC with a soluble TM-thrombin complex yielded a 2,000-fold faster rate compared with that by free thrombin at the same (physiological) calcium level. In contrast, the acidic mutants DED and EEE exhibited virtually no TM-mediated increase in activation rate, while the neutral mutant GGG was somewhat intermediate with a 30-fold stimulation of activation rate. These reductions in activation rate were independent of the presence of chondroitin sulfate on TM. Our observations represent the first identification of residues whose mutation essentially uncouples activation by the TM-thrombin complex without affecting activation by free thrombin. Further, our results suggest that VR1 residues within the zymogen form of a serine protease can be important for recognition by physiological activators.     

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.     

Role of carboxyl residues surrounding heme of human cytochrome b5 in the electrostatic interaction with NADH-cytochrome b5 reductase

To identify the cytochrome b5 residues responsible for the electrostatic interaction with NADH-cytochrome b5 reductase (b5R), we prepared and characterized the cytochrome b5 mutants in which Glu41, Glu42, Glu63, Asp70, and Glu73 were replaced by Ala, utilizing site-directed mutagenesis and the expression system for cytochrome b5 in Escherichia coli. Apparent Km values of the wild type b5R for Glu42Ala cytochrome b5 and Asp70Ala cytochrome b5 were approximately three-fold and six-fold higher than that for the wild type cytochrome b5, respectively, while the kcat values for those mutants were not remarkably affected. In contrast, Glu41Ala, Glu63Ala, and Glu73Ala cytochrome b5 showed almost the same kinetic properties as the wild type cytochrome b5. Furthermore, kinetic studies on combinations of the cytochrome b5 and b5R mutants suggested the interaction between Glu42 and Asp70 of cytochrome b5 and Lys125 and Lys41 of b5R, respectively, in the reaction.     

Engineering of cytochrome P450 2B1 specificity. Conversion of an androgen 16 beta-hydroxylase to a 15 alpha-hydroxylase

Six site-directed mutants of cytochrome P450 2B1 were constructed, and function was evaluated in COS cell microsomes by monitoring testosterone and androstenedione hydroxylation and inactivation by chloramphenicol. Mutants Ile-114-->Val and Ile-114-->Ala exhibited marked decreases in androgen 16 beta-OH:16 alpha-OH ratios and increases in 15 alpha-OH:16-OH ratios. Since substitution of Gly-478 with Ala or Ser reduces 16 beta-hydroxylation in favor of 15 alpha-hydroxylation, four double mutants containing Val or Ala at position 114 and Ala or Ser at position 478 were examined. For any given residue at position 114 (Ile, Val, or Ala), the 15 alpha-OH:16-OH ratio increased as residue 478 was changed from Gly to Ala to Ser, and for any residue at position 478, this ratio increased as residue 114 was changed from Ile to Val to Ala. As a consequence, the Ile-114-->Ala, Gly-478-->Ser mutant displayed an approximately 1000-fold higher androgen 15 alpha-OH:16-OH ratio compared with the parental enzyme and functionally resembles mouse P450 2A4 much more closely than P450 2B1. All three mutants with Val at position 114 retained susceptibility to inactivation by chloramphenicol, whereas inactivation was suppressed by Ala at this position. The results suggest the feasibility of an empirical approach to P450 engineering involving the appropriate combination of residues at a few critical sites to confer new regio- and stereoselectivity with retention of overall monooxygenase activity.     

Construction of the plasmid PMEX8-HAK1 and random site-directed mutagenesis of human cytosolic adenylate kinase

The pMEX8-hAK1 vector was devised from the pAK plasmid (Kim J. H. et al., 1989, Protein Engineering 5, 379-386), which could directly express human adenylate kinase proteins without recombination and its single strand DNA could be withdrawn with helper phage for random site-directed mutagenesis. The conserved key residues at Lys21, Lys27, and Thr39 were engineered to obtain mutants for kinetic analysis. Three mutants were obtained as K21P, K27R, and T39S, their specific activities were strikingly reduced compared to those of wild type adenylate kinase. This pMEX8-hAK1 will be a powerful tool for site-directed mutagenesis to detect the substrate-enzyme interaction for human adenylate kinase including various other enzymes.     

