Genetic alterations enhancing cell survival and suppressing apoptosis are hallmarks of cancer that significantly reduce the efficacy of chemotherapy or radiotherapy. The Inhibitor of Apoptosis ...Protein (IAP) family hosts conserved proteins in the apoptotic pathway whose over-expression, frequently found in tumours, potentiates survival and resistance to anticancer agents. In humans, IAPs comprise eight members hosting one or more structural Baculoviral IAP Repeat (BIR) domains. Cellular IAPs (cIAP1 and 2) indirectly inhibit caspase-8 activation, and regulate both the canonical and the non-canonical NF-κB signaling pathways. In contrast to cIAPs, XIAP (X chromosome-linked Inhibitor of Apoptosis Protein) inhibits directly the effector caspases-3 and -7 through its BIR2 domain, and initiator caspase-9 through its BIR3 domain; molecular docking studies suggested that Smac/DIABLO antagonizes XIAP by simultaneously targeting both BIR2 and BIR3 domains. Here we report analytical gel filtration, crystallographic and SAXS experiments on cIAP1-BIR3, XIAP-BIR3 and XIAP-BIR2BIR3 domains, alone and in the presence of compound 9a, a divalent homodimeric Smac mimetic. 9a is shown to bind two BIR domains inter- (in the case of two BIR3) and intra-molecularly (in the case of XIAP-BIR2BIR3), with higher affinity for cIAP1-BIR3, relative to XIAP-BIR3. Despite the different crystal lattice packing, 9a maintains a right handed helical conformation in both cIAP1-BIR3 and XIAP-BIR3 crystals, that is likely conserved in solution as shown by SAXS data. Our structural results demonstrate that the 9a linker length, its conformational degrees of freedom and its hydrophobicity, warrant an overall compact structure with optimal solvent exposure of its two active moieties for IAPs binding. Our results show that 9a is a good candidate for pre-clinical and clinical studies, worth of further investigations in the field of cancer therapy.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
By providing predicted protein structures from nearly all known protein sequences, the artificial intelligence program AlphaFold (AF) is having a major impact on structural biology. While a stunning ...accuracy has been achieved for many folding units, predicted unstructured regions and the arrangement of potentially flexible linkers connecting structured domains present challenges. Focusing on single‐chain structures without prosthetic groups, an earlier comparison of features derived from small‐angle X‐ray scattering (SAXS) data taken from the Small‐Angle Scattering Biological Data Bank (SASBDB) is extended to those calculated using the corresponding AF‐predicted structures. Selected SASBDB entries were carefully examined to ensure that they represented data from monodisperse protein solutions and had sufficient statistical precision and q resolution for reliable structural evaluation. Three examples were identified where there is clear evidence that the single AF‐predicted structure cannot account for the experimental SAXS data. Instead, excellent agreement is found with ensemble models generated by allowing for flexible linkers between high‐confidence predicted structured domains. A pool of representative structures was generated using a Monte Carlo method that adjusts backbone dihedral allowed angles along potentially flexible regions. A fast ensemble modelling method was employed that optimizes the fit of pair distance distribution functions P(r) versus r and intensity profiles I(q) versus q computed from the pool to their experimental counterparts. These results highlight the complementarity between AF prediction, solution SAXS and molecular dynamics/conformational sampling for structural modelling of proteins having both structured and flexible regions.
A rapid ensemble modelling method that optimizes the fit to the small‐angle X‐ray scattering (SAXS)‐derived pair‐wise distance distribution function P(r) versus r and the measured intensity profile I(q) versus q has been used to account for differences between AlphaFold‐predicted and experimental SAXS profiles. By considering the confidence levels that come with the predicted structures, a conformational ensemble with potentially flexible linkers between stable folded domains can be optimized to provide representative structures.
Small angle x-ray scattering was used to follow changes in the conformation and interactions of nucleosome core particles (NCP) as a function of the monovalent salt concentration
C
s. The maximal ...extension (
D
max) of the NCP (145
±
3-bp DNA) increases from 137
±
5
Å to 165
±
5
Å when
C
s rises from 10 to 50
mM and remains constant with further increases of
C
s up to 200
mM. In view of the very weak increase of the
R
g value in the same
C
s range, we attribute this
D
max variation to tail extension, a proposal confirmed by simulations of the entire
I(
q) curves, considering an ideal solution of particles with tails either condensed or extended. This tail extension is observed at higher salt values when particles contain longer DNA fragments (165
±
10
bp). The maximal extension of the tails always coincides with the screening of repulsive interactions between particles. The second virial coefficient becomes smaller than the hard sphere virial coefficient and eventually becomes negative (net attractive interactions) for NCP
145. Addition of salt simultaneously screens Coulombic repulsive interactions between NCP and Coulombic attractive interactions between tails and DNA inside the NCP. We discuss how the coupling of these two phenomena may be of biological relevance.
