Identification of Essential Residues for the C-Terminal Cleavage of the Mammalian LC3: A Lesson from Yeast Atg8

Atg8, a member of an evolutionarily conserved ubiquitin-like protein family, is involved in multiple membrane trafficking pathways including autophagy. In a recent study, we have identified two functional sites in the yeast Saccharomyces cerevisiae Atg8, one involving residues Tyr49 and Leu50, and the other—located on the opposite side of the molecule—residues Phe77 and Phe79. Here we extended our studies to the mammalian system and report that in LC3 residues Phe80 and Leu82, the equivalents of Phe77 and Phe79 in Atg8, are essential for its C-terminal cleavage. We propose that these residues are part of the Atg4 recognition site.

Addendum to:

Two Newly Identified Sites in the Ubiquitin-Like Protein Atg8 Are Essential for Autophagy

N. Amar, G. Lustig, Y. Ichimura, Y. Ohsumi and Z. Elazar

EMBO Rep 2006; 7:635-42     

Two newly identified sites in the ubiquitin-like protein Atg8 are essential for autophagy

Atg8, a member of a novel ubiquitin-like protein family, is an essential component of the autophagic machinery in yeast. This protein undergoes reversible conjugation to phosphatidylethanolamine through a multistep process in which cleavage of Atg8 by a specific protease is followed by ubiquitin-like conjugation processes. Here, we identify two essential sites in Atg8, one of them involving residues Phe 77 and Phe 79 and the other, located on the opposite surface of Atg8, residues Tyr 49 and Leu 50. We show that these two sites are associated with different functions of Atg8: Phe 77 and Phe 79 seem to be part of the recognition site for Atg4, a cystein protease that acts also as a deubiquitination enzyme, whereas Tyr 49 and Leu 50 act downstream of the lipidation step. These two newly identified distinct sites that are essential for Atg8 activity provide an explanation for the many protein-protein interactions of this low-molecular-weight protein.     

The carboxy terminus of yeast Atg13 binds phospholipid membrane via motifs that overlap with the Vac8-interacting domain

ABSTRACT

Macroautophagy/autophagy is a conserved catabolic recycling pathway involving the sequestration of cytoplasmic components within double-membrane vesicles termed autophagosomes. The autophagy-related (Atg) protein Atg13 is a key member of the autophagy initiation complex. The Atg13 C terminus is an intrinsically disordered region (IDR) harboring a binding site for the vacuolar membrane protein Vac8. Recent reports suggest Atg13 acts as a hub to assemble the initiation complex, and also participates in membrane recognition. Here we show that the Atg13 C terminus directly binds to lipid membranes via electrostatic interactions between positively charged residues in Atg13 and negatively charged phospholipids as well as a hydrophobic insertion of a Phe residue. We identified 2 sets of residues in the Atg13 IDR that affect its phospholipid-binding properties; these residues overlap with the Vac8-binding domain of Atg13. Our data indicate that Atg13 binding to phospholipids and Vac8 is mutually exclusive, and both are required for efficient autophagy.     

Introduction of a mutation in the shutter region of antithrombin (Phe77 --> Leu) increases affinity for heparin and decreases thermal stability

The shutter region of serpins consists of a number of highly conserved residues that are critical for both stability and function. Several variants of antithrombin with substitutions in this region are unstable and predispose the carrier to thrombosis. Although most mutations in the shutter region investigated to date are deleterious with respect to serpin stability and function, the substitution of Phe51 by Leu in alpha(1)-antitrypsin results in enhanced stability. Here, we have investigated the effects of introducing an analogous mutation into antithrombin (Phe 77 to Leu). The mutation did not affect the kinetics of interaction with proteases. Strikingly, however, the thermostability of the protein was markedly decreased, with the serpin displaying a 13 degrees C decrease in melting temperature as compared to wild-type recombinant antithrombin. Further studies revealed that in contrast to wild-type antithrombin, the mutant adopted the latent (inactive) conformation upon mild heating. Previous studies on shutter region mutations that destabilize antithrombin revealed that such variants possess enhanced affinity for both heparin pentasaccharide and full-length heparin. The N135A/F77L mutant had unchanged affinity for heparin pentasaccharide, but the affinity for full-length heparin was increased. We suggest that the Phe77Leu mutation causes conformational changes around the top of the D-helix in antithrombin, in particular, to the arginine 132 and 133 residues that may mediate additional antithrombin/heparin interactions. This paper also demonstrates that there are major differences between the shutter regions of antithrombin and alpha(1)-antitrypsin since a stabilizing mutation in antitrypsin has the converse effect in antithrombin.     

