Matthew C. J. Wilce

CD1d–lipid-antigen recognition by the semi-invariant NKT T-cell receptor

The CD1 family is a large cluster of non-polymorphic, major histocompatibility complex (MHC) class-I-like molecules that bind distinct lipid-based antigens that are recognized by T cells. The most studied group of T cells that interact with lipid antigens are natural killer T (NKT) cells, which characteristically express a semi-invariant T-cell receptor (NKT TCR) that specifically recognizes the CD1 family member, CD1d. NKT-cell-mediated recognition of the CD1d–antigen complex has been implicated in microbial immunity, tumour immunity, autoimmunity and allergy. Here we describe the structure of a human NKT TCR in complex with CD1d bound to the potent NKT-cell agonist α-galactosylceramide, the archetypal CD1d-restricted glycolipid. In contrast to T-cell receptor–peptide-antigen–MHC complexes, the NKT TCR docked parallel to, and at the extreme end of the CD1d-binding cleft, which enables a lock-and-key type interaction with the lipid antigen. The structure provides a basis for the interaction between the highly conserved NKT TCR α-chain and the CD1d–antigen complex that is typified in innate immunity, and also indicates how variability of the NKT TCR β-chain can impact on recognition of other CD1d–antigen complexes. These findings provide direct insight into how a T-cell receptor recognizes a lipid-antigen-presenting molecule of the immune system.

Ab5 Subtilase Cytotoxin Inactivates the Endoplasmic Reticulum Chaperone Bip

AB5 toxins are produced by pathogenic bacteria and consist of enzymatic A subunits that corrupt essential eukaryotic cell functions, and pentameric B subunits that mediate uptake into the target cell. AB5 toxins include the Shiga, cholera and pertussis toxins and a recently discovered fourth family, subtilase cytotoxin, which is produced by certain Shiga toxigenic strains of Escherichia coli. Here we show that the extreme cytotoxicity of this toxin for eukaryotic cells is due to a specific single-site cleavage of the essential endoplasmic reticulum chaperone BiP/GRP78. The A subunit is a subtilase-like serine protease; structural studies revealed an unusually deep active-site cleft, which accounts for its exquisite substrate specificity. A single amino-acid substitution in the BiP target site prevented cleavage, and co-expression of this resistant protein protected transfected cells against the toxin. BiP is a master regulator of endoplasmic reticulum function, and its cleavage by subtilase cytotoxin represents a previously unknown trigger for cell death.

T cell allorecognition via molecular mimicry.

T cells often alloreact with foreign human leukocyte antigens (HLA). Here we showed the LC13 T cell receptor (TCR), selected for recognition on self-HLA-B( *)0801 bound to a viral peptide, alloreacts with B44 allotypes (HLA-B( *)4402 and HLA-B( *)4405) bound to two different allopeptides. Despite extensive polymorphism between HLA-B( *)0801, HLA-B( *)4402, and HLA-B( *)4405 and the disparate sequences of the viral and allopeptides, the LC13 TCR engaged these peptide-HLA (pHLA) complexes identically, accommodating mimicry of the viral peptide by the allopeptide. The viral and allopeptides adopted similar conformations only after TCR ligation, revealing an induced-fit mechanism of molecular mimicry. The LC13 T cells did not alloreact against HLA-B( *)4403, and the single residue polymorphism between HLA-B( *)4402 and HLA-B( *)4403 affected the plasticity of the allopeptide, revealing that molecular mimicry was associated with TCR specificity. Accordingly, molecular mimicry that is HLA and peptide dependent is a mechanism for human T cell alloreactivity between disparate cognate and allogeneic pHLA complexes.

