Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point to a prominent role of this loop for cytotoxicity

Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240-residue A chain and five 103-residue B subunits. The B pentamer is responsible for G_M1 receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, G_Sα, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47–56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild-type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single-site substitutions have been made in the LT-A subunit 47–56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild-type residues Thr-50 and Val-53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active-site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine-substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild-type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild-type LT. These results lend support for assignment of a prominent role to loop 47–56 in catalysis by LT and CT.     

Identification of three amino acid residues in the B subunit of Shiga toxin and Shiga-like toxin type II that are essential for holotoxin activity.

Shiga toxin of Shigella dysenteriae type I and Shiga-like toxins I and II (SLT-I and SLT-II, respectively) of enterohemorrhagic Escherichia coli are functionally similar protein cytotoxins. These toxin molecules have a bipartite molecular structure which consists of an enzymatically active A subunit that inhibits protein synthesis in eukaryotic cells and an oligomeric B subunit that binds to globotriaosylceramide glycolipid receptors on eukaryotic cells. Regionally directed chemical mutagenesis of the B subunit of SLT-II was used to identify amino acids in the B subunit that are critical for SLT-II holotoxin cytotoxic activity. Three noncytotoxic mutants were isolated, and their mutations were mapped. The substitutions of arginine with cysteine at codon 32, alanine with threonine at codon 42, and glycine with aspartic acid at codon 59 in the 70-amino-acid mature SLT-II B polypeptide resulted in the complete abolition of cytotoxicity. The analogous arginine, alanine, and glycine residues were conserved at codons 33, 43, and 60 in the 69-amino-acid mature B polypeptide of Shiga toxin. Comparable mutations induced in the B-subunit gene of Shiga toxin by oligonucleotide-directed, site-specific mutagenesis resulted in drastically decreased cytotoxicity (10(3)- to 10(6)-fold) as compared with that of wild-type Shiga toxin. The mutant SLT-II and Shiga toxin B subunits were characterized for stability, receptor binding, immunoreactivity, and ability to be assembled into holotoxin.     

Intersubunit communication in the dihydroorotase–aspartate transcarbamoylase complex of Aquifex aeolicus

Aspartate transcarbamoylase and dihydroorotase, enzymes that catalyze the second and third step in de novo pyrimidine biosynthesis, are associated in dodecameric complexes in Aquifex aeolicus and many other organisms. The architecture of the dodecamer is ideally suited to channel the intermediate, carbamoyl aspartate from its site of synthesis on the ATC subunit to the active site of DHO, which catalyzes the next step in the pathway, because both reactions occur within a large, internal solvent‐filled cavity. Channeling usually requires that the reactions of the enzymes are coordinated so that the rate of synthesis of the intermediate matches its rate of utilization. The linkage between the ATC and DHO subunits was demonstrated by showing that the binding of the bisubstrate analog, N‐phosphonacetyl‐L ‐aspartate to the ATC subunit inhibits the activity of the distal DHO subunit. Structural studies identified a DHO loop, loop A, interdigitating between the ATC domains that would be expected to interfere with domain closure essential for ATC catalysis. Mutation of the DHO residues in loop A that penetrate deeply between the two ATC domains inhibits the ATC activity by interfering with the normal reciprocal linkage between the two enzymes. Moreover, a synthetic peptide that mimics that part of the DHO loop that binds between the two ATC domains was found to be an allosteric or noncompletive ATC inhibitor (K_i = 22 μM). A model is proposed suggesting that loop A is an important component of the functional linkage between the enzymes.     

Cytotoxicity of the Vibrio vulnificus MARTX toxin Effector DUF5 is linked to the C2A Subdomain