Importance of each residue within secretin for receptor binding and biological activity

Secretin is a linear 27-residue peptide hormone that stimulates pancreatic and biliary ductular bicarbonate and water secretion by acting at its family B G protein-coupled receptor. While, like other family members, the carboxyl-terminal region of secretin is most important for high affinity binding and its amino-terminal region is most important for receptor selectivity and receptor activation, determinants for these activities are distributed throughout the entire length of this peptide. In this work, we have systematically investigated changing each residue within secretin to alanine and evaluating the impact on receptor binding and biological activity. The residues most critical for receptor binding were His1, Asp3, Gly4, Phe6, Thr7, Ser8, Leu10, Asp15, Leu19, and Leu23. The residues most critical for biological activity included His1, Gly4, Thr7, Ser8, Glu9, Leu10, Leu19, Leu22, and Leu23, with Asp3, Phe6, Ser11, Leu13, Asp15, Leu26, and Val27 also contributing. While the importance of residues in positions analogous to His1, Asp3, Phe6, Thr7, and Leu23 is conserved for several closely related members of this family, Leu19 is uniquely important for secretin. We, therefore, have further studied this residue by molecular modeling and molecular dynamics simulations. Indeed, the molecular dynamics simulations showed that mutation of Leu19 to alanine was destabilizing, with this effect greater than that observed for the analogous position in the other close family members. This could reflect reduced contact with the receptor or an increase in the solvent-accessible surface area of the hydrophobic residues in the carboxyl terminus of secretin as bound to its receptor.     

Alanine scanning mutagenesis of a type 1 insulin-like growth factor receptor ligand binding site

The high resolution crystal structure of an N-terminal fragment of the IGF-I receptor, has been reported. While this fragment is itself devoid of ligand binding activity, mutational analysis has indicated that its N terminus (L1, amino acids 1-150) and the C terminus of its cysteine-rich domain (amino acids 190-300) contain ligand binding determinants. Mutational analysis also suggests that amino acids 692-702 from the C terminus of the alpha subunit are critical for ligand binding. A fusion protein, formed from these fragments, binds IGF-I with an affinity similar to that of the whole extracellular domain, suggesting that these are the minimal structural elements of the IGF-I binding site. To further characterize the binding site, we have performed structure directed and alanine-scanning mutagenesis of L1, the cysteine-rich domain and amino acids 692-702. Alanine mutants of residues in these regions were transiently expressed as secreted recombinant receptors and their affinity was determined. In L1 alanine mutants of Asp(8), Asn(11), Tyr(28), His(30), Leu(33), Leu(56), Phe(58), Arg(59), and Trp(79) produced a 2- to 10-fold decrease in affinity and alanine mutation of Phe(90) resulted in a 23-fold decrease in affinity. In the cysteine-rich domain, mutation of Arg(240), Phe(241), Glu(242), and Phe(251) produced a 2- to 10-fold decrease in affinity. In the region between amino acids 692 and 702, alanine mutation of Phe(701) produced a receptor devoid of binding activity and alanine mutations of Phe(693), Glu(693), Asn(694), Leu(696), His(697), Asn(698), and Ile(700) exhibited decreases in affinity ranging from 10- to 30-fold. With the exception of Trp(79), the disruptive mutants in L1 form a discrete epitope on the surface of the receptor. Those in the cysteine-rich domain essential for intact affinity also form a discrete epitope together with Trp(79).     

