Dichotomy in TCR V-domain dynamics binding the opposed inclined planes of pMHC-II and pMHC-I α-helices.

Ligand recognition by the human α/ß T-cell antigen receptor (TCR) heterodimer protein, unlike the surface immunoglobulin (sIg) B-cell receptor, is not governed by relative binding affinity. Its interaction with the peptide (p) plus major histocompatibility complex (MHC) protein (abbrev. pMHC) likely involves some different molecular mechanism linking pMHC binding to T-cell functions. Recent analytical geometry of TCR:pMHC-II solved crystallographic structures (n = 40) revealed that each variable (V)-domain is bound in similar, yet mathematically unique orientations to its target pMHC groove. The relative position of the central cysteine of each V-domain was examined by multivariable calculus in spherical coordinates, where a simple volume element (dV) was found to describe clonotypic geometry with pMHC-II. Here, the study was expanded to include TCR:pMHC-I structures, and to model a physical mechanism, specifically involving the two directionally opposed inclined planes (IP) manifest by the two major α-helices prominent in both MHC-I and MHC-II proteins. Calculations for rotational torque of each V-domain, together with acceleration up and down the slopes of both MHC α-helices were used to estimate the time a given V-domain spends sliding down its cognate MHC IP. This V-domain rotation/sliding mechanism appears to be quantitatively unique for each TCR:pMHC V-domain (n = 40). However, there is an apparent and common dichotomy between the mobility of each V-domain with respect to the two classes of MHC proteins. Evolutionary motifs in the MHC helices support that the V-domains negotiate the opposed inclined planes of pMHC ligands in clonotypic fashion. Thus, this model is useful in understanding how mechanical forces are linked to TCR function.

CDR3 binding chemistry controls TCR V-domain rotational probability and germline CDR2 scanning of polymorphic MHC.

The mechanism which adapts the T-cell antigen receptor (TCR) within a given major histocompatibility complex (MHC/HLA) genotype is essential for protection against pathogens. Historically attributed to relative affinity, genetically vast TCRs are surprisingly focused towards a micromolar affinity for their respective peptide (p) plus MHC (pMHC) ligands. Thus, the somatic diversity of the TCR with respect to MHC-restriction, and (ultimately) to pathogens, remains enigmatic. Here, we derive a triple integral solution (from fixed geometry) for any given V domain in TCR bound to pMHC. Solved complexes involving HLA-DR and HLA-DQ, where genetic linkage to the TCR is most profound, were examined in detail. Certain V domains displayed rare geometry within this panel-specifying a restricted rotational probability/volumetric density (dV). Remarkably, hydrogen (H) bond charge-relays distinguished these structures from the others; suggesting that CDR3 binding chemistry dictates CDR2 contacts on the opposite MHC-II alpha helix. Together, these data suggest that TCR recapitulate dV and specialise target pMHC recognition. As such, there are implications for the design of TCR-based therapeutics.

TE-domestication and horizontal transfer in a putative Nef-AP1mu mimic of HLA-A cytoplasmic domain re-trafficking.

Genes of the major histocompatibility complex (MHC; also called HLA in human) are polymorphic elements in the genomes of sharks to humans. Class-I and class-II MHC loci appear responsible for much of the genetic linkage to myriad disease states via the capacity to bind short (~8-15 a.a.) peptides of a given pathogen's proteome, or in some cases, the altered proteomes of cancerous cells, and even (in autoimmunity) certain nominal 'self' peptides (Janeway, 2004).(1) Unfortunately, little is known about how the canonical structure of the MHC-I/-II peptide-presenting gene evolved, particularly since beyond ~500 Mya (sharks) no paralogs exist.(2,3) We previously reported that HLA-A isotype alleles with the α1-helix, R65 motif, are wide-spread in phylogeny, but that the α 2-helix, H151R motif, has apparently segregated out of most species. Surprisingly, an uncharacterized orf in T. syrichta (Loc-103275158) encoded R151, but within a truncated A-23 like gene containing 5'- and 3'- footprints of the transposon (TE), tigger-1; the extant tarsier A-23 allele is totally missing exon-3 and part-of exon-4; together, suggesting TE-mediated inactivation of an intact/ancestral A-23 allele (Murray, 2015a).(4) The unique Loc-103275158 orf encodes a putative 15-exon transcript with no apparent paralogs throughout phylogeny. However, an HLA-A11 like gene in M. leucophaeus with a shortened C-terminal domain, and an HLA-A like orf in C. atys with two linked α1/α2/α3 domains, both contain a second transmembrane segment, which is conserved in Loc-103275158. Thus, we could model the putative protein with its Nef-like tail domain docked to its MHC-I like α3 domain (i.e., on the same side of a membrane). This modeled tertiary structure is strikingly similar to the solved structure of the Nef:MHC-I CD:AP1mu transporter (Jia, 2012).(5) Nef:AP1mu binds the CD of MHC-I in trafficking MHC-I away from the trans-golgi and into the endocytic pathway in HIV-1 infected cells. The CD loop of the Loc-103275158 provisional protein conserved the nominal MHC-I CD tyrosine phosphorylation site, and it has an N-terminal SH3 domain that we docked in one conformation to its internal Nef-like domain. Here, we suggest that phosphorylation of the protein's CD-loop signals an exchange between the internal Nef-like domain and a lentiviral-Nef for binding the N-terminal SH3 domain - freeing the Nef-like domain to bind MHC-I CD. Since the 5'-tigger sequence encodes part of the pseudo α1/α2 MHC-I domain, and the 3'-tigger part of the Nef-like domain, we speculate that transposition proceeded phylogenetically disparate horizontal transfers, involving adjacent 5'- and 3'- parasitic footprints, which we also found in the Loc-103275158 orf.