Core mutants of the immunoglobulin binding domain of streptococcal protein G: stability and structural integrity

A library of core mutants of the GB1 domain of streptococcal protein G was created, and the structure and stability of selected members was assessed by 1H-15N heteronuclear correlation NMR spectroscopy and fluorescence. All mutants comprised changes in beta-sheet residues, with sidechains at positions 5 (Leu), 7 (Leu), 52 (Phe) and 54 (Val) forming the beta-sheet side of the sheet-helix core interface. A solvent exposed position Ile-6 was chosen as a control. Randomization of bases at codon positions 1 and 3 with thymine at position 2 introduces five possible hydrophobic amino acids, namely Leu, Val, Ile, Phe, and Met. The distribution of encoded amino acids at all five positions is approximately as expected theoretically and indicates that no major bias was introduced towards particular residues. The overall structural integrity of several mutants, as assessed by NMR, ranges from very close to wild type to fully unfolded. Interestingly, the stability of the mutants is not strictly correlated with the number of changes or residue volume.     

A small engineered protein lacks structural uniqueness by increasing the side-chain conformational entropy

A small globular protein, the third repeat of the c-Myb DNA-binding domain, which is composed of 54 amino acid residues, was engineered so as to understand the structural uniqueness of native proteins. This small protein has three alpha-helices that form a helix-turn-helix structure, which is maintained by the hydrophobic core with three Ile residues. One of the mutant proteins, with two of the buried Ile (Ile-155 and Ile-181) substituted with Leu residues, showed multiple conformations, as monitored by heteronuclear magnetic resonance spectroscopy for 13C- and 15N-labeled proteins. The increase in the side-chain conformational entropy, caused by changing the Ile to a Leu residue on an alpha-helix, could engender the lack of structural uniqueness. In native proteins, the conformations of not only the beta-branched side chains, but also those of the neighboring bulky side chains, can be greatly restricted, depending upon the local backbone structure.     

Mapping surface sequences of the tubulin dimer and taxol-induced microtubules with limited proteolysis

Native tubulin alpha beta dimers and microtubules have been subjected to limited proteolysis with trypsin, chymotrypsin, elastase, clostripain, proteinase lysine-C, thermolysin, protease V8, papain, subtilisin, proteinase K, proteinase aspartic-N, and bromelain. Eighty nicking points have been mapped onto the alpha- and beta-tubulin sequences with the aid of site-directed antibodies, of which 18 sites have been exactly determined by N-terminal sequencing, and the probable position of 6 others deduced from protease specificities. Proteolytic sites cluster into five characteristic zones, including the C termini of both chains. Residues accessible to proteases in the tubulin dimer include alpha-tubulin Lys40-Thr41-Ile42, Glu168-Phe169-Ser170, Ser178-Thr179-Ala180-Val181, Lys280-Ala281, Glu290-Ile291, Ala294-Cys295, Arg339-Ser340 (plus probably Lys60-His61 and Glu183-Pro184) and beta-tubulin Gly93-Gln94, Lys174-Val175, Gly277-Ser278, Tyr281-Arg282-Ala283, Cys354-Asp355 (plus probably Arg121-Lys122, Phe167-Ser168, Tyr183-Asn184, and Glu426-Asp427 or Ala430-Asp431). While the majority of these sites remain accessible at the outer surface of taxol-induced microtubules, alpha-tubulin Lys280-Ala281, Arg339-Ser340 and beta-tubulin Tyr281-Arg282-Ala283 (and probably Arg121-Lys122) become protected from limited proteolysis, suggesting that they are close to or at intermolecular contacts in the assembled structure. The protease nicking points constitute sets of surface constraints for any three-dimensional model structures of tubulin and microtubules. The dimer tryptic site at alpha-tubulin 339-340 jumps approximately 12-22 residues upstream (probably to Lys326-Asp327 or Lys311-Tyr312) in taxol microtubules, suggesting a tertiary structural change. The cleavage of the approximately 10 C-terminal residues of alpha-tubulin by protease V8, papain, and subtilisin is inhibited in taxol microtubules compared to tubulin dimers, while the approximately 20 C-terminal residues of beta-tubulin are similarly accessible to protease V8, subtilisin, proteinase K, proteinase AspN, and bromelain and show enhanced papain cleavage. This is consistent with models in which the alpha-tubulin C-terminal zone is near the interdimer contact zone along the protofilaments, whereas the C terminus of beta is near the interface between both subunits.     

