STRUCTURES, DYNAMICS AND INTERACTIONS OF EPHA5, EPHA7 AND NOGO-66 INVOLVED IN CNS REGNERATION INHIBITION

Ph.DDOCTOR OF PHILOSOPH

Unique Structure and Dynamics of the EphA5 Ligand Binding Domain Mediate Its Binding Specificity as Revealed by X-ray Crystallography, NMR and MD Simulations

10.1371/journal.pone.0074040PLoS ONE89-POLN

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

Abstract 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)

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 α-β-β-β-α topology and resembles the structural similarity to the double-stranded RNA-binding domain. Notably, the N-terminal α helix of DUF283 runs cross over the C-terminal α 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

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.Singapore Food AgencyPublished versionThis research is supported by the Singapore Food Agency under the Singapore Food Story ("SFS") R&D Programme 1st Grant Call (Theme 1 "Sustainable Urban Food Production") (SFS_RND_SUFP_001_01)

The structure of the unbound EphA5 LBD resembles that of other Eph receptors bound to ephrin ligands.

<p>(a) Superimposition of the LBD structures of unbound EphA5 (red), EphA2 (green, 3C8X), EphA4 (yellow, 3CKH) and EphB2 (blue, 3ETP). A short β-sheet is formed by the EphA4 and EphB2 residues corresponding to EphA5 residues Ala179-Ser182 and Gly189-M193 in the J–K loop (orange arrows). (b) Superimposition of the LBD structures of the unbound EphA5 (red), EphA2 in complex with ephrin-A2 (green, 3CZU), EphA4 with ephrin-A2 (cyan, 3WO3), EphA4 with ephrin-B2 (blue, 3GXU), EphB2 with ephrin-B2 (pink, 1KGY) and EphB4 with ephrin-B2 (yellow, 2HLE).</p

Sequence-structure relationship for the EphA5 and EphA4 LBDs.

<p>(a) Alignment of the sequences of the EphA5 and EphA4 LBDs. Identical residues are colored in blue, homologous in green and different in black. Residues in the D and E β-strands are highlighted in yellow and residues in the J–K loop in pink. Two residues that are in close contact in the EphA4 structure (Ile in the D strand and Asp in the J–K-loop) and the corresponding residues in the EphA5 LBD are boxed. (b) Structure of the EphA5 LBD with spheres for Asp190 in the J–K-loop, and Glu80 in the D strand which corresponds to a Ile in the structure of the EphA4 LBD (c).</p

Distinctive dynamic behaviors of the EphA5 LBD as revealed by molecular dynamics simulations.

<p>(a) Trajectories of root-mean-square deviations (RMSD) of heavy atoms in three independent molecular dynamics simulations. (b) Trajectories of root-mean-square fluctuations (RMSF) of the Cα atoms computed for three independent simulations, with average values and standard deviations calculated over 30 ns for each simulation. (c) EphA5 LBD structure with the residues having RMSF >average in green and those >2-fold the average in red.</p

¹⁵N backbone dynamics for the EphA5 LBD on the ps-ns time scale.

<p>(a) Generalized squared order parameter (S²) derived from the Model-free analysis of the relaxation data for EphA5. Red indicates residues with S² < the average value. (b) Residue-specific Rex derived from Model-free analysis of relaxation data for EphA5 (green) and EphA4 (red and light brown). Red indicates EphA4 residues in the D and E strands as well as D–E and J–K loops while light brown for the other EphA4 residues. (c) EphA5 LBD structure with residues having S² < the average value (0.7) colored in green and those with S² < the average – STD (0.5) in red. (d) EphA5 LBD structure with residues having Rex >2 Hz colored in cyan and those >5 Hz colored in red.</p

Unique ligand-binding specificity of the EphA5 LBD.

<p>(a) Isothermal titration calorimetry profiles for the interaction of the EphA5 LBD with the WDC peptide (upper panel) and plots of the integrated values for the reaction heats (after blank subtraction and normalization to the amount of the peptide injected) versus EphA5 to WDC molar ratio (lower panel). The thermodynamic binding parameters are shown in the lower panel. (b) Inhibition of ephrin-A5 alkaline phosphatase (AP) binding to immobilized EphA5 Fc by increasing concentrations of WDC in ELISAs. Bound ephrin-A5 AP represents the ratio of the OD at 405 nm for ephrin-A5 AP bound to EphA5 Fc in the presence of the indicated concentrations of the WDC peptide and in the absence of peptide. (c) Inhibition of ephrin-A5 AP binding to EphA receptors and ephrin-B2 AP binding to EphB receptors by 100 μM WDC. Bound ephrin AP represents the ratio of the OD at 405 nm for ephrin-A5 AP or ephrin-B2 AP bound to different Eph receptor Fc proteins in the presence of WDC peptide and in the absence of peptide. The peptide substantially inhibits ephrin binding only to EphA5. Averages and standard errors from triplicate measurements are shown. (d) Superimposition of the NMR HSQC spectra of the EphA5 LBD in the absence (blue) and in the presence (red) of WDC at a molar ratio of 1:3 (EphA5:WDC). (e) Superimposition of the NMR HSQC spectra of the EphA5 LBD in the absence (blue) and in the presence (red) of C1 at a molar ratio of 1:20 (EphA5:C1).</p