An old Twist in HLA-A: CDR3α Hook up at an R65-joint.

T-cell ontogeny optimizes the α/ß T-cell receptor (TCR) repertoire for recognition of major histocompatibility complex (MHC) class-I/II genetic polymorphism, and co-evolution of TCR germline V-gene segments and the MHC must entail somatic diversity generated in the third complimentary determining regions (CDR3α/ß); however, it is still not clear how. Herein, a conspicuous structural link between the V-Jα used by several different TCR [all in complex with the same MHC molecule (HLA-A2)], and a conserved MHC motif (a.a., R65-X-X-K-A-X-S-Q72) is described. We model this R65-joint in detail, and show that the same TCR's CDR3α loop maintains its CDR2α loop at a distance of ~4 Å from polymorphic amino acid (a.a.) positions of the α-2 helix in all but one of the analyzed crystal structures. Indeed, the pitch of docked TCRs varies as their twist/tilt/sway maintains the R65-joint and peptide contacts. Thus, the R65-joint appears to have poised the HLA-A lineage toward alloreactivity.

Transposon-mediated death of an ancestral A-23-like allele: evolution of TCR-positioning motifs in the HLA-A lineage.

HLA-A alleles are characterized by tandem arginine and histidine/arginine motifs (i.e., R65 and H151R motifs) present on the α1- and α2-helix, respectively. In crystallographic structures, α/ß T-cell receptors (TCR) contact both motifs and appear to be geometrically positioned for alloreactivity. Herein, bioinformatics of "dual-motif" MHC A-like alleles were investigated across phylogeny. While A-like alleles with the R65 motif are widespread, the H151R motif has segregated out of most species. Surprisingly, an uncharacterized orf in tarsiers (Loc-103275158) encodes R151 within a truncated A-23-like gene, which is in frame with short footprints of Tc5 and Tigger transposons (TE); the extant tarsier A-23 allele is totally missing exon-3 and part of exon-4; together, suggesting TE-mediated inactivation of an intact/ancestral A-23 allele. Since the only other (non-human) dual-motif A-like alleles are in gorilla, chimpanzee, and the Florida manatee, we speculate that dual-motif A alleles first emerged in the Afrotherian lineage and reappeared during the evolution of higher primates.

Suppression of type 1 diabetes in NOD mice by bifunctional peptide inhibitor: modulation of the immunological synapse formation.