Cysteine-scanning mutagenesis of helix IV and the adjoining loops in the lactose permease of Escherichia coli: Glu126 and Arg144 are essential. off

Cys-scanning mutagenesis has been applied to the remaining 45 residues in lactose permease that have not been mutagenized previously (from Gln100 to Arg144 which comprise helix IV and adjoining loops). Of the 45 single-Cys mutants, 26 accumulate lactose to > 75% of the steady state observed with Cys-less permease, and 14 mutants exhibit lower but significant levels of accumulation (35-65% of Cys-less permease). Permease with Phe140-->Cys or Lys131-->Cys exhibits low activity (15-20% of Cys-less permease), while mutants Gly115-->Cys, Glu126-->Cys and Arg144-->Cys are completely unable to accumulate the dissacharide. However, Cys-less permease with Ala or Pro in place of Gly115 is highly active, and replacement of Lys131 or Phe140 with Cys in wild-type permease has a less deleterious effect on activity. In contrast, mutant Glu126-->Cys or Arg144-->Cys is inactive with respect to both uphill and downhill transport in either Cys-less or wild-type permease. Furthermore, mutants Glu126-->Ala or Gln and Arg144-->Ala or Gln are also inactive in both backgrounds, and activity is not rescued by double neutral replacements or inversion of the charged residues at these positions. Finally, a mutant with Lys in place of Arg144 accumulates lactose to about 25% of the steady state of wild-type, but at a slow rate. Replacement of Glu126 with Asp, in contrast, has relatively little effect on activity. None of the effects can be attributed to decreased expression of the mutants, as judged by immunoblot analysis. Although the activity of most of the single-Cys mutants is unaffected by N-ethylmaleimide, Cys replacement at three positions (Ala127, Val132, or Phe138) renders the permease highly sensitive to alkylation. The results indicate that the cytoplasmic loop between helices IV and V, where insertional mutagenesis has little effect on activity [McKenna, E., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11954-11958], contains residues that play an important role in permease activity and that a carboxyl group at position 126 and a positive charge at position 144 are absolutely required.     

Folding of the presequence of yeast pAPI into an amphipathic helix determines transport of the protein from the cytosol to the vacuole

To investigate the role of the 17 residues long presequence (p17) in the transport of the precursor of yeast API (pAPI) from the cytosol to the vacuole we have studied the effects of point mutations upon its conformation and on the process of transport. 1H NMR analysis of p17 indicates that in aqueous solution 26% of the molecules have the 4-12 segment folded into an helix. The hydrophobic environment provided by SDS micelles promotes the folding of 54% of the p17 molecules into a 5-16 amphipathic alpha-helix. Both Schiffer-Edmunson helical wheel analysis of segment 4-12 and residue hydrophobic moments calculated considering all possible side-chain orientations between 80 and 120 degrees, indicate the amphipathic character of the helixes assembled in water and detergent. Charge interactions between the dipole pairs N-Glu2Glu3 and C-Lys12Lys13 are essential for helix stability and condition pAPI transport. Substitution of either Pro2Pro3 or Lys2Lys3 for Glu2Glu3, results in moderate destabilization of the helix, decreases protein targeting to the vacuolar membrane and partly inhibits translocation of the protein to the vacuolar lumen. Replacement of either Pro12Pro13 or Glu12Glu13 for Lys12Lys13, causes a major disruption of the helix, decreases protein targeting and blocks completely the translocation of the protein to the vacuolar lumen. Replacement of Gly7 for Ile7, a substitution which is known to destabilize alpha-helixes in peptides and proteins as a result of the peptide bond to the solvent at Gly residues, produces similar effects as the substitutions for the K12K13 pair. The effects of Gly7 on helix stability and protein transport are partly reversed by introduction of Asp residues at positions 2 and 3 and Ala at position 4. Replacements such as Arg2 for Glu2, or Arg6 for Glu6, which change the net and local charges of the presequence without altering its conformation, have no effect on the protein transport. These results provide direct evidence of the involvement of the presequence in the transport of pAPI from the cytosol to the vacuole. They show that folding of the pAPI presequence is conditioned by the physical/chemical properties of the environment and is critical for targeting the protein to the vacuolar membrane and for its translocation to the vacuolar lumen.     