The intrinsic fluorescence of the recombinant human leukocyte interferon-alpha A and fibroblast interferon-beta 1

The environment of Trp residues of the recombinant human interferons has been studied by the analysis of the emission spectra of native and denatured proteins at pH 1.5-8.5 and temperature 10-65 degrees C in the presence and absence of the anionic, cationic and neutral to charge contact quenchers--KI, CsCl and acrylamide, respectively. The obtained data allow to suppose that in IFN-alpha A and IFN-beta 1 Trp141 interacts with Arg145 and one or several from the following residues--Phe124, Ile127, Tyr130, Leu131, whereas Trp77--with Arg33 and Phe36, Phe78, Leu81 or Leu82 (Ile81 or Val82 for IFN-beta 1).     

Functional role of putative critical residues in Mycobacterium tuberculosis RNase P protein

RNase P is involved in processing the 5⿲ end of pre-tRNA molecules. Bacterial RNase P contains a catalytic RNA subunit and a protein subunit. In this study, we have analyzed the residues in RNase P protein of M. tuberculosis that differ from the residues generally conserved in other bacterial RNase Ps. The residues investigated in the current study include the unique residues, Val27, Ala70, Arg72, Ala77, and Asp124, and also Phe23 and Arg93 which have been found to be important in the function of RNase P protein components of other bacteria. The selected residues were individually mutated either to those present in other bacterial RNase P protein components at respective positions or in some cases to alanine. The wild type and mutant M. tuberculosis RNase P proteins were expressed in E. coli, purified, used to reconstitute holoenzymes with wild type RNA component in vitro, and functionally characterized. The Phe23Ala and Arg93Ala mutants showed very poor catalytic activity when reconstituted with the RNA component. The catalytic activity of holoenzyme with Val27Phe, Ala70Lys, Arg72Leu and Arg72Ala was also significantly reduced, whereas with Ala77Phe and Asp124Ser the activity of holoenzyme was similar to that with the wild type protein. Although the mutants did not suffer from any binding defects, Val27Phe, Ala70Lys, Arg72Ala and Asp124Ser were less tolerant towards higher temperatures as compared to the wild type protein. The K_m of Val27Phe, Ala70Lys, Arg72Ala and Ala77Phe were >2-fold higher than that of the wild type, indicating the substituted residues to be involved in substrate interaction. The study demonstrates that residues Phe23, Val27 and Ala70 are involved in substrate interaction, while Arg72 and Arg93 interact with other residues within the protein to provide it a functional conformation.     

Guinea pig chymase is leucine-specific: a novel example of functional plasticity in the chymase/granzyme family of serine peptidases

To explore guinea pigs as models of chymase biology, we cloned and expressed the guinea pig ortholog of human chymase. In contrast to rats and mice, guinea pigs appear to express just one chymase, which belongs to the alpha clade, like primate chymases and mouse mast cell protease-5. The guinea pig enzyme autolyzes at Leu residues in the loop where human chymase autolyzes at Phe. In addition, guinea pig alpha-chymase selects P1 Leu in a combinatorial peptide library and cleaves Ala-Ala-Pro-Leu-4-nitroanilide but has negligible activity toward substrates with P1 Phe and does not cleave angiotensin I. This contrasts with human chymase, which cleaves after Phe or Tyr, prefers P1 Phe in peptidyl 4-nitroanilides, and avidly hydrolyzes angiotensin I at Phe8 to generate bioactive angiotensin II. The guinea pig enzyme also is inactivated more effectively by alpha1-antichymotrypsin, which features P1 Leu in the reactive loop. Unlike mouse, rat, and hamster alpha-chymases, guinea pig chymase lacks elastase-like preference for P1 Val or Ala. Partially humanized A216G guinea pig chymase acquires human-like P1 Phe- and angiotensin-cleaving capacity. Molecular models suggest that the wild type active site is crowded by the Ala216 side chain, which potentially blocks access by bulky P1 aromatic residues. On the other hand, the guinea pig pocket is deeper than in Val-selective chymases, explaining the preference for the longer aliphatic side chain of Leu. These findings are evidence that chymase-like peptidase specificity is sensitive to small changes in structure and provide the first example of a vertebrate Leu-selective peptidase.     

