Leucyl-tRNA synthetase from the hyperthermophilic bacterium Aquifex aeolicus recognizes minihelices

Aminoacylation of the minihelix mimicking the amino acid acceptor arm of tRNA has been demonstrated in more than 10 aminoacyl-tRNA synthetase systems. Although Escherichia coli or Homo sapiens cytoplasmic leucyl-tRNA synthetase (LeuRS) is unable to charge the cognate minihelix or microhelix, we show here that minihelix(Leu) is efficiently charged by Aquifex aeolicus synthetase, the only known heterodimeric LeuRS (alpha beta-LeuRS). Aminoacylation of minihelices is strongly dependent on the presence of the A73 identity nucleotide and greatly stimulated by destabilization of the first base pair as reported for the E. coli isoleucyl-tRNA synthetase and methionyl-tRNA synthetase systems. In the E. coli LeuRS system, the anticodon of tRNA(Leu) is not important for recognition by the synthetase. However, the addition of RNA helices that mimic the anticodon domain stimulates minihelix(Leu) charging by alpha beta-LeuRS, indicating possible domain-domain communication within alpha beta-LeuRS. The leucine-specific domain of alpha beta-LeuRS is responsible for minihelix recognition. To ensure accurate translation of the genetic code, LeuRS functions to hydrolyze misactivated amino acids (pretransfer editing) and misaminoacylated tRNA (posttransfer editing). In contrast to tRNA(Leu), minihelix(Leu) is unable to induce posttransfer editing even upon the addition of the anticodon domain of tRNA. Therefore, the context of tRNA is crucial for the editing of mischarged products. However, the minihelix(Leu) cannot be misaminoacylated, perhaps because of the tRNA-independent pretransfer editing activity of alpha beta-LeuRS.     

Leucyl-tRNA synthetase consisting of two subunits from hyperthermophilic bacteria Aquifex aeolicus

In a hyperthermophilic bacterium, Aquifex aeolicus, leucyl-tRNA synthetase (LeuRS) consists of two non-identical polypeptide subunits (alpha and beta), different from the canonical LeuRS, which has a single polypeptide chain. By PCR, using genome DNA of A. aeolicus as a template, genes encoding the alpha and beta subunits were amplified and cloned in Escherichia coli. The alpha subunit could not be expressed stably in vivo, whereas the beta subunit was overproduced and purified by a simple procedure. The beta subunit was inactive in catalysis but was able to bind tRNA(Leu). Interestingly, the heterodimer alphabeta-LeuRS could be overproduced in E. coli cells containing both genes and was purified to 95% homogeneity as a hybrid dimer. The kinetics of A. aeolicus LeuRS in pre-steady and steady states and cross-recognition of LeuRS and tRNA(Leu) from A. aeolicus and E. coli were studied. Magnesium concentration, pH value, and temperature aminoacylation optima were determined to be 12 mm, 7.8, and 70 degrees C, respectively. Under optimal conditions, A. aeolicus alphabeta-LeuRS is stable up to 65 degrees C.     

Enzymes assembled from Aquifex aeolicus and Escherichia coli leucyl-tRNA synthetases

Aquifex aeolicus alphabeta-LeuRS is the only known heterodimeric LeuRS, while Escherichia coli LeuRS is a canonical monomeric enzyme. By using the genes encoding A. aeolicus and E. coli LeuRS as PCR templates, the genes encoding the alpha and beta subunits from A. aeolicus alphabeta-LeuRS and the equivalent amino- and carboxy-terminal parts of E. coli LeuRS (identified as alpha' and beta') were amplified and recombined using suitable plasmids. These recombinant plasmids were transformed or cotransformed into E. coli to produce five monomeric and five heterodimeric LeuRS mutants. Seven of these were successfully overexpressed in vivo and purified, while three dimeric mutants with the beta' part of E. coli LeuRS were not successfully expressed. The seven purified mutants catalyzed amino acid activation, although several exhibited reduced aminoacylation properties. Removal of the last 36 residues of the alpha subunit of the A. aeolicus enzyme was determined to be deleterious for tRNA charging. Indeed, subunit exchange showed that the cross-species-specific recognition of A. aeolicus tRNA(Leu) occurs at the alpha subunit. None of the mixed E. coli-A. aeolicus enzymes were as thermostable as the native alphabeta-LeuRS. However, the fusion of the two alpha and beta peptides from A. aeolicus as a single chain analogous to canonical LeuRS resulted in a product more resistant to heat denaturation than the original enzyme.     