The multifunctional‐autoprocessing repeats‐in‐toxin (MARTX) toxins are bacterial protein toxins that serve as delivery platforms for cytotoxic effector domains. The domain of unknown function in position 5 (DUF5) effector domain is present in at least six different species' MARTX toxins and as a hypothetical protein in Photorhabdus spp. Its presence increases the potency of the Vibrio vulnificus MARTX toxin in mouse virulence studies, indicating DUF5 directly contributes to pathogenesis. In this work, DUF5 is shown to be cytotoxic when transiently expressed in HeLa cells. DUF5 localized to the plasma membrane dependent upon its C1 domain and the cells become rounded dependent upon its C2 domain. Both full‐length DUF5 and the C2 domain caused growth inhibition when expressed in Saccharomyces cerevisiae. A structural model of DUF5 was generated based on the structure of Pasteurella multocida toxin facilitating localization of the cytotoxic activity to a 186 amino acid subdomain termed C2A. Within this subdomain, an alanine scanning mutagenesis revealed aspartate‐3721 and arginine‐3841 as residues critical for cytotoxicity. These residues were also essential for HeLa cell intoxication when purified DUF5 fused to anthrax toxin lethal factor was delivered cytosolically. Thermal shift experiments indicated that these conserved residues are important to maintain protein structure, rather than for catalysis. The Aeromonas hydrophila MARTX toxin DUF5_Ah domain was also cytotoxic, while the weakly conserved C1–C2 domains from P. multocida toxin were not. Overall, this study is the first demonstration that DUF5 as found in MARTX toxins has cytotoxic activity that depends on conserved residues in the C2A subdomain. Proteins 2014; 82:2643–2656. © 2014 Wiley Periodicals, Inc.     

Determinants for cleavage of the chlorophyll a/b binding protein precursor: a requirement for a basic residue that is not universal for chloroplast imported proteins

We demonstrate that the precursor of the major light-harvesting chlorophyll a/b binding protein (LHCP of Photosystem II), encoded by a Type I gene, contains distinct determinants for processing at two sites during in vitro import into the chloroplast. Using precursors from both pea and wheat, it is shown that primary site processing, and release of a approximately 26-kD peptide, depends on an amino-proximal basic residue. Substitution of an arginine at position -4 resulted in an 80% reduction in processing, with the concomitant accumulation of a high molecular weight intermediate. Cleavage occurred normally when arginine was changed to lysine. The hypothesis that a basic residue is a general requirement for transit peptide removal was tested. We find that the precursors for the small subunit of Rubisco and Rubisco activase do not require a basic residue within seven amino acids of the cleavage site for maturation. In the wheat LHCP precursor, determinants for efficient cleavage at a secondary site were identified carboxy to the primary site, beyond what is traditionally called the transit peptide, within the sequence ala-lys-ala-lys (residues 38-41). Introduction of this sequence into the pea precursor, which has the residues thr-thr-lys-lys in the corresponding position, converted it to a substrate with an efficiently recognized secondary site. Our results indicate that two different forms of LHCP can be produced with distinct NH2-termini by selective cleavage of a single precursor polypeptide.     

Replacement of a conserved arginine in the assembly domain of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit interferes with holoenzyme formation.

In higher plants the small subunit (S) of ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose-P2 carboxylase, EC 4.1.1.39) contains a segment of 16 amino acids which is absent from cyanobacterial S. This segment connecting two beta sheets has been shown, by crystallographic analysis, to form a hairpin loop. The quaternary structure of ribulose-P2 carboxylase indicates several S to large subunit (L) interactions. Eleven of 22 residues within the loop form the interface with 20 residues from two different L dimers. Eight of the loop residues are involved in hydrogen bonds, salt links, and hydrophobic interactions. To test the hypothesis, whether this loop had a function in the assembly of L and S into the hexadecameric enzyme, 6 amino acids within the loop were modified by site-directed mutagenesis of the pea rbcS-3A gene. All substituted S were imported by isolated chloroplasts from pea with wild type efficiency. Mutants E54-R, H55-A, P59-A, D63-G, D63-L, and Y66-A were assembly-competent, indicating that changes of side chains at these positions are tolerated. Replacement of arginine 53, whose side chain forms H-bonds with L residues Y226 and G261, with glutamate completely abolished assembly into holoenzyme. We suggest that arginine 53 in S is essential for ribulose-P2 carboxylase quaternary structure in higher plants.     

Homologous sequences in cholera toxin A and B subunits to peptide domains in myelin basic protein

Recent reports that myelin basic protein (MBP) can be ADP-ribosylated and contains specific sites that bind GTP and GM1 ganglioside, have suggested an analogy to the properties of cholera toxin. Comparisons of pairs of sequences between these two proteins yielded two regions of homology between MBP and the cholera toxin B (chol B) subunit, and one region of homology with the cholera toxin A (chol A) subunit. The matching sites within chol B consisted of a 17 amino acid residue sequence (residues 30-46 in chol B and residues 102-118 in human-MBP, hMBP, p less than 0.0007) and an 11 residue span (residues 31-41 in chol B and sequence 29-39 in hMBP, p less than 0.0004). The homologous site within chol A corresponded to an 11 residue span (residues 130-140 in chol A and 67-77 in hMBP sequence, p less than 0.00007). Since portions of the cholera toxin sequence are virtually identical to sections of the sequence in E. coli toxin, the homology is also valid for the same sequences in this toxin. The highly antigenic behavior of MBP that is related to the induction of experimental allergic encephalomyelitis may be paralleled by comparable neural pathology from the homologous regions of cholera toxin.     