The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity.

The hydrophobic S1' subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1' position. Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1' position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S1' subsite. Thermolysin and TLP-ste variants with identical S1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P1' position.     

Role of strong anchor residues in the effective binding of 10-mer and 11-mer peptides to HLA-A*2402 molecules

The binding capacity of one-hundred-and-seventy-two 8-mer to 11-mer peptides carrying HLA-A24 anchor residues to HLA-A^*2402 molecules was analyzed by using a HLA class I stabilization assay. Most (76.2%) of these peptides bound to HLA-A^*2402 molecules. These results confirmed previous findings that Tyr and Phe at P2 as well as Phe, Trp, Ile, and Leu at the C-terminus were main anchor residues for HLA-A^*2402. Tyr at P2 was a stronger anchor residue than Phe, while bulky aromatic hydrophobic residues Phe and Trp at the C-terminus are stronger anchors than aliphatic hydrophobic residues Ile and Leu. These results were also supported by an analysis using a panel of mutated 9-mer peptides at P2 and P9. Taken together, these results suggest that HLA-A^*2402 molecules have deep B- and F-pockets because they favor peptides carrying bulky aromatic hydrophobic residues at P2 and the C-terminus. The affinity of 8-mer peptides was significantly lower than that of 9-mer to 11-mer peptides, while there was no difference in affinity between 9-mer, 10-mer, and 11-mer peptides. The affinity of peptides carrying bulky aromatic hydrophobic residues at the C-terminus was higher than that of peptides carrying aliphatic hydrophobic residues in each of the 8-mer to 11-mer peptides, though the greatest difference in affinity was observed in 11-mer peptides. The strong interaction of side chains of these anchor residues with the corresponding pockets may permit the effective binding of 10-mer and 11-mer peptides to HLA-A^*2402 molecules.     

Characterization of the functional insulin binding epitopes of the full-length insulin receptor

Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the alpha subunit (amino acids 705-715). To evaluate the physiological significance of this ligand binding site, we have transiently expressed cDNAs encoding full-length receptors with alanine mutations of the residues forming the functional epitopes of this binding site and determined their insulin binding properties. Insulin bound to wild-type receptors with complex kinetics, which were fitted to a two-component sequential model; the Kd of the high affinity component was 0.03 nM and that of the low affinity component was 0.4 nM. Mutations of Arg14, Phe64, Phe705, Glu706, Tyr708, Asn711, and Val715 inactivated the receptor. Alanine mutation of Asn15 resulted in a 20-fold decrease in affinity, whereas mutations of Asp12, Gln34, Leu36, Leu37, Leu87, Phe89, Tyr91, Lys121, Leu709, and Phe714 all resulted in 4-10-fold decreases. When the effects of the mutations were compared with those of the same mutations of the secreted recombinant receptor, significant differences were observed for Asn15, Leu37, Asp707, Leu709, Tyr708, Asn711, Phe714, and Val715, suggesting that the molecular basis for the interaction of each form of the receptor with insulin differs. We also examined the effects of alanine mutations of Asn15, Gln34, and Phe89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC50 commensurate with their effect on the affinity of the receptor for insulin.     

On the structural role of the aromatic residue environment of the chlorophyll a in the cytochrome b6f complex