Two distinct domains of the beta subunit of Aquifex aeolicus leucyl-tRNA synthetase are involved in tRNA binding as revealed by a three-hybrid selection

The Aquifex aeolicus alphabeta-LeuRS is the only known heterodimeric class Ia aminoacyl-tRNA synthetase. In this study, we investigated the function of the beta subunit which is believed to bind tRNA(Leu). A yeast three-hybrid system was constructed on the basis of the interaction of the beta subunit with its cognate tRNA(Leu). Then, seven mutated beta subunits exhibiting impaired tRNA binding capacities were selected out from a randomly mutated library. Two mutations were identified in the class Ia-helix-bundle-domain, which might interact with the D-hairpin of the tRNA analogous to other class Ia tRNA:synthetases complexes. The five other mutations were found in the LeuRS-specific C-terminal domain of which the folding is still unknown. tRNA affinity measurements and kinetic analyses performed on the isolated beta subunits and on the co-expressed alphabeta-heterodimers showed for all the mutants an effect in tRNA affinity in the ground state. In addition, an effect on the transition state of the aminoacylation reaction was observed for a 21-residues deletion mutant of the C-terminal end. These results show that the genetic approach of the three hybrid system is widely applicable and is a powerful tool for the investigation of tRNA:synthetase interactions.     

Discrimination of tRNA(Leu) isoacceptors by the mutants of Escherichia coli leucyl-tRNA synthetase in editing

Leucyl-tRNA synthetase (LeuRS), one of the class Ia aminoacyl-tRNA synthetases, joins Leu to tRNA(Leu) and excludes noncognate amino acids in protein synthesis. In this study, Escherichia coli LeuRS mutants at amino acid E292, which was located in the connective polypeptide 1 insertion region, were synthesized. Although mutated LeuRS showed little change in structure compared with wild-type LeuRS, the mutants were impaired in activity to varying extents. It was also showed that mutations did not affect the adenylation reaction. However, mutated LeuRS can mischarge tRNA(Leu) isoacceptors tRN or tRN with isoleucine to different extents. Isoleucylation of tRN was more than that of tRN. The mutant LeuRS-E292S, which was picked out as an example for the investigation of the relationship between tRNA(Leu) isoacceptors and editing function, can discriminate the Watson-Crick base pair of the first base pair of tRNA(Leu) from the wobble base pair. The tRNA(Leu) with the Watson-Crick base pair may result in more isoleucylated product than that with the wobble base pair. The same phenomenon happened to another mutant, LeuRS-A293D. It seems that the flexibility of the first base pair affects the editing reaction of LeuRS. The results indicate that the flexibility of the first base pair of tRNA(Leu) may probably affect the mischarged 3'-end of tRNA(Leu) shuttling from synthetic site to editing site and that the transferred acceptor arm of tRNA(Leu) may interact with LeuRS in the region around E292.     

The C-terminal domain of the archaeal leucyl-tRNA synthetase prevents misediting of isoleucyl-tRNA(Ile)

In the archaeal leucyl-tRNA synthetase (LeuRS), the C-terminal domain recognizes the long variable arm of tRNA(Leu) for aminoacylation, and the so-called editing domain deacylates incorrectly formed Ile-tRNA(Leu). We previously reported, for Pyrococcus horikoshii LeuRS, that a deletion mutant lacking the C-terminal domain (LeuRS_delta(811-967)) retains normal editing activity, but has severely reduced aminoacylation activity. In this study, we found that LeuRS_delta(811-967), but not the wild-type LeuRS, exhibited surprisingly robust deacylation activity against Ile-tRNA(Ile), correctly formed by isoleucyl-tRNA synthetase ("misediting"). Structural superposition of tRNA(Ile) onto the LeuRS x tRNA(Leu) complex indicated that Ile911, Lys912, and Glu913 of the LeuRS C-terminal domain clash with U20 of tRNA(Ile), which is bulged out as compared to the corresponding nucleotide of tRNA(Leu). The deletion of amino acid residues 911-913 of LeuRS enhanced the Ile-tRNA(Ile) deacylation activity, without affecting the Ile-tRNA(Leu) deacylation activity. These results demonstrate that the clashing between U20 of tRNA(Ile) and residues 911-913 of the LeuRS C-terminal domain is the structural mechanism that prevents misediting. In contrast, the deletion of the C-terminal domains of the isoleucyl- and valyl-tRNA synthetases impaired both the aminoacylation (Ile-tRNA(Ile) and Val-tRNA(Val) formation, respectively) and editing (Val-tRNA(Ile) and Thr-tRNA(Val) deacylation, respectively) activities, and did not cause misediting (Val-tRNA(Val) and Thr-tRNA(Thr) deacylation, respectively) activity. Thus, the requirement of the C-terminal domain for misediting prevention is unique to LeuRS, which does not recognize the anticodon of the cognate tRNA, unlike the common aminoacyl-tRNA synthetases.     