Chemical modification studies of gelonin: Involvement of arginine residues in biological activity

Gelonin inhibits protein synthesis by inactivating the eukaryotic 60 S ribosomal subunit by an unknown mechanism. The protein was purified in high yield by a new method using Cibacron blue F₃GA-Sepharose. Chemical modification studies reveal that arginine residues are essential for biological activity.     

Role of cysteine 41 of the A subunit of pertussis toxin

The 2 cysteine residues present in the A subunit of pertussis toxin form a disulfide bond in the conformation of the toxin secreted from the bacteria. Previous studies have shown that reduction of this bond is necessary for activation of the enzyme. We have found that reduction of this bond also alters the conformation of the A subunit such that it no longer readily associates with the B oligomer of the toxin, a finding which may have implications concerning the form of the toxin found within the eukaryotic cell. In addition, we have demonstrated that reduction of the disulfide bond of the purified A subunit followed by treatment with sulfhydryl-modifying reagents such as N-ethylmaleimide or 5,5'-dithiobis-(2-nitrobenzoic acid) results in inhibition of the NAD glycohydrolase activity of the protein. When a tryptic fragment of the A subunit which contains only 1 of the cysteine residues (Cys-41) of the native protein was reacted with N-ethylmaleimide, the NAD glycohydrolase activity of this fragment was substantially reduced. These data indicate that Cys-41 may be in a region of the molecule which is critical for the enzymatic activity of the toxin.     

Functional complexity of the twin-arginine translocase TatC component revealed by site-directed mutagenesis

The Escherichia coli Tat apparatus is a membrane-bound protein translocase that serves to export folded proteins synthesized with N-terminal twin-arginine signal peptides. The essential TatC component of the Tat translocase is an integral membrane protein probably containing six transmembrane helices. Sequence analysis identified conserved TatC amino acid residues, and the role of these side-chains was assessed by single alanine substitution. This approach identified three classes of TatC mutants. Class I mutants included F94A, E103A and D211A, which were completely devoid of Tat-dependent protein export activity and thus represented residues essential for TatC function. Cross-complementation experiments with class I mutants showed that co-expression of D211A with either F94A or E103A regenerated an active Tat apparatus. These data suggest that different class I mutants may be blocked at different steps in protein transport and point to the co-existence of at least two TatC molecules within each Tat translocon. Class II mutations identified residues important, but not essential, for Tat activity, the most severely affected being L99A and Y126A. Class III mutants showed no significant defects in protein export. All but three of the essential and important residues are predicted to cluster around the cytoplasmic N-tail and first cytoplasmic loop regions of the TatC protein.     

Purification and Characterization of Enterotoxin Produced by Aeromonas sobria

We purified the toxin of Aeromonas sobria capable of inducing a positive response in the mouse intestinal loop assay. The purified toxin showed a positive response not only in the loop assay but also in a hemolytic assay. Subsequently, we cloned the toxin gene and demonstrated that the product of this gene possessed both hemolytic and enterotoxic activities. These results showed that the enterotoxin of A. sobria possesses hemolytic activity. Nucleotide sequence determination of the toxin gene and amino acid sequence analysis of the purified toxin revealed that it is synthesized as a precursor composed of 488 amino acid residues, and that the 24 amino-terminal amino acid residues of the precursor is removed in the mature toxin. As antiserum against the purified toxin neutralized the fluid accumulation induced by living cells not only of A. sobria but also of A. hydrophila, this and antigenically related toxin(s) are thought to play an essential role in the induction of diarrhea by these organisms. The toxin-injured Chinese hamster ovary (CHO) cells induced the release of intracellular lactose dehydrogenase (LDH). The release of LDH from CHO cells and the lysis of erythrocytes by the toxin were repressed by the addition of dextran to the reaction solution, indicating that the toxin forms pores in the membranes and that the cells were injured by the osmotic gradient developed due to pore formation. However, the histopathological examination of intestinal cells exposed to the toxin showed that it caused fluid accumulation in the mouse intestinal loop without causing cellular damage.     