Large conformational changes of the ɛ subunit in the bacterial F1F0 ATP synthase provide a ratchet action to regulate this rotary motor enzyme

The F1F0 ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the γ and ɛ subunits in the F1 part and c subunit ring in the F0 part, rotates relative to a stator composed of α3β3δab2 during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the ɛ subunit plays a key role by acting as a switch of this motor. Two different arrangements of the ɛ subunit have been visualized recently. The first has been observed in beef heart mitochondrial F1-ATPase where the C-terminal portion is arranged as a two-α-helix hairpin structure that extends away from the α3β3 region, and toward the position of the c subunit ring in the intact F1F0. The second arrangement was observed in a structure determination of a complex of the γ and ɛ subunits of the Escherichia coli F1-ATPase. In this, the two C-terminal helices are apart and extend along the γ to interact with the α and β subunits in the intact complex. We have been able to trap these two arrangements by cross-linking after introducing appropriate Cys residues in E. coli F1F0, confirming that both conformations of the ɛ subunit exist in the enzyme complex. With the C-terminal domain of ɛ toward the F0, ATP hydrolysis is activated, but the enzyme is fully coupled in both ATP hydrolysis and synthesis. With the C-terminal domain toward the F1 part, ATP hydrolysis is inhibited and yet the enzyme is fully functional in ATP synthesis; i.e., it works in one direction only. These results help explain the inhibitory action of the ɛ subunit in the F1F0 complex and argue for a ratchet function of this subunit.

GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop.

Gamma-aminobutyric acid (GABA) is synthesized by two isoforms of the pyridoxal 5′-phosphate–dependent enzyme glutamic acid decarboxylase (GAD65 and GAD67). GAD67 is constitutively active and is responsible for basal GABA production. In contrast, GAD65, an autoantigen in type I diabetes, is transiently activated in response to the demand for extra GABA in neurotransmission, and cycles between an active holo form and an inactive apo form. We have determined the crystal structures of N-terminal truncations of both GAD isoforms. The structure of GAD67 shows a tethered loop covering the active site, providing a catalytic environment that sustains GABA production. In contrast, the same catalytic loop is inherently mobile in GAD65. Kinetic studies suggest that mobility in the catalytic loop promotes a side reaction that results in cofactor release and GAD65 autoinactivation. These data reveal the molecular basis for regulation of GABA homeostasis.

Structure of the gamma-epsilon complex of ATP synthase.

ATP synthases (F1Fo-ATPases) use energy released by the movement of protons down a transmembrane electrochemical gradient to drive the synthesis of ATP, the universal biological energy currency. Proton flow through Fo drives rotation of a ring of c-subunits and a complex of the γ and ɛ-subunits, causing cyclical conformational changes in F1 that are required for catalysis. The crystal structure of a large portion of F1 has been resolved. However, the structure of the central portion of the enzyme, through which conformational changes in Fo are communicated to F1, has until now remained elusive. Here we report the crystal structure of a complex of the ɛ-subunit and the central domain of the γ-subunit refined at 2.1 Å resolution. The structure reveals how rotation of these subunits causes large conformational changes in F1, and thereby provides new insights into energy coupling between Fo and F1.

Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin

AB5 toxins comprise an A subunit that corrupts essential eukaryotic cell functions, and pentameric B subunits that direct target-cell uptake after binding surface glycans. Subtilase cytotoxin (SubAB) is an AB5 toxin secreted by Shiga toxigenic Escherichia coli (STEC), which causes serious gastrointestinal disease in humans. SubAB causes haemolytic uraemic syndrome-like pathology in mice through SubA-mediated cleavage of BiP/GRP78, an essential endoplasmic reticulum chaperone. Here we show that SubB has a strong preference for glycans terminating in the sialic acid N-glycolylneuraminic acid (Neu5Gc), a monosaccharide not synthesized in humans. Structures of SubB-Neu5Gc complexes revealed the basis for this specificity, and mutagenesis of key SubB residues abrogated in vitro glycan recognition, cell binding and cytotoxicity. SubAB specificity for Neu5Gc was confirmed using mouse tissues with a human-like deficiency of Neu5Gc and human cell lines fed with Neu5Gc. Despite lack of Neu5Gc biosynthesis in humans, assimilation of dietary Neu5Gc creates high-affinity receptors on human gut epithelia and kidney vasculature. This, and the lack of Neu5Gc-containing body fluid competitors in humans, confers susceptibility to the gastrointestinal and systemic toxicities of SubAB. Ironically, foods rich in Neu5Gc are the most common source of STEC contamination. Thus a bacterial toxins receptor is generated by metabolic incorporation of an exogenous factor derived from food.