Modulation of the catalytic rate of Cu,Zn superoxide dismutase in single and double mutants of conserved positively and negatively charged residues

The catalytic rate of four single and three double mutants of Xenopus laevis Cu,Zn superoxide dismutase B, neutralized at Lys120, Asp130, Glu131, and Lys134, has been determined by pulse radiolysis as a function of ionic strength. Neutralization of Glu131 increases the catalytic rate by 80% at low ionic strength, but the effect is reduced to 50% at physiological ionic strength. The rate is unperturbed upon neutralization of Asp130, while neutralization of either of the two lysines drastically decreases the enzyme activity. The Lys120Leu-Lys134Thr and Lys134Thr-Asp130Gln double mutations have an additive and a compensative effect, respectively, on the activity values, while neutralization of the Glu131-Lys134 pair, which also has a compensative effect, gives rise to a faster enzyme at any ionic strength value. The effects observed in the single Asp130Gln and Lys120Leu mutants differ from those reported on human or bovine enzymes [Getzoff et al. (1992) Nature (London) 358, 347-351; Sines et al. (1990) Biochemistry 29, 9403-9412], indicating that some residues occupying the same position in the linear sequence of different Cu,Zn superoxide dismutases have a different functional weight. Our results also suggest that the strategy of multiple charge mutation may be a promising approach in order to increase the catalytic rate of Cu,Zn SODs independently of ionic strength.     

The insulin-like growth factor (IGF)binding protein 1 binding epitope on IGF-I probed by heteronuclear NMR spectroscopy and mutational analysis

NMR spectroscopy studies and biosensor interaction analysis of native and site-directed mutants of insulin-like growth factor I (IGF-I) was applied to identify the involvement of individual residues in IGF-I binding to IGF-binding protein 1 (IGFBP-1). Backbone NMR chemical shifts were found to be affected by IGFBP-1 binding in the following residues: Pro2, Glu3, Cys6, Gly7, Gly19, Pro28-Gly30, Gly32, Arg36, Arg37, Gln40-Gly42, Pro63, Lys65, Pro66, and Lys68-Ala70. Three IGF-I arginine side chains were identified by NMR to participate in IGFBP-1 binding. All IGF-I arginine residues were replaced by alanines, using site-directed mutagenesis, in four single substituted variants, IGF-I(R21A), IGF-I(R50A), IGF-I(R55A), and IGF-I(R56A), and one double replacement mutant, IGF-I(R36A/R37A). Biosensor interaction analysis binding studies demonstrate the involvement of Arg36-Arg37 and Arg50 in IGFBP-1 binding, while experiments with the IGF-I receptor implicate Arg21, Arg36-Arg37, and Arg56 as part of the receptor binding epitope. These overlapping binding surfaces explain why IGF-I receptor and IGFBP-1 binding to IGF-I is competitive. The C terminus of free, but not IGFBP-1-bound, IGF-I is found to exist in two distinct, NMR-detectable conformations at 30 degreesC. One possible explanation for this structural heterogeneity could be cis-trans isomerization of the Cys6-Cys48 disulfide bond.     