Mechanism of action of choleragen

Choleragen exerts its effect on cells through activation of adenylate cyclase. Choleragen initially interacts with cells through binding of the B subunit of the toxin to the ganglioside G_M1 on the cell surface. Subsequent events are less clear. Patching or capping of toxin on the cell surface may be an obligatory step in choleragen action. Studies in cell-free systems have demonstrated that activation of adenylate cyclase by choleragen requires NAD. In addition to NAD, requirements have been observed for ATP, GTP, and calcium-dependent regulatory protein. GTP also is required for the expression of choleragen-activated adenylate cyclase. In preparations from turkey erythrocytes, choleragen appears to inhibit an isoproterenol-stimulated GTPase. It has been postulated that by decreasing the activity of a specific GTPase, choleragen would stabilize a GTP-adenylate cyclase complex and maintain the cyclase in an activated state. Although the holotoxin is most effective in intact cells, with the A subunit having 1/20th of its activity and the B subunit (choleragenoid) being inactive, in cell-free systems the A subunit, specifically the A₁ fragment, is required for adenylate cyclase activation. The B protomer is inactive. Choleragen, the A subunit, or A₁ fragment under suitable conditions hydrolyzes NAD to ADP-ribose and nicotinamide (NAD glycohydrolase activity) and catalyzes the transfer of the ADP-ribose moiety of NAD to the guandino group of arginine (ADP-ribosyltransferase activity). The NAD glycohydrolase activity is similar to that exhibited by other NAD-dependent bacterial toxins (diphtheria toxin, Pseudomonas exotoxin A), which act by catalyzing the ADP-ribosylation of a specific acceptor protein. If the ADP-ribosylation of arginine is a model for the reaction catalyzed by choleragen in vivo, then arginine is presumably an analog of the amino acid which is ADP-ribosylated in the acceptor protein. It is postulated that choleragen exerts its effects on cells through the NAD-dependent ADP-ribosylation of an arginine or similar amino acid in either the cyclase itself or a regulatory protein of the cyclase system.     

On the probable involvement of arginine residues in the bile-salt-binding site of human pancreatic carboxylic ester hydrolase

Modification of arginine residues with 2,3-butanedione inhibits the carboxylic-ester hydrolase activity on soluble and emulsified substrates when assayed with bile salts. The alpha-dicarbonyl reagent modifies seven of the nineteen arginine residues present per enzyme molecule. Nevertheless the inactivation with butanedione is greatly diminished when the protein is in the presence of negatively charged micellar bile salt. In these conditions we observe the protection of one arginine residue by sodium taurodeoxycholate and of two arginine residues by sodium cholate. This suggests that the carboxylic-ester hydrolase from human pancreatic juice contains at least two arginine residues essential for the activation by bile salts. All our data confirm the presence of two bile-salt-binding sites on the enzyme in which one arginine per site is involved and plays the general role of an anionic binding site. This study provides evidence that arginine residues may play an essential role in the interaction between bile salts and protein.     

Autophosphorylation of the catalytic subunit of the DNA-dependent protein kinase is required for efficient end processing during DNA double-strand break repair

The DNA-dependent protein kinase (DNA-PK) plays an essential role in nonhomologous DNA end joining (NHEJ) by initially recognizing and binding to DNA breaks. We have shown that in vitro, purified DNA-PK undergoes autophosphorylation, resulting in loss of activity and disassembly of the kinase complex. Thus, we have suggested that autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) may be critical for subsequent steps in DNA repair. Recently, we defined seven autophosphorylation sites within DNA-PKcs. Six of these are tightly clustered within 38 residues of the 4,127-residue protein. Here, we show that while phosphorylation at any single site within the major cluster is not critical for DNA-PK's function in vivo, mutation of several sites abolishes the ability of DNA-PK to function in NHEJ. This is not due to general defects in DNA-PK activity, as studies of the mutant protein indicate that its kinase activity and ability to form a complex with DNA-bound Ku remain largely unchanged. However, analysis of rare coding joints and ends demonstrates that nucleolytic end processing is dramatically reduced in joints mediated by the mutant DNA-PKcs. We therefore suggest that autophosphorylation within the major cluster mediates a conformational change in the DNA-PK complex that is critical for DNA end processing. However, autophosphorylation at these sites may not be sufficient for kinase disassembly.     

