Allopurinol non-covalently facilitates binding of unconventional peptides to HLA-B*58:01.

Allopurinol, widely used in gout treatment, is the most common cause of severe cutaneous adverse drug reactions. The risk of developing such life-threatening reactions is increased particularly for HLA-B*58:01 positive individuals. However the mechanism of action between allopurinol and HLA remains unknown. We demonstrate here that a Lamin A/C peptide KAGQVVTI which is unable to bind HLA-B*58:01 on its own, is enabled to form a stable peptide-HLA complex only in the presence of allopurinol. Crystal structure analysis reveal that allopurinol non-covalently facilitated KAGQVVTI to adopt an unusual binding conformation, whereby the C-terminal isoleucine does not engage as a PΩ that typically fit deeply in the binding F-pocket. A similar observation, though to a lesser degree was seen with oxypurinol. Presentation of unconventional peptides by HLA-B*58:01 aided by allopurinol contributes to our fundamental understanding of drug-HLA interactions. The binding of peptides from endogenously available proteins such as self-protein lamin A/C and viral protein EBNA3B suggest that aberrant loading of unconventional peptides in the presence of allopurinol or oxypurinol may be able to trigger anti-self reactions that can lead to Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS).

Molecular Characterization of MHC Class I Alpha 1 and 2 Domains in Asian Seabass (Lates calcarifer).

The Asian seabass is of importance both as a farmed and wild animal. With the emergence of infectious diseases, there is a need to understand and characterize the immune system. In humans, the highly polymorphic MHC class I (MHC-I) molecules play an important role in antigen presentation for the adaptive immune system. In the present study, we characterized a single MHC-I gene in Asian seabass (Lates calcarifer) by amplifying and sequencing the MHC-I alpha 1 and alpha 2 domains, followed by multi-sequence alignment analyses. The results indicated that the Asian seabass MHC-I α1 and α2 domain sequences showed an overall similarity within Asian seabass and retained the majority of the conserved binding residues of human leukocyte antigen-A2 (HLA-A2). Phylogenetic tree analysis revealed that the sequences belonged to the U lineage. Mapping the conserved binding residue positions on human HLA-A2 and grass carp crystal structure showed a high degree of similarity. In conclusion, the availability of MHC-I α1 and α2 sequences enhances the quality of MHC class I genetic information in Asian seabass, providing new tools to analyze fish immune responses to pathogen infections, and will be applicable in the study of the phylogeny and the evolution of antigen-specific receptors.

Crystal structure of suboptimal viral fragments of Epstein Barr Virus Rta peptide-HLA complex that stimulate CD8 T cell response.

Peptides presented by Human leukocyte antigen (HLA) class-I molecules are generally 8-10 amino acids in length. However, the predominant pool of peptide fragments generated by proteasomes is less than 8 amino acids in length. Using the Epstein - Barr virus (EBV) Rta-epitope (ATIGTAMYK, residues 134-142) restricted by HLA-A*11:01 which generates a strong immunodominant response, we investigated the minimum length of a viral peptide that can constitute a viral epitope recognition by CD8 T cells. The results showed that Peripheral blood mononuclear cells (PBMCs) from healthy donors can be stimulated by a viral peptide fragment as short as 4-mer (AMYK), together with a 5-mer (ATIGT) to recapitulate the full length EBV Rta epitope. This was confirmed by generating crystals of the tetra-complex (2 peptides, HLA and ß2-microglobulin). The solved crystal structure of HLA-A*11:01 in complex with these two short peptides revealed that they can bind in the same orientation similar to parental peptide (9-mer) and the free ends of two short peptides acquires a bulged conformation that is directed towards the T cell receptor. Our data shows that suboptimal length of 4-mer and 5-mer peptides can complement each other to form a stable peptide-MHC (pMHC) complex.

Crystallization and preliminary X-ray crystallographic analysis of a putative nonribosomal peptide synthase AmbB from Pseudomonas aeruginosa.

AmbB is a putative nonribosomal peptide synthase from Pseudomonas aeruginosa, which is involved in the production of IQS, a potent cell-cell communication signal molecule that integrates the quorum-sensing mechanism and stress response. It consists of 1249 amino acids and contains an AMP-binding domain, a phosphopantetheine-binding (PB) domain and a condensation (C) domain. In this report, a truncated form of AmbB that contains the PB domain and the condensation domain was overexpressed with an N-terminal GST tag in Escherichia coli and purified as a monomer using affinity and size-exclusion chromatography. The recombinant AmbBc (comprising residues 727-1249 of full-length AmbB) was crystallized using the hanging-drop vapour-diffusion method and a full data set was collected to 2.45 Šresolution using a synchrotron-radiation source. The crystals belonged to space group P6122 or P6522, with unit-cell parameters a = b = 87.81, c = 286.8 Å, α = 90, ß = 90, γ = 120°, and contained one molecule per asymmetric unit.