Binding ATP to tryptophanyl-tRNA synthetase (TrpRS) in a catalytically competent configuration for amino acid activation destabilizes the enzyme structure prior to forming the transition state. This ...conclusion follows from monitoring the titration of TrpRS with ATP by small angle solution X-ray scattering, enzyme activity, and crystal structures. ATP induces a significantly smaller radius of gyration at pH=7 with a transition midpoint at ∼8
mM. A non-reciprocal dependence of Trp and ATP dissociation constants on concentrations of the second substrate show that Trp binding enhances affinity for ATP, while the affinity for Trp falls with the square of the ATP over the same concentration range (∼5
mM) that induces the more compact conformation. Two distinct TrpRS:ATP structures have been solved, a high-affinity complex grown with 1
mM ATP and a low-affinity complex grown at 10
mM ATP. The former is isomorphous with unliganded TrpRS and the Trp complex from monoclinic crystals. Reacting groups of the two individually-bound substrates are separated by 6.7
Å. Although it lacks tryptophan, the low-affinity complex has a closed conformation similar to that observed in the presence of both ATP and Trp analogs such as indolmycin, and resembles a complex previously postulated to form in the closely-related TyrRS upon induced-fit active-site assembly, just prior to catalysis. Titration of TrpRS with ATP therefore successively produces structurally distinct high- and low-affinity ATP-bound states. The higher quality X-ray data for the closed ATP complex (2.2
Å) provide new structural details likely related to catalysis, including an extension of the KMSKS loop that engages the second lysine and serine residues, K195 and S196, with the α and γ-phosphates; interactions of the K111 side-chain with the γ-phosphate; and a water molecule bridging the consensus sequence residue T15 to the β-phosphate. Induced-fit therefore strengthens active-site interactions with ATP, substantially intensifying the interaction of the KMSKS loop with the leaving PP
i group. Formation of this conformation in the absence of a Trp analog implies that ATP is a key allosteric effector for TrpRS. The paradoxical requirement for high ATP implies that Gibbs binding free energy is stored in an unfavorable protein conformation and can then be recovered for useful purposes, including catalysis in the case of TrpRS.
Ceruloplasmin is a copper protein found in vertebrate plasma, which belongs to the family of multicopper oxidases. Like transferrin of the blood plasma, lactoferrin, the iron-containing protein of ...human milk, saliva, tears, seminal plasma and of neutrophilic leukocytes tightly binds two ferric ions. Human lactoferrin and ceruloplasmin have been previously shown to interact both in vivo and in vitro forming a complex. Here we describe a study of the conformation of the human lactoferrin/ceruloplasmin complex in solution using small angle X-ray scattering. Our ab initio structural analysis shows that the complex has a 1:1 stoichiometry and suggests that complex formation occurs without major conformational rearrangements of either protein. Rigid-body modeling of the mutual arrangement of proteins in the complex essentially yields two families of solutions. Final discrimination is possible when integrating in the modeling process extra information translating into structural constraints on the interaction between the two partners.
Agonist-induced conformational changes in the ligand-binding domains (LBD) of glutamate receptor ion channels provide the driving force for molecular rearrangements that mediate channel opening and ...subsequent desensitization. The resulting regulated transmembrane ion fluxes form the basis for most excitatory neuronal signaling in the brain. Crystallographic analysis of the GluR2 LBD core has revealed a ligand-binding cleft located between two lobes. Channel antagonists stabilize an open cleft, whereas agonists stabilize a closed cleft. The crystal structure of the apo form is similar to the antagonist-bound, open state. To understand the conformational behavior of the LBD in the absence of crystal lattice constraints, and thus better to appreciate the thermodynamic constraints on ligand binding, we have undertaken a solution x-ray scattering study using two different constructs encoding either the core or an extended LBD. In agreement with the GluR2 crystal structures, the LBD is more compact in the presence of agonist than it is in the presence of antagonist. However, the time-averaged conformation of the ligand-free core in solution is intermediate between the open, antagonist-bound state and the closed, agonist-bound state, suggesting a conformational equilibrium. Addition of peptide moieties that connect the core domain to the other functional domains in each channel subunit appears to constrain the conformational equilibrium in favor of the open state.