Because light is not required for catalytic turnover of the cytochrome b 6 f complex, the role of the single chlorophyll a in the structure and function of the complex is enigmatic. Photodamage from this pigment is minimized by its short singlet excited-state lifetime ( approximately 200 ps), which has been attributed to quenching by nearby aromatic residues ( Dashdorj et al., 2005). The crystal structure of the complex shows that the fifth ligand of the chlorophyll a contains two water molecules. On the basis of this structure, the properties of the bound chlorophyll and the complex were studied in the cyanobacterium, Synechococcus sp. PCC 7002, through site-directed mutagenesis of aromatic amino acids in the binding niche of the chlorophyll. The b 6 f complex was purified from three mutant strains, a double mutant Phe133Leu/Phe135Leu in subunit IV and single mutants Tyr112Phe and Trp125Leu in the cytochrome b 6 subunit. The purified b 6 f complex from Tyr112Phe or Phe133Leu/Phe135Leu mutants was characterized by (i) a loss of bound Chl and b heme, (ii) a shift in the absorbance peak and increase in bandwidth, (iii) multiple lifetime components, including one of 1.35 ns, and (iv) relatively small time-resolved absorbance anisotropy values of the Chl Q y band. A change in these properties was minimal in the Trp125Leu mutant. In vivo, no decrease in electron-transport efficiency was detected in any of the mutants. It was concluded that (a) perturbation of its aromatic residue niche influences the stability of the Chl a and one or both b hemes in the monomer of the b 6 f complex, and (b) Phe residues (Phe133/Phe135) of subunit IV are important in maintaining the short lifetime of the Chl a singlet excited state, thereby decreasing the probability of singlet oxygen formation.     

Specific transformations at the N-terminal region of phospholipase A2.

Treatment of porcine pancreatic prophospholipase A2 with methyl acetimidate converted all lysine residues into epsilon-acetimidolysine residues. Enzymatically active epsilon-amidinated phospholipase A2 (AMPA) was obtained from the epsilon-amidinated zymogen by limited tryptic proteolysis cleaving the Arg7-Ala8 bond. AMPA was used to prepare des-Ala8-, des-(Ala8,Leu9)- and des-(ALa8),Leu9,Trp10)-AMP by successive Edman degradations, and des-(A la 8-Arg13)-AMPA by selective splitting of the Arg13-Ser14 bond by trypsin. Structural analogues of AMPA with different N-terminal amino acid residues, viz., D-Ala, beta-Ala, and Gly, have been prepared by reacting des-Ala8-AMPA with the corresponding N-t-Boc-N-hydroxysuccinimide esters of these amino acids. Similarly, the only Trp10 residue has been substituted for Phe by coupling of des-(Ala8-,Leu9,Trp10)-AMPA with N-t-Boc-L-Ala-L-Leu-L-Phe-N-hydroxysuccinimide ester. The feasibility of these substitutions has been proven unambiguously by the retroconversion of des-Ala8-AMPA and of [Ala7]AMPA into AMPA having identical enzymatic activity as the starting AMPA. The single Trp10 residue in native phospholipase A2 and its zymogen was specifically sulfenylated using 0-nitrophenyl-sulfenyl chloride. The homogenous proteins were kinetically analyzed using short-chain lecithins in the monomeric and micellar region. All modified AMPA analogues, except those in which two or more of the N-terminal amino acid residues are removed, show enzymatic activities toward monermic substrate comparable to that of AMPA, indicating that the active site region is still intact. Only [Gly8]-, [beta-Ala8]-, and [Ala8,Leu9,Phe10]AMPA exhibit a dramatic increase in enzymatic activity similar to that of AMPA upon passing the critical micellar concentration (cmc) of the substrate. From these results it can be concluded that the N-terminal region of the enzyme requires a very precise architecture in order to interact with lipid-water interfaces and consequently to display its full enzymatic activity.     

Conserved residues and their role in the structure, function, and stability of acyl-coenzyme A binding protein