Structure-function analysis of inositol hexakisphosphate-induced autoprocessing of the Vibrio cholerae multifunctional autoprocessing RTX toxin

Vibrio cholerae secretes a large virulence-associated multifunctional autoprocessing RTX toxin (MARTX(Vc)). Autoprocessing of this toxin by an embedded cysteine protease domain (CPD) is essential for this toxin to induce actin depolymerization in a broad range of cell types. A homologous CPD is also present in the large clostridial toxin TcdB and recent studies showed that inositol hexakisphosphate (Ins(1,2,3,4,5,6)P(6) or InsP(6)) stimulated the autoprocessing of TcdB dependent upon the CPD (Egerer, M., Giesemann, T., Jank, T., Satchell, K. J., and Aktories, K. (2007) J. Biol. Chem. 282, 25314-25321). In this work, the autoprocessing activity of the CPD within MARTX(Vc) is similarly found to be inducible by InsP(6). The CPD is shown to bind InsP(6) (K(d), 0.6 microm), and InsP(6) is shown to stimulate intramolecular autoprocessing at both physiological concentrations and as low as 0.01 microm. Processed CPD did not bind InsP(6) indicating that, subsequent to cleavage, the activated CPD may shift to an inactive conformation. To further pursue the mechanism of autoprocessing, conserved residues among 24 identified CPDs were mutagenized. In addition to cysteine and histidine residues that form the catalytic site, 2 lysine residues essential for InsP(6) binding and 5 lysine and arginine residues resulting in loss of activity at low InsP(6) concentrations were identified. Overall, our data support a model in which basic residues located across the CPD structure form an InsP(6) binding pocket and that the binding of InsP(6) stimulates processing by altering the CPD to an activated conformation. After processing, InsP(6) is shown to be recycled, while the cleaved CPD becomes incapable of further binding of InsP(6).     

Ubiquitous SPRY domains and their role in the skeletal type ryanodine receptor

We recently identified the second of three SPRY domains in the skeletal muscle ryanodine receptor type 1 (RyR1) as a potential binding partner in the RyR1 ion channel for the recombinant II–III loop of the skeletal muscle dihydropyridine receptor, for a scorpion toxin, Imperatoxin A and for an interdomain interaction within RyR1. SPRY domains are structural domains that were first described in the fungal Dictyostelium discoideum tyrosine kinase spore lysis A and all three isoforms of the mammalian ryanodine receptor (RyR). Our studies are the first to assign a function to any of the three SPRY domains in the RyR. However, in other systems SPRY domains provide binding sites for regulatory proteins or intramolecular binding sites that maintain the structural integrity of a protein. In this article, we review the general characteristics of a range of SPRY domains and discuss evidence that the SPRY2 domain in RyR1 supports interactions with binding partners that contain a structural surface of aligned basic residues.     

Alkylation of cysteine 41, but not cysteine 200, decreases the ADP-ribosyltransferase activity of the S1 subunit of pertussis toxin

Sulfhydryl-alkylating reagents are known to inactivate the NAD glycohydrolase and ADP-ribosyltransferase activities of the S1 subunit of pertussis toxin, a protein which contains two cysteines at positions 41 and 200. It has been proposed that NAD can retard alkylation of one of the two cysteines of this protein (Kaslow, H.R., and Lesikar, D.D. (1987) Biochemistry 26, 4397-4402). We now report that NAD retards the ability of these alkylating reagents to inactivate the S1 subunit. In order to determine which cysteine is protected by NAD, we used site-directed mutagenesis to construct analogs of the toxin with serines at positions 41 and/or 200. Sulfhydryl-alkylating reagents reduced the ADP-ribosyltransferase activity of the analog with a single cysteine at position 41; NAD retarded this inactivation. In contrast, sulfhydryl-alkylating reagents did not inactivate analogs with serine at position 41. An analog with alanine at position 41 possessed substantial ADP-ribosyltransferase activity. We conclude that alkylation of cysteine 41, and not cysteine 200, inactivates the S1 subunit of pertussis toxin, but that the sulfhydryl group of cysteine 41 is not essential for the ADP-ribosyltransferase activity of the toxin. These results suggest that the region near cysteine 41 contributes to features of the S1 subunit important for ADP-ribosyltransferase activity. Using site-directed mutagenesis, we found that changing aspartate 34 to asparagine, arginine 39 to lysine, and glutamine 42 to glutamate had little effect on ADP-ribosyltransferase activity. However, substituting an asparagine for the histidine at position 35 markedly decreased, but did not eliminate, ADP-ribosyltransferase activity. Chou-Fasman analysis predicted no significant modifications in secondary structure of the S1 peptide with the change of histidine 35 to asparagine. Thus, histidine 35 may interact with a substrate of the S1 subunit without being essential for catalysis.     

