Structural Analysis of the UBA Domain of X-linked Inhibitor of Apoptosis Protein Reveals Different Surfaces for Ubiquitin-Binding and Self-Association

BACKGROUND: Inhibitor of apoptosis proteins (IAPs) belong to a pivotal antiapoptotic protein family that plays a crucial role in tumorigenesis, cancer progression, chemoresistance and poor patient-survival. X-linked inhibitor of apoptosis protein (XIAP) is a prominent member of IAPs attracting intense research because it has been demonstrated to be a physiological inhibitor of caspases and apoptosis. Recently, an evolutionarily conserved ubiquitin-associated (UBA) domain was identified in XIAP and a number of RING domain-bearing IAPs. This has placed the IAPs in the group of ubiquitin binding proteins. Here, we explore the three-dimensional structure of the XIAP UBA domain (XIAP-UBA) and how it interacts with mono-ubiquitin and diubiquitin conjugates. PRINCIPAL FINDINGS: The solution structure of the XIAP-UBA domain was determined by NMR spectroscopy. XIAP-UBA adopts a typical UBA domain fold of three tightly packed alpha-helices but with an additional N-terminal 3(10) helix. The XIAP-UBA binds mono-ubiquitin as well as Lys48-linked and linear-linked diubiquitins at low-micromolar affinities. NMR analysis of the XIAP-UBA-ubiquitin interaction reveals that it involves the classical hydrophobic patches surrounding Ile44 of ubiquitin and the conserved MGF/LV motif surfaces on XIAP-UBA. Furthermore, dimerization of XIAP-UBA was observed. Mapping of the self-association surface of XIAP-UBA reveals that the dimerization interface is formed by residues in the N-terminal 3(10) helix, helix alpha1 and helix alpha2, separate from the ubiquitin-binding surface. CONCLUSION: Our results provide the first structural information of XIAP-UBA and map its interaction with mono-ubiquitin, Lys48-linked and linear-linked diubiquitins. The notion that XIAP-UBA uses different surfaces for ubiquitin-binding and self-association provides a plausible model to explain the reported selectivity of XIAP in binding polyubiquitin chains with different linkages.published_or_final_versio

Quantification of H/D isotope effects on protein hydrogen-bonds by h3JNC' and 1JNC' couplings and peptide group 15N and 13C' chemical shifts

The effect of hydrogen/deuterium exchange on protein hydrogen bond coupling constants (h3)J(NC') has been investigated in the small globular protein ubiquitin. The couplings across deuterated or protonated hydrogen bonds were measured by a long-range quantitative HA(CACO)NCO experiment. The analysis is combined with a determination of the H(N)/D(N) isotope effect on the amide group (1)J(NC') couplings and the (15)N and (13)C' chemical shifts. On average, H-bond deuteration exchange weakens (h3)J(NC') and strengthens (1)J(NC') couplings. A correlation is found between the size of the (15)N isotope shift, the (15)N chemical shift, and the (h3)J(NC') coupling constants. The data are consistent with a reduction of donor-acceptor overlap as expected from the classical Ubbelohde effect and the common understanding that H(N)/D(N) exchange leads to a shortening of the N-hydron bond length

Assessment of cancer and virus antigens for cross-reactivity in human tissues.

MOTIVATION: Cross-reactivity or invocation of autoimmune side effects in various tissues has important safety implications in adoptive immunotherapy directed against selected antigens. The ability to predict cross-reactivity (on-target and off-target toxicities) may help in the early selection of safer therapeutically relevant target antigens. RESULTS: We developed a methodology for the calculation of quantitative cross-reactivity for any defined peptide epitope. Using this approach, we performed assessment of four groups of 283 currently known human MHC-class-I epitopes including differentiation antigens, overexpressed proteins, cancer-testis (CT) antigens, and mutations displayed by tumor cells. In addition, 89 epitopes originating from viral sources were investigated. The natural occurrence of these epitopes in human tissues was assessed based on proteomics abundance data, while the probability of their presentation by MHC-class-I molecules was modeled by the method of Kesmir et al. (2002), which combines proteasomal cleavage, TAP affinity and MHC-binding predictions. The results of these analyses for many previously defined peptides are presented as cross-reactivity indices and tissue profiles. The methodology thus allows for quantitative comparisons of epitopes, and is suggested to be suited for the assessment of epitopes of candidate antigens in an early stage of development of adoptive immunotherapy. AVAILABILITY: Our method is implemented as a Java program, with curated datasets stored in a MySQL database. It predicts all naturally possible self-antigens for a given sequence of a therapeutic antigen (or epitope), and after filtering for predicted immunogenicity outputs results as an index and profile of cross-reactivity to the self-antigens in 22 human tissues

Observation of the closing of individual hydrogen bonds during TFE-induced helix formation in a peptide

Helix formation of an S-peptide analog, comprising the first 20 residues of Ribonuclease A and two additional N-terminal residues, was studied by measuring hydrogen bond (H-bond) (h3)J(NC') scalar couplings as a function of 2,2,2-trifluoroethanol (TFE) concentration. The (h3)J(NC') couplings give direct evidence for the closing of individual backbone N-H***O = C H-bonds during the TFE-induced formation of secondary structure. Whereas no (h3)J(NC') correlations could be detected without TFE, alpha-helical (i,i +4) H-bond correlations were observed for the amides of residues A5 to M15 in the presence of TFE. The analysis of individual coupling constants indicates that alpha-helix formation starts at the center of the S-peptide around residue E11 and proceeds gradually from there to both peptide ends as the TFE concentration is increased. At 60% to 90% TFE, well-formed alpha-helical H-bonds were observed for the amides hydrogens of residues K9 to Q13, whereas H-bonds of residues T5 to A8, H14, and M15 are affected by fraying. No intramolecular backbone H-bonds are present at and beyond the putative helix stop signal D16. As the (h3)J(NC') constants represent ensemble averages and the dependence of (h3)J(NC') on H-bond lengths is very steep, the size of the individual (h3)J(NC') coupling constants can be used as a measure for the population of a closed H-bond. These individual populations are in agreement with results derived from the Lifson-Roig theory for coil-to-helix transitions. The present work shows that the closing of individual H-bonds during TFE-induced helix formation can be monitored by changes in the size of H-bond scalar couplings

Peak picking NMR spectral data using non-negative matrix factorization

Background: Simple peak-picking algorithms, such as those based on lineshape fitting, perform well when peaks are completely resolved in multidimensional NMR spectra, but often produce wrong intensities and frequencies for overlapping peak clusters. For example, NOESY-type spectra have considerable overlaps leading to significant peak-picking intensity errors, which can result in erroneous structural restraints. Precise frequencies are critical for unambiguous resonance assignments. Results: To alleviate this problem, a more sophisticated peaks decomposition algorithm, based on non-negative matrix factorization (NMF), was developed. We produce peak shapes from Fourier-transformed NMR spectra. Apart from its main goal of deriving components from spectra and producing peak lists automatically, the NMF approach can also be applied if the positions of some peaks are known a priori, e.g. from consistently referenced spectral dimensions of other experiments. Conclusions: Application of the NMF algorithm to a three-dimensional peak list of the 23 kDa bi-domain section of the RcsD protein (RcsD-ABL-HPt, residues 688-890) as well as to synthetic HSQC data shows that peaks can be picked accurately also in spectral regions with strong overlap