Expression, purification, and characterization of a new heterotetramer structure of leucyl-tRNA synthetase from Aquifex aeolicus in Escherichia coli

Aminoacyl-tRNA synthetases are key players in the interpretation of the genetic code. They constitute a textbook example of multi-domain proteins including insertion and terminal functional modules appended to one of the two class-specific active site domains. The non-catalytic domains usually have distinct roles in the aminoacylation reaction. Aquifex aeolicus leucyl-tRNA synthetase (LeuRS) is composed of a separated catalytic site and tRNA anticodon-binding site, which would represent one of the closest relics of the primordial aminoacyl-tRNA synthetase. Moreover, the essential catalytic site residues are split into the two different subunits. In all other class-I aminoacyl-tRNA synthetases, those two functional polypeptides are nowadays fused into a single protein chain. In this work, we report the isolation and the characterization, in Escherichia coli, of a novel oligomeric form (alphabeta)2 for A. aeolicus LeuRS, which is present in addition to the alphabeta heterodimer. A. aeolicus (alphabeta)2 LeuRS has been characterized by biochemical and biophysical methods. Native gel electrophoresis, mass spectrometry, analytical ultracentrifugation, and kinetic analysis confirmed that the (alphabeta)2 enzyme was a stable and active entity. By mass spectrometry we confirmed that the heterodimer alphabeta can bind one tRNALeu molecule whereas the heterotetramer (alphabeta)2 can bind two tRNALeu molecules. Active site titration and aminoacylation assays showed that two functional active sites are found per heterotetramer, suggesting that this molecular species might exist and be active in vivo. All those data suggest that the existence of the heterotetramer is certainly not an artifact of overexpression in E. coli.     

Leucyl-tRNA synthetase from the ancestral bacterium Aquifex aeolicus contains relics of synthetase evolution

The editing reactions catalyzed by aminoacyl-tRNA synthetases are critical for the faithful protein synthesis by correcting misactivated amino acids and misaminoacylated tRNAs. We report that the isolated editing domain of leucyl-tRNA synthetase from the deep-rooted bacterium Aquifex aeolicus (alphabeta-LeuRS) catalyzes the hydrolytic editing of both mischarged tRNA(Leu) and minihelix(Leu). Within the domain, we have identified a crucial 20-amino-acid peptide that confers editing capacity when transplanted into the inactive Escherichia coli LeuRS editing domain. Likewise, fusion of the beta-subunit of alphabeta-LeuRS to the E. coli editing domain activates its editing function. These results suggest that alphabeta-LeuRS still carries the basic features from a primitive synthetase molecule. It has a remarkable capacity to transfer autonomous active modules, which is consistent with the idea that modern synthetases arose after exchange of small idiosyncratic domains. It also has a unique alphabeta-heterodimeric structure with separated catalytic and tRNA-binding sites. Such an organization supports the tRNA/synthetase coevolution theory that predicts sequential addition of tRNA and synthetase domains.     