The Role of β20–β21 Loop Structure in Insecticidal Activity of Cry1Ac Toxin from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The β20–β21 loop is a unique structure in the domain III of Bacillus thuringiensis Cry proteins. In this study, the role of β20–β21 loop on insecticidal activity of Cry1Ac toxin was investigated. 10 residues in β20–β21 loop were substituted with alanine using PCR-based site-directed mutagenesis. All mutants were capable of producing diamond-shaped crystal and expressing a protein sized 130 kDa. The mutants S581A and I585A enhanced toxicity against Helicoverpa armigera larvae dramatically, while most of the rest mutants possess a reduced toxicity at different degrees. Indoor bioassay result revealed that mutants S581A and I585A had a 1.72- and 1.89-fold increasing in toxicity against Helicoverpa armigera larvae compared with the wild-type strain, respectively; On the contrary, G583A experienced a significant reduced insecticidal activity. Three-dimensional analysis of Cry1Ac5 protein demonstrated that the side chain of residues T579, S580, L582, and I585 extended to the surface of the protein, and might participate in the interaction between the protein and its receptor, whereas side chain of residues N576, F578, S581, N584, and V586 preferred the inside of the protein, and which might be critical to the stability of the protein structure. Our study for the first time clarified the special properties and the functions of the β20–β21 loop in domain III of Cry1Ac5. These findings also provided the latest biological evidence for the recognition and binding mechanism of the domain III in Cry1Ac, and its role in maintaining the structure stability of Cry1Ac.     

The selectivity of cathepsin D suggests an involvement of the enzyme in the generation of T-cell epitopes

The selectivity of cathepsin D, a mammalian intracellular aspartyl proteinase involved in the degradation of endocytosed proteins, was studied. For this purpose, several proteins of known primary structure were subjected to mild proteolysis by the enzyme, and the preferentially cleaved peptide bonds were identified. Comparison of the primary structures around these sites indicates that cathepsin D shows a strong preference for peptide bonds within a distinct sequence pattern of amino acids extending over 7 residues. In general, this pattern is most likely to occur within amphipathic alpha-helical structures. These findings and their possible implications are discussed together with additional evidence suggesting an important role for cathepsin D in the processing of protein antigens, an essential step for their recognition by T-cells. Accordingly, it is proposed that the proteolytic activity of cathepsin D is crucial in selecting processing sites and hence the location and structural context of T-cell epitopes for the majority of protein antigens.     

Characterization of the membrane domain subunit NuoK (ND4L) of the NADH-quinone oxidoreductase from Escherichia coli

The ND4L subunit is the smallest mitochondrial DNA-encoded subunit of the proton-translocating NADH-quinone oxidoreductase (complex I). In an attempt to study the functional and structural roles of the NuoK subunit (the Escherichia coli homologue of ND4L) of the bacterial NADH-quinone oxidoreductase (NDH-1), we have performed a series of site-specific mutations on the nuoK gene of the NDH-1 operon by using the homologous recombination technique. The amino acid residues we targeted included two highly conserved glutamic acids that are presumably located in the middle of the membrane and several arginine residues that are predicted to be on the cytosolic side. All point mutants examined had fully assembled NDH-1 as detected by blue-native gel electrophoresis and immunostaining. Mutations of nearly perfectly conserved Glu-36 lead to almost null activities of coupled electron transfer with a concomitant loss of generation of electrochemical gradient. A significant diminution of the coupled activities was also observed with mutations of another highly conserved residue, Glu-72. These results may suggest that both membrane-embedded acidic residues are important for the coupling mechanism of NDH-1. Furthermore, a severe impairment of the coupled activities occurred when two vicinal arginine residues on a cytosolic loop were simultaneously mutated. Possible roles of these arginine residues and other conserved residues in the NuoK subunit for NDH-1 function were discussed.