The NADPH oxidase complex is involved in the destruction of phagocytosed pathogens through the production of reactive oxygen species. This activatable complex consists of a membranous heterodimeric ...flavocytochrome b, a small G-protein Rac1/Rac2 and cytosolic factors, p47(phox), p67(phox) and p40(phox). p67(phox), due to its modular structure, is the NADPH oxidase component for which global structure information is most scarce despite its mandatory role in activation and its central position in the whole complex organization. Indeed, p67(phox) is the only factor establishing interaction with all others. In this study, we report the SAXS analysis of p67(phox). Our data reveals that p67(phox) behaves as a multidomain protein with semi-flexible linkers. On the one hand, it appears to be a very elongated molecule with its various domains organized as beads on a string. Linkers are predicted to be partially or mainly unstructured and features of our experimental data do point towards inter-domain flexibility. On the other hand, our work also suggests that the protein is not as extended as unstructured linkers could allow, thereby implying the existence of intra-molecular interactions within p67(phox). We suggest that the dual character of p67(phox) conformation in solution is central to ensure the numerous interactions to be accommodated.
In 2012, preliminary guidelines were published addressing sample quality, data acquisition and reduction, presentation of scattering data and validation, and modelling for biomolecular small‐angle ...scattering (SAS) experiments. Biomolecular SAS has since continued to grow and authors have increasingly adopted the preliminary guidelines. In parallel, integrative/hybrid determination of biomolecular structures is a rapidly growing field that is expanding the scope of structural biology. For SAS to contribute maximally to this field, it is essential to ensure open access to the information required for evaluation of the quality of SAS samples and data, as well as the validity of SAS‐based structural models. To this end, the preliminary guidelines for data presentation in a publication are reviewed and updated, and the deposition of data and associated models in a public archive is recommended. These guidelines and recommendations have been prepared in consultation with the members of the International Union of Crystallography (IUCr) Small‐Angle Scattering and Journals Commissions, the Worldwide Protein Data Bank (wwPDB) Small‐Angle Scattering Validation Task Force and additional experts in the field.
Updated guidelines are presented for publishing biomolecular small‐angle scattering (SAS) experiments so that readers can independently assess the quality of the data and models presented. The focus is on solution scattering experiments with either X‐rays (SAXS) or neutrons (SANS), where the primary goal is the generation and testing of three‐dimensional models, particularly in the context of integrative/hybrid structural modelling.
DC-SIGN is a C-type lectin receptor of dendritic cells and is involved in the early stages of numerous infectious diseases. DC-SIGN is organized into a tetramer enabling multivalent interaction with ...pathogens. Once formed, the DC-SIGN-pathogen complex can be internalized into compartments of increasing acidity. We have studied the pH dependence of the oligomerization state and conformation of the entire extracellular domain and neck region. We present evidence for equilibrium between the monomeric and tetrameric states of the extracellular domain, which exhibits a marked dependence with respect to both pH and ionic strength. Using solution x-ray scattering we have obtained a molecular envelope of the extracellular domain in which a model has been built. Our results highlight the central role of the neck domain in the pH-sensitive control of the oligomerization state, in the extended conformation of the protein, and in carbohydrate recognition domain organization and presentation. This work opens new insight into the molecular mechanism of ligand release and points to new avenues to block the first step of this important infection pathway.
Fibrinogen is a large heterogeneous aggregation/degradation‐prone protein playing a central role in blood coagulation and associated pathologies, whose structure is not completely resolved. When a ...high‐molecular‐weight fraction was analyzed by size‐exclusion high‐performance liquid chromatography/small‐angle X‐ray scattering (HPLC‐SAXS), several composite peaks were apparent and because of the stickiness of fibrinogen the analysis was complicated by severe capillary fouling. Novel SAS analysis tools developed as a part of the UltraScan Solution Modeler (US‐SOMO; http://somo.uthscsa.edu/), an open‐source suite of utilities with advanced graphical user interfaces whose initial goal was the hydrodynamic modeling of biomacromolecules, were implemented and applied to this problem. They include the correction of baseline drift due to the accumulation of material on the SAXS capillary walls, and the Gaussian decomposition of non‐baseline‐resolved HPLC‐SAXS elution peaks. It was thus possible to resolve at least two species co‐eluting under the fibrinogen main monomer peak, probably resulting from in‐column degradation, and two others under an oligomers peak. The overall and cross‐sectional radii of gyration, molecular mass and mass/length ratio of all species were determined using the manual or semi‐automated procedures available within the US‐SOMO SAS module. Differences between monomeric species and linear and sideways oligomers were thus identified and rationalized. This new US‐SOMO version additionally contains several computational and graphical tools, implementing functionalities such as the mapping of residues contributing to particular regions of P(r), and an advanced module for the comparison of primary I(q) versus q data with model curves computed from atomic level structures or bead models. It should be of great help in multi‐resolution studies involving hydrodynamics, solution scattering and crystallographic/NMR data.
PDF