Cloning and expression of human phenylalanyl-tRNA synthetase in Escherichia coli: comparative study of purified recombinant enzymes

Human phenylalanyl-tRNA synthetase (PheRS) was cloned and expressed in Escherichia coli. The cDNAs of the alpha and beta subunits were cloned into pET-21b(+) and pET-28b(+) vectors. The 6x histidine-tagged (HT) plasmids pET-21_HTbeta, pET-28_HTalpha, and pET-28_HTbeta were constructed. Three different types of (alphabeta)(2) heterodimers of human PheRS carrying HT at the N-terminus of either of two alpha or beta subunits or simultaneously on both of them were overproduced and purified. The heterodimeric protein with HT appended to the N-terminus of the beta subunit revealed no activity in the aminoacylation reaction as opposed to those with HT on the alpha subunit. It is known from the structure of the Thermus thermophilus Phe system that the N-terminal coiled-coil domain of the alpha subunit is involved in the binding of cognate tRNA(Phe). Our data demonstrate that a histidine-tagged N-terminal extension appended to the alpha subunit does not affect the kinetic parameters of tRNA(Phe) aminoacylation. Elimination of the HT from the alpha subunit by thrombin cleavage leads to nonspecific splitting of the enzyme that occurs in parallel to the main reaction. In addition to the tagged proteins the properly assembled heterodimer containing intact alpha and beta subunits free of HT was overproduced and purified. Aminoacylation activity of the overproduced human PheRS in the crude bacterial extract is two orders of magnitude higher than the corresponding activity in human placenta and the yield of the recombinant enzyme overproduced in E. coli is five times higher.     

The unexpected structural role of glutamate synthase [4Fe-4S](+1,+2) clusters as demonstrated by site-directed mutagenesis of conserved C residues at the N-terminus of the enzyme beta subunit

Azospirillum brasilense glutamate synthase (GltS) is a complex iron-sulfur flavoprotein whose catalytically active alphabeta protomer (alpha subunit, 162kDa; beta subunit, 52.3 kDa) contains one FAD, one FMN, one [3Fe-4S](0,+1), and two [4Fe-4S](+1,+2) clusters. The structure of the alpha subunit has been determined providing information on the mechanism of ammonia transfer from L-glutamine to 2-oxoglutarate through a 30 A-long intramolecular tunnel. On the contrary, details of the electron transfer pathway from NADPH to the postulated 2-iminoglutarate intermediate through the enzyme flavin co-factors and [Fe-S] clusters are largely indirect. To identify the location and role of each one of the GltS [4Fe-4S] clusters, we individually substituted the four cysteinyl residues forming the first of two conserved C-rich regions at the N-terminus of GltS beta subunit with alanyl residues. The engineered genes encoding the beta subunit variants (and derivatives carrying C-terminal His6-tags) were co-expressed with the wild-type alpha subunit gene. In all cases the C/A substitutions prevented alpha and beta subunits association to yield the GltS alphabeta protomer. This result is consistent with the fact that these residues are responsible for the formation of glutamate synthase [4Fe-4S](+1,+2) clusters within the N-terminal region of the beta subunit, and that these clusters are implicated not only in electron transfer between the GltS flavins, but also in alphabeta heterodimer formation by structuring an N-terminal [Fe-S] beta subunit interface subdomain, as suggested by the three-dimensional structure of dihydropyrimidine dehydrogenase, an enzyme containing an N-terminal beta subunit-like domain.     

The crystal structure of leucyl-tRNA synthetase complexed with tRNALeu in the post-transfer-editing conformation

Leucyl-tRNA synthetase (LeuRS) has a specific post-transfer editing activity directed against mischarged isoleucine and similar noncognate amino acids. We describe the post-transfer-editing and product complexes of Thermus thermophilus LeuRS (LeuRSTT) with tRNA(Leu) at 2.9- to 3.3-A resolution. In the post-transfer-editing configuration, A76 binds in the editing active site exactly as previously found for the adenosine moiety of a small-molecule editing-substrate analog. The 60 C-terminal residues of LeuRSTT, unseen in previous structures, fold into a compact domain flexibly linked to the rest of the molecule and interacting with the G19-C56 tertiary base pair of tRNA(Leu). LeuRS recognition of tRNA(Leu) depends essentially on tRNA shape rather than base-specific interactions. The structures show that considerable domain rotations, notably of the editing domain, accompany the tRNA-3' end dynamics associated successively with aminoacylation, post-transfer editing and product release.     