In the family of acyl-coenzyme A binding proteins, a subset of 26 sequence sites are identical in all eukaryotes and conserved throughout evolution of the eukaryotic kingdoms. In the context of the bovine protein, the importance of these 26 sequence positions for structure, function, stability, and folding has been analyzed using single-site mutations. A total of 28 mutant proteins were analyzed which covered 17 conserved sequence positions and three nonconserved positions. As a first step, the influence of the mutations on the protein folding reaction has been probed, revealing a folding nucleus of eight hydrophobic residues formed between the N- and C-terminal helices [Kragelund, B. B., et al. (1999) Nat. Struct. Biol. (In press)]. To fully analyze the role of the conserved residues, the function and the stability have been measured for the same set of mutant proteins. Effects on function were measured by the extent of binding of the ligand dodecanoyl-CoA using isothermal titration calorimetry, and effects on protein stability were measured with chemical denaturation followed by intrinsic tryptophan and tyrosine fluorescence. The sequence sites that have been conserved for direct functional purposes have been identified. These are Phe5, Tyr28, Tyr31, Lys32, Lys54, and Tyr73. Binding site residues are mainly polar or charged residues, and together, four of these contribute approximately 8 kcal mol-1 of the total free energy of binding of 11 kcal mol-1. The sequence sites conserved for stability of the structure have likewise been identified and are Phe5, Ala9, Val12, Leu15, Leu25, Tyr28, Lys32, Gln33, Tyr73, Val77, and Leu80. Essentially, all of the conserved residues that maintain the stability are hydrophobic residues at the interface of the helices. Only one conserved polar residue, Gln33, is involved in stability. The results indicate that conservation of residues in homologous proteins may result from a summed optimization of an effective folding reaction, a stable native protein, and a fully active binding site. This is important in protein design strategies, where optimization of one of these parameters, typically function or stability, may influence any of the others markedly.     

Kinetic studies on the oxidation of cytochrome b 5 Phe35 mutants with cytochrome c, plastocyanin and inorganic complexes

To illustrate the functions of the aromatic residue Phe35 of cytochrome b(5) and to give further insight into the roles of the Phe35-containing hydrophobic patch and/or aromatic channel of cytochrome b(5), we studied electron transfer reactions of cytochrome b(5) and its Phe35Tyr and Phe35Leu variants with cytochrome c, with the wild-type and Tyr83Phe and Tyr83Leu variants of plastocyanin, and with the inorganic complexes [Fe(EDTA)](-), [Fe(CDTA)](-) and [Ru(NH(3))(6)](3+). The changes at Phe35 of cytochrome b(5) and Tyr83 of plastocyanin do not affect the second-order rate constants for the electron transfer reactions. These results show that the invariant aromatic residues and aromatic patch/channel are not essential for electron transfer in these systems.     

Molecular dynamics simulation and steered molecular dynamics simulation on irisin dimers

Irisin is found closely associated with promoting the browning of beige fat cells in white adipose tissue. The crystal structure reveals that irisin forms a continuous inter-subunit β-sheet dimer. Here, molecular dynamics (MD) simulation and steered molecular dynamics (SMD) simulation were performed to investigate the dissociation process and the intricate interactions between the two irisin monomers. In the process of MD, the interactions between the monomers were roughly analyzed through the average numbers of both hydrophobic contacts and H-bonds. Then, SMD was performed to investigate the accurate interaction energy between the monomers. By the analysis of dissociation energy, the van der Waals (vdW) force was identified as the major energy to maintain the dimer structure, which also verified the results of MD simulation. Meanwhile, 11 essential residues were discovered by the magnitude of rupture force during dissociation. Among them, residues Arg75, Glu79, Ile77, Ala88, and Trp90 were reported in a previous study using the method of mutagenesis and size exclusion chromatography, and several new important residues (Arg72, Leu74, Phe76, Gln78, Val80, and Asp91) were also identified. Interestingly, the new important residues that we discovered and the important residues that were reported are located in the opposite side of the β-sheet of the dimer.     

Identification by comprehensive chimeric analysis of a key residue responsible for high affinity glucose transport by yeast HXT2