Structure-function studies on positions 17, 18, and 21 replacement analogues of glucagon: the importance of charged residues and salt bridges in glucagon biological activity

We have designed and synthesized eight compounds 2-9 which incorporate various amino acid residues in positions 17, 18, and 21 of the glucagon molecule: 2, [Lys17]glucagon amide; 3, [Lys18]glucagon amide; 4, [Nle17,Lys18,Glu21]glucagon amide; 5, [Orn17,18, Glu21]glucagon amide; 6, [d-Arg17]glucagon; 7, [d-Arg18]glucagon; 8, [d-Phe17]glucagon; and 9, [d-Phe18]glucagon. Compared to glucagon (IC50 = 1.5 nM), analogues 2-9 were found to have binding affinity IC50 values (in nM) of 0.7, 4.1, 1.0, 2.0, 5.0, 25.0, 43.0, and 32.0, respectively. When these compounds were tested for their ability to stimulate adenylate cyclase (AC) activity, they were found to be full or partial agonists having maximum stimulation values of 100, 100, 100, 100, 87, 78, 94, and 100%, respectively. On the basis of the X-ray crystal structure of [Lys17,18,Glu21]glucagon amide reported here, the ability to form a salt bridge between Lys18 and Glu21 is probably key to their increased binding and second messenger activities. Among the eight analogues synthesized here, only analogue 4 preserves the ability to form a salt bridge between Lys18 and Glu21. However, since these modifications are minor they do not seem to change the amphiphilic character of the C-terminus, allowing these analogues to reach 78-100% stimulation in the adenylate cyclase assay. Biological data from analogues 6-9 supports the idea that position 18 of glucagon may influence binding only, while position 17 may influence both receptor recognition and transduction.     

Effects of mutations in the gamma-phosphate binding site of myosin on its motor function

The role of the highly conserved residues in the gamma-phosphate binding site of myosin upon myosin motor function was studied. Each of five residues (Ser181, Lys185, Asn235, Ser236, and Arg238) in smooth muscle myosin was mutated. K185Q has neither a steady state ATPase nor an initial Pi burst. Although ATP and actin bind to K185Q, it is not dissociated from actin by ATP. These results indicate that the hydrolysis of bound ATP by K185Q is inhibited. S236T has nearly normal basal Mg2+-ATPase activity, initial Pi burst, ATP-induced enhancement of intrinsic tryptophan fluorescence, and ATP-induced dissociation from actin. However, the actin activation of the Mg2+-ATPase activity and actin translocation of S236T were blocked. In contrast S236A has nearly normal enzymatic properties and actin-translocating activity. These results indicate that 1) the hydroxyl group of Ser236 is not critical as an intermediary of proton transfer during the ATP hydrolysis step, and 2) the bulk of the extra methyl group of the threonine residue in S236T blocks the acceleration of product release from the active site by actin. Arg238, which interacts with Glu459 at the Switch II region, was mutated to Lys and Ile, respectively. R238K has essentially normal enzymatic activity and motility. In contrast, R238I does not hydrolyze ATP or support motility, although it still binds ATP. These results indicate that the charge interaction between Glu459 and Arg238 is critical for ATP hydrolysis by myosin. Other mutants, S181A, S181T, and N235I, showed nearly normal enzymatic and motile activity.     

Purification and characterization of human topoisomerase I mutants

A system for rapid purification and characterization of eukaryotic topoisomerase-I mutants has been developed. The system utilizes six-histidine tagging of human topoisomerase I expressed in Saccharomyces cerevisiae to enable purification by nickel-affinity chromatography. Virtually homogenous mutant proteins are then tested for their ability to relax supercoiled DNA plasmids and their capacity for binding, cleaving and religating short defined DNA substrates. Relaxation-deficient mutants were obtained by site-directed mutagenesis of selected highly conserved amino acids. The mutants Tyr723Phe (active site mutation), Arg488Gln and Lys532Glu were inert in relaxation of DNA, whereas Lys720Glu showed a 50-fold reduction in specific relaxation activity. Accordingly, only Lys720Glu showed low, but detectable cleavage activity on suicide DNA substrates, uncoupling the cleavage and religation events of topoisomerase I. The relative religation efficiency of Lys720Glu comparable to that of wild-type topoisomerase I, indicating that Lys720 is involved in interactions important for normal DNA cleavage, but not for the religation reaction. All mutants could be cross linked by ultraviolet light to bromo-dUTP-substituted DNA oligonucleotides carrying a topoisomerase-I-binding site, indicating that the deficiency of Tyr723Phe, Arg488Gln and Lys532Glu in DNA relaxation and cleavage is not due to an inability of these mutants to bind DNA non-covalently.