Amphipathic alpha-helix mediates the heterodimerization of soluble guanylyl cyclase

Soluble guanylyl cyclase (soluble GC) is an enzyme consisting of alpha and beta subunits and catalyzes the conversion of GTP to cGMP. The formation of the heterodimer is essential for the activity of soluble GC. Each subunit of soluble GC has been shown to comprize three functionally different parts: a C-terminal catalytic domain, a central dimerization domain, and an N-terminal regulatory domain. The central dimerization domain of the beta(1) subunit, which contains an N-terminal binding site (NBS) and a C-terminal binding site (CBS), has been postulated to be responsible for the formation of alpha/ beta heterodimer. In this study, we analyzed heterodimerization by the pull-down assay using the affinity between a histidine tag and Ni(2+) Sepharose after co-expression of various N- and C-terminally truncated FLAG-tagged mutants of the alpha(1) subunit and the histidine-tagged wild type of the beta(1) subunit in the vaculovirus/Sf9 system, and demonstrated that the CBS-like sequence of the alpha(1) subunit is critical for the formation of the heterodimer with the beta(1) subunit and the NBS-like sequence of the alpha(1) subunit is essential for the formation of the enzymatically active heterodimer, although this particular sequence was not involved in heterodimerization. The analysis of the secondary structure of the alpha(1) subunit predicted the existence of an amphipathic alpha-helix in residues 431-464. Experiments with site-directed alpha(1) subunit mutant proteins demonstrated that the amphipathicity of the alpha-helix is important for the formation of the heterodimer, and Leu(463) in the alpha-helix region plays a critical role in the formation of a properly arranged active center in the dimer.     

High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus

Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5 kDa, and named alphabeta-LeuRS. The gene encoding alpha subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of alpha subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNA(Arg)(AGA/AGG). The argU gene helps A. aeolicus LeuRS, which contains AGA/AGG codons in exceptionally high frequency, express well in E. coli. The gene encoding beta subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce alphabeta-LeuRS with a His6 tag at the N-terminus of alpha subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7 mg purified alphabeta-LeuRS was obtained from 250 ml culture. The His6-tag at the N-terminus did not affect the aminoacylation activity of the enzyme.     

Release of a damaged cofactor from a coenzyme B12-dependent enzyme: X-ray structures of diol dehydratase-reactivating factor

The crystal structures of ADP bound and nucleotide-free forms of molecular chaperone-like diol dehydratase-reactivating factor (DDR) were determined at 2.0 and 3.0 A, respectively. DDR exists as a dimer of heterodimer (alphabeta)2. The alpha subunit has four domains: ATPase domain, swiveling domain, linker domain, and insert domain. The beta subunit, composed of a single domain, has a similar fold to the beta subunit of diol dehydratase (DD). The binding of an ADP molecule to the nucleotide binding site of DDR causes a marked conformational change of the ATPase domain of the alpha subunit, which would weaken the interactions between the DDR alpha and beta subunits and make the displacement of the DDR beta subunit by DD through the beta subunit possible. The binding of the DD beta subunit to the DDR alpha subunit induces steric repulsion between the DDR alpha and DD alpha subunits that would lead to the release of a damaged cofactor from inactivated holoDD.     

Functional implications of the unstructured loop in the (beta/alpha)(8) barrel structure of the bacterial luciferase alpha subunit.

Bacterial luciferase catalyzes the conversion of FMNH(2), a long-chain aliphatic aldehyde, and molecular oxygen to FMN, the corresponding carboxylic acid, and H(2)O with the emission of light. The light-emitting species is an enzyme-bound excited state flavin. The enzyme is a heterodimer (alphabeta) of homologous subunits each with an (beta/alpha)(8) barrel structure. A portion of the loop in the alpha subunit that connects beta strand 7 to alpha helix 7 is disordered in the crystal structure. To test the hypothesis that this loop closes over the active site during catalysis and protects the active site from bulk solvent, a mutant was constructed in which the 29 residues that are disordered in the 2.4 A crystal structure were deleted. Deletion of this loop results in a heterodimer with a subunit equilibrium dissociation constant of 1.32 +/- 1.25 microM, whereas the wild-type heterodimer shows no measurable subunit dissociation. This mutant retains its ability to bind substrate flavin and aldehyde with wild-type affinity and can carry out the chemistry of the bioluminescence reaction with nearly wild-type efficiency. However, the bioluminescent quantum yield of the reaction is reduced nearly 2 orders of magnitude from that of the wild-type enzyme.     