Hxt2 and Hxt1 are, respectively, high affinity and low affinity facilitative glucose transporter paralogs of Saccharomyces cerevisiae. We have previously investigated which amino acid residues of Hxt2 are important for high affinity transport activity. Studies with all the possible combinations of 12 transmembrane segments (TMs) of Hxt2 and Hxt1 revealed that TMs 1, 5, 7, and 8 of Hxt2 are necessary for high affinity transport. Systematic shuffling of the 20 amino acid residues that differ between Hxt2 and Hxt1 in these TMs subsequently identified 5 residues as important for such activity: Leu(59) and Leu(61) (TM1), Leu(201) (TM5), Asn(331) (TM7), and Phe(366) (TM8). We have now studied the relative importance of these 5 residues by individually replacing them with each of the other 19 residues. Replacement of Asn(331) yielded transporters with various affinities, with those of the Ile(331), Val(331), and Cys(331) mutants being higher than that of the wild type. Replacement of the Hxt2 residues at the other four sites yielded transporters with affinities similar to that of the wild type but with various capacities. A working homology model of the chimeric transporters containing Asn(331) or its 19 replacement residues indicated that those residues at this site that yield high affinity transporters (Ile(331), Val(331), Cys(331)) face the central cavity and are within van der Waals distances of Phe(208) (TM5), Leu(357) (TM8), and Tyr(427) (TM10). Interactions via these residues of the four TMs, which compose a half of the central pore, may thus play a pivotal role in formation of a core structure for high affinity transport.     

Residues Leu261, Trp264, and Phe265 account for apolipoprotein E-induced dyslipidemia and affect the formation of apolipoprotein E-containing high-density lipoprotein

Overexpression of apolipoprotein E (apoE) induces hypertriglyceridemia in apoE-deficient mice, which is abrogated by deletion of the carboxy-terminal segment of residues 260-299. We have used adenovirus-mediated gene transfer in apoE-/- and apoA-I-/- mice to test the effect of three sets of apoE mutations within the region of residues 261-265 on the induction of hypertriglyceridemia, the esterification of cholesterol of very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL), and the formation of spherical or discoidal apoE-containing HDL. A single-amino acid substitution (apoE4[Phe265Ala]) induced hypertriglyceridemia in apoE-/- or apoA-I-/- mice, promoted the accumulation of free cholesterol in the very low-density lipoprotein (VLDL) and HDL region, and decreased HDL cholesterol levels. A double substitution (apoE4[Leu261Ala/Trp264Ala]) induced milder hypertriglyceridemia and increased HDL cholesterol levels. A triple substitution (apoE4[Leu261Ala/Trp264Ala/Phe265Ala] or apoE2[Leu261Ala/Trp264Ala/Phe265Ala]) did not induce hypertriglyceridemia and increased greatly the HDL cholesterol levels. Electron microscopy (EM) analysis of the HDL fractions showed that apoE4[Leu261Ala/Trp264Ala/Phe265Ala] and apoE2[Leu261Ala/Trp264Ala/Phe265Ala] contained spherical HDL, apoE4[Leu261Ala/Trp264Ala] contained mostly spherical and few discoidal HDL particles, and apoE4[Phe265Ala] contained discoidal HDL. We conclude that residues Leu261, Trp264, and Phe265 play an important role in apoE-induced hypertriglyceridemia, the accumulation of free cholesterol in VLDL and HDL, and the formation of discoidal HDL. Substitution of these residues with Ala improves the apoE functions by preventing hypertriglyceridemia and promoting formation of spherical apoE-containing HDL.     

Functional implications of the proximal site hydrogen bonding network in Vitreoscilla hemoglobin (VHb): role of Tyr95 (G5) and Tyr126 (H12)

Although Vitreoscilla hemoglobin (VHb) carries a conventional globin fold, its proximal site geometry is unique in having a hydrogen-bonding network between proximal site residues, HisF8-TyrG5-GluH23 and TyrG5-TyrH12. TyrG5 and TyrH12 were mutated to study their relevance in VHb function. VHb G5 mutants (Tyr95Phe and Tyr95Leu showed no stable oxyform and nitric oxide dioxygenase activity, whereas, VHb H12 mutants (Tyr126Phe and Tyr126Leu) displayed little change in their oxygen affinity indicating a crucial role of Tyr95 in protein function. The VHb H12 mutant, Tyr126Leu, enhanced the intracellular pool of oxygen and cell growth better than VHb. Molecular modeling suggests that the replacement of tyrosine with leucine in Tyr126Leu creates an opening on the protein surface that may facilitate oxygen diffusion and accumulation.