CD94-NKG2A recognition of human leukocyte antigen (HLA)-E bound to an HLA class I leader sequence

The recognition of human leukocyte antigen (HLA)-E by the heterodimeric CD94-NKG2 natural killer (NK) receptor family is a central innate mechanism by which NK cells monitor the expression of other HLA molecules, yet the structural basis of this highly specific interaction is unclear. Here, we describe the crystal structure of CD94-NKG2A in complex with HLA-E bound to a peptide derived from the leader sequence of HLA-G. The CD94 subunit dominated the interaction with HLA-E, whereas the NKG2A subunit was more peripheral to the interface. Moreover, the invariant CD94 subunit dominated the peptide-mediated contacts, albeit with poor surface and chemical complementarity. This unusual binding mode was consistent with mutagenesis data at the CD94-NKG2A–HLA-E interface. There were few conformational changes in either CD94-NKG2A or HLA-E upon ligation, and such a “lock and key” interaction is typical of innate receptor–ligand interactions. Nevertheless, the structure also provided insight into how this interaction can be modulated by subtle changes in the peptide ligand or by the pairing of CD94 with other members of the NKG2 family. Differences in the docking strategies used by the NKG2D and CD94-NKG2A receptors provided a basis for understanding the promiscuous nature of ligand recognition by NKG2D compared with the fidelity of the CD94-NKG2 receptors.

Natural micropolymorphism in human leukocyte antigens provides a basis for genetic control of antigen recognition

Human leukocyte antigen (HLA) gene polymorphism plays a critical role in protective immunity, disease susceptibility, autoimmunity, and drug hypersensitivity, yet the basis of how HLA polymorphism influences T cell receptor (TCR) recognition is unclear. We examined how a natural micropolymorphism in HLA-B44, an important and large HLA allelic family, affected antigen recognition. T cell–mediated immunity to an Epstein-Barr virus determinant (EENLLDFVRF) is enhanced when HLA-B*4405 was the presenting allotype compared with HLA-B*4402 or HLA-B*4403, each of which differ by just one amino acid. The micropolymorphism in these HLA-B44 allotypes altered the mode of binding and dynamics of the bound viral epitope. The structure of the TCR–HLA-B*4405EENLLDFVRF complex revealed that peptide flexibility was a critical parameter in enabling preferential engagement with HLA-B*4405 in comparison to HLA-B*4402/03. Accordingly, major histocompatibility complex (MHC) polymorphism can alter the dynamics of the peptide-MHC landscape, resulting in fine-tuning of T cell responses between closely related allotypes.

Antigen Ligation Triggers a Conformational Change within the Constant Domain of the αβ T Cell Receptor

Ligation of the alphabeta T cell receptor (TCR) by a specific peptide-loaded major histocompatibility complex (pMHC) molecule initiates T cell signaling via the CD3 complex. However, the initial events that link antigen recognition to T cell signal transduction remain unclear. Here we show, via fluorescence-based experiments and structural analyses, that MHC-restricted antigen recognition by the alphabeta TCR results in a specific conformational change confined to the A-B loop within the alpha chain of the constant domain (Calpha). The apparent affinity constant of this A-B loop movement mirrored that of alphabeta TCR-pMHC ligation and was observed in two alphabeta TCRs with distinct pMHC specificities. The Ag-induced A-B loop conformational change could be inhibited by fixing the juxtapositioning of the constant domains and was shown to be reversible upon pMHC disassociation. Notably, the loop movement within the Calpha domain, although specific for an agonist pMHC ligand, was not observed with a pMHC antagonist. Moreover, mutagenesis of residues within the A-B loop impaired T cell signaling in an in vitro system of antigen-specific TCR stimulation. Collectively, our findings provide a basis for the earliest molecular events that underlie Ag-induced T cell triggering.