Protein engineering of homodimeric tyrosyl-tRNA synthetase to produce active heterodimers

Heterodimers of tyrosyl-tRNA synthetase from Bacillus stearothermophilus have been produced by mutagenesis at the subunit interface. Oppositely charged groups have been engineered into the subunits so that they can form a complementary pair. Wild-type tyrosyl-tRNA synthetase is a symmetrical dimer in which the side chains of the 2 Phe-164 residues interact at the subunit interface. Phe-164 was mutated to Asp in tyrosyl-tRNA synthetase and to Lys in a truncated enzyme (des-(321-419)tyrosyl-tRNA synthetase) which lacks the two tRNA-binding sites, but which can catalyze pyrophosphate exchange. The size difference allows subunit association to be studied by gel filtration chromatography. These changes induce reversible dissociation from active dimers into inactive monomers at pH values which favor ionization at position 164. A mixture of the two mutants near neutral pH is apparently fully active in pyrophosphate exchange and consists of a heterodimer of [Asp164]tyrosyl-tRNA synthetase and [Lys164]des-(321-419)tyrosyl-tRNA synthetase. Despite having only one binding site for tRNA, heterodimer has full aminoacylation activity at high concentrations of tyrosine. We have therefore produced a family of dimers that differ in stability near neutral pH. This novel approach using protein engineering allows specific dimerization of subunits of the same size that have different defined mutations, each subunit being tagged by the charge. Such hybrid proteins can be used to study subunit interaction.     

The peptide bond between E292-A293 of Escherichia coli leucyl-tRNA synthetase is essential for its activity.

Escherichia coli leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that contains a large connecting polypeptide (CP1) inserted into its nucleotide binding fold, or active site. In this study, purified leucyl-tRNA synthetase was found to be cleaved between E292 and A293 in its CP1 domain. SDS-PAGE analysis showed peptides of 63 and 34 kDa in addition to the native 97.3 kDa synthetase. By internal complementation, the two peptides could form a 97.3 kDa complex similar to the native LeuRS. This complex could support the ATP approximately PP(i) exchange activity of LeuRS, but could not complement for aminoacylation. To study the function of the region around the bond of E292 and A293, four pairs of peptides resulting from different cleavage sites in CP1 were reconstituted in vivo. With the exception of the enzyme assembled from the E292-A293 cleavage site, all the reassembled LeuRSs catalyzed the aminoacylation of tRNA(Leu). Although the E292-A293-cleaved LeuRS could not catalyze aminoacylation, fluorescence titration revealed that its tRNA binding ability was almost identical to that of wild-type LeuRS. These results suggest that the region around E292-A293 may be responsible for maintaining the proper conformation of LeuRS required for the tRNA charging activity.     

CD8alphabeta has two distinct binding modes of interaction with peptide-major histocompatibility complex class I

Interaction of CD8 (CD8alphaalpha or CD8alphabeta) with the peptide-major histocompatibility complex (MHC) class I (pMHCI) is critical for the development and function of cytolytic T cells. Although the crystal structure of CD8alphaalpha.pMHCI complex revealed that two symmetric CD8alpha subunits interact with pMHCI asymmetrically, with one subunit engaged in more extensive interaction than the other, the details of the interaction between the CD8alphabeta heterodimer and pMHCI remained unknown. The Ig-like domains of mouse CD8alphabeta and CD8alphaalpha are similar in the size, shape, and surface electrostatic potential of their pMHCI-binding regions, suggesting that their interactions with pMHCI could be very similar. Indeed, we found that the CD8alpha variants CD8alpha(R8A) and CD8alpha(E27A), which were functionally inactive as homodimers, could form an active co-receptor with wild-type (WT) CD8beta as a CD8alpha(R8A)beta or CD8alpha(E27A)beta heterodimer. We also identified CD8beta variants that could form active receptors with WT CD8alpha but not with CD8alpha(R8A). This observation is consistent with the notion that the CD8beta subunit may replace either CD8alpha subunit in CD8alphaalpha.pMHCI complex. In addition, we showed that both anti-CD8alpha and anti-CD8beta antibodies were unable to completely block the co-receptor activity of WT CD8alphabeta. We propose that CD8alphabeta binds to pMHCI in at least two distinguishable orientations.