Unique structure and dynamics of the EphA5 ligand binding domain mediate its binding specificity as revealed by X-ray crystallography, NMR and MD simulations.

The 16 EphA and EphB receptors represent the largest family of receptor tyrosine kinases, and their interactions with 9 ephrin-A and ephrin-B ligands initiate bidirectional signals controlling many physiological and pathological processes. Most interactions occur between receptor and ephrins of the same class, and only EphA4 can bind all A and B ephrins. To understand the structural and dynamic principles that enable Eph receptors to utilize the same jellyroll ß-sandwich fold to bind ephrins, the VAPB-MSP domain, peptides and small molecules, we have used crystallography, NMR and molecular dynamics (MD) simulations to determine the first structure and dynamics of the EphA5 ligand-binding domain (LBD), which only binds ephrin-A ligands. Unexpectedly, despite being unbound, the high affinity ephrin-binding pocket of EphA5 resembles that of other Eph receptors bound to ephrins, with a helical conformation over the J-K loop and an open pocket. The openness of the pocket is further supported by NMR hydrogen/deuterium exchange data and MD simulations. Additionally, the EphA5 LBD undergoes significant picosecond-nanosecond conformational exchanges over the loops, as revealed by NMR and MD simulations, but lacks global conformational exchanges on the microsecond-millisecond time scale. This is markedly different from the EphA4 LBD, which shares 74% sequence identity and 87% homology. Consequently, the unbound EphA5 LBD appears to comprise an ensemble of open conformations that have only small variations over the loops and appear ready to bind ephrin-A ligands. These findings show how two proteins with high sequence homology and structural similarity are still able to achieve distinctive binding specificities through different dynamics, which may represent a general mechanism whereby the same protein fold can serve for different functions. Our findings also suggest that a promising strategy to design agonists/antagonists with high affinity and selectivity might be to target specific dynamic states of the Eph receptor LBDs.

Structure of the Arabidopsis thaliana DCL4 DUF283 domain reveals a noncanonical double-stranded RNA-binding fold for protein-protein interaction.

Dicer or Dicer-like (DCL) protein is a catalytic component involved in microRNA (miRNA) or small interference RNA (siRNA) processing pathway, whose fragment structures have been partially solved. However, the structure and function of the unique DUF283 domain within dicer is largely unknown. Here we report the first structure of the DUF283 domain from the Arabidopsis thaliana DCL4. The DUF283 domain adopts an alpha-beta-beta-beta-alpha topology and resembles the structural similarity to the double-stranded RNA-binding domain. Notably, the N-terminal alpha helix of DUF283 runs cross over the C-terminal alpha helix orthogonally, therefore, N- and C-termini of DUF283 are in close proximity. Biochemical analysis shows that the DUF283 domain of DCL4 displays weak dsRNA binding affinity and specifically binds to double-stranded RNA-binding domain 1 (dsRBD1) of Arabidopsis DRB4, whereas the DUF283 domain of DCL1 specifically binds to dsRBD2 of Arabidopsis HYL1. These data suggest a potential functional role of the Arabidopsis DUF283 domain in target selection in small RNA processing.

Structural characterization of the EphA4-Ephrin-B2 complex reveals new features enabling Eph-ephrin binding promiscuity.

EphA and EphB receptors preferentially bind ephrin-A and ephrin-B ligands, respectively, but EphA4 is exceptional for its ability to bind all ephrins. Here, we report the crystal structure of the EphA4 ligand-binding domain in complex with ephrin-B2, which represents the first structure of an EphA-ephrin-B interclass complex. A loose fit of the ephrin-B2 G-H loop in the EphA4 ligand-binding channel is consistent with a relatively weak binding affinity. Additional surface contacts also exist between EphA4 residues Gln(12) and Glu(14) and ephrin-B2. Mutation of Gln(12) and Glu(14) does not cause significant structural changes in EphA4 or changes in its affinity for ephrin-A ligands. However, the EphA4 mutant has approximately 10-fold reduced affinity for ephrin-B ligands, indicating that the surface contacts are critical for interclass but not intraclass ephrin binding. Thus, EphA4 uses different strategies to bind ephrin-A or ephrin-B ligands and achieve binding promiscuity. NMR characterization also suggests that the contacts of Gln(12) and Glu(14) with ephrin-B2 induce dynamic changes throughout the whole EphA4 ligand-binding domain. Our findings shed light on the distinctive features that enable the remarkable ligand binding promiscuity of EphA4 and suggest that diverse strategies are needed to effectively disrupt different Eph-ephrin complexes.