The aim of this work was to design and utilize a bifunctional peptide inhibitor called glutamic acid decarboxylase-bifunctional peptide inhibitor to suppress the progression of type 1 diabetes in non-obese diabetic mice. The hypothesis is that glutamic acid decarboxylase-bifunctional peptide inhibitor binds simultaneously to major histocompatibility complex-II and intercellular adhesion molecule type 1 on antigen-presenting cell and inhibits the immunological synapse formation during T-cell-antigen-presenting cell interactions. Glutamic acid decarboxylase-bifunctional peptide inhibitor was composed of a major epitope of the type 1 diabetes-associated antigen, glutamic acid decarboxylase 65 kDa, covalently linked to a peptide derived from CD11a of lymphocyte function-associated antigen-1. The suppression of insulitis and type 1 diabetes was evaluated using non-obese diabetic and non-obese diabetic severe combined immunodeficiency mice. Glutamic acid decarboxylase-bifunctional peptide inhibitor had the capacity to suppress invasive insulitis in non-obese diabetic mice. CD4+ T-cells isolated from glutamic acid decarboxylase-bifunctional peptide inhibitor treated mice also suppressed insulitis and hyperglycemia when transferred with diabetogenic non-obese diabetic spleen cells into non-obese diabetic severe combined immunodeficiency recipients. As predicted, the glutamic acid decarboxylase-bifunctional peptide inhibitor cross-linked a significant fraction of major histocompatibility complex class-II molecules to intercellular adhesion molecule type 1 molecules on the surface of live antigen-presenting cell. Intravenous injection of the glutamic acid decarboxylase-bifunctional peptide inhibitor elicited interleukin-4-producing T-cells in non-obese diabetic mice primed against the glutamic acid decarboxylase-epitope peptide. Together, the results indicate that glutamic acid decarboxylase-bifunctional peptide inhibitor induces interleukin-4-producing regulatory cells but does not expand the glutamic acid decarboxylase-specific Th2 population. Given that Th2 effector cells can cause pathology, the glutamic acid decarboxylase-bifunctional peptide inhibitor may represent a novel mechanism to induce interleukin-4 without Th2-associated pathology.

N-cadherin involvement in the heterotypic adherence of malignant T-cells to epithelia.

N-cadherin, a cell adhesion molecule normally found in neural cell tissue, has been found recently to be expressed on the surface of malignant T-cells. The function of N-cadherin on these cells remains unclear. Heterotypic assays between Molt-3 T lymphoblastic leukemia cells and Caco-2 epithelial monolayers were examined under different conditions to assess the functional role of N-cadherin. The results indicate that adherence of Molt-3 cells to Caco-2 monolayers was reduced significantly following pretreatment of Molt-3 cells with 100 microM of an N-cadherin-derived antagonist decapeptide. In contrast, pretreatment of Molt-3 cells with an anti-N-cadherin antibody raised against the first 20 amino acids of N-cadherin sequence led to a surprisingly marked enhancement of Molt-3 cell adherence to Caco-2 monolayers. In addition, the presence of anti-N-cadherin antibody neutralized the inhibitory effect of anti-ICAM-1 on Molt-3 adhesion to Caco-2 monolayers. This novel finding demonstrates that external stimulus through the N-cadherin amino terminus can modulate adhesion of malignant T-cells to epithelia and may promote their ability to invade or metastasize to inflammatory sites.

Inhibition of LFA-1/ICAM-1 and VLA-4/VCAM-1 as a therapeutic approach to inflammation and autoimmune diseases.

This review focuses on providing insights into the structural basis and clinical relevance of LFA-1 and VLA-4 inhibition by peptides and small molecules as adhesion-based therapeutic strategies for inflammation and autoimmune diseases. Interactions of cell adhesion molecules (CAM) play central roles in mediating immune and inflammatory responses. Leukocyte function-associated antigen (LFA-1, alpha(L)beta(2), and CD11a/CD18) and very late antigen (VLA-4, alpha(4)beta(1), and CD49d/CD29) are members of integrin-type CAM that are predominantly involved in leukocyte trafficking and extravasation. LFA-1 is exclusively expressed on leukocytes and interacts with its ligands ICAM-1, -2, and -3 to promote a variety of homotypic and heterotypic cell adhesion events required for normal and pathologic functions of the immune systems. VLA-4 is expressed mainly on lymphocyte, monocytes, and eosinophils, but is not found on neutrophils. VLA-4 interacts with its ligands VCAM-1 and fibronectin (FN) CS1 during chronic inflammatory diseases, such as rheumatoid arthritis, asthma, psoriasis, transplant-rejection, and allergy. Blockade of LFA-1 and VLA-4 interactions with their ligands is a potential target for immunosuppression. LFA-1 and VLA-4 antagonists (antibodies, peptides, and small molecules) are being developed for controlling inflammation and autoimmune diseases. The therapeutic intervention of mostly mAb-based has been extensively studied. However, due to the challenging relative efficacy/safety ratio of mAb-based therapy application, especially in terms of systemic administration and immunogenic potential, strategic alternatives in the forms of peptide, peptide mimetic inhibitors, and small molecule non-peptide antagonists are being sought. Linear and cyclic peptides derived from the sequences of LFA-1, ICAM-1, ICAM-2, VCAM-1, and FN C1 have been shown to have inhibitory effects in vitro and in vivo. Finally, understanding the mechanism of LFA-1 and VLA-4 binding to their ligands has become a fundamental basis in developing therapeutic agents for inflammation and autoimmune diseases.