Zika virus (ZIKV), a mosquito-borne human flavivirus that causes microcephaly and other neurological disorders, has been a recent focus for the development of flavivirus vaccines and therapeutics. We ...report here a 4.0 Å resolution structure of the mature ZIKV in complex with ADI-30056, a ZIKV-specific human monoclonal antibody (hMAb) isolated from a ZIKV infected donor with a prior dengue virus infection. The structure shows that the hMAb interactions span across the E protein dimers on the virus surface, inhibiting conformational changes required for the formation of infectious fusogenic trimers similar to the hMAb, ZIKV-117. Structure-based functional analysis, and structure and sequence comparisons, identified ZIKV residues essential for neutralization and crucial for the evolution of highly potent E protein crosslinking Abs in ZIKV. Thus, this epitope, ZIKV's "Achilles heel", defined by the contacts between ZIKV and ADI-30056, could be a suitable target for the design of therapeutic antibodies.
The allosteric mechanism of one of the best characterized bacterial transcription regulators, tetracycline repressor (TetR), has recently been questioned. Tetracycline binding induces cooperative ...folding of TetR, as suggested by recent unfolding studies, rather than switching between two defined conformational states, namely a DNA-binding-competent conformation and a non-DNA-binding conformation. Upon ligand binding, a host of near-native multiconformational structures collapse into a single, highly stabilized protein conformation that is no longer able to bind DNA. Here, structure–function studies performed with four synthetic peptides that bind to TetR and mimic the function of low-molecular-weight effectors, such as tetracyclines, provide new means to discriminate between different allosteric models. Whereas two inducing peptides bind in an extended β-like conformation, two anti-inducing peptides form an α-helix in the effector binding site of TetR. This exclusive bimodal interaction mode coincides with two distinct overall conformations of TetR, namely one that is identical with induced TetR and one that mirrors the DNA-bound state of TetR. Urea-induced unfolding studies show no increase in thermodynamic stability for any of the peptide complexes, although fluorescence measurements demonstrate peptide binding to TetR. This strongly suggests that, at least for these peptide effectors, a classical two-state allosteric model best describes TetR function.
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► The origin of allostery in proteins is under constant debate. ► Allostery in the bacterial regulator TetR has recently been questioned. ► Here, we investigated TetR in complex with four synthetic peptides. ► The peptides trigger different functions but induce only two structural states. ► We conclude that allostery in TetR adheres to a classical two-state model.
PML nuclear bodies (PML-NBs) are enigmatic structures of the cell nucleus that act as key mediators of intrinsic immunity against viral pathogens. PML itself is a member of the E3-ligase TRIM family ...of proteins that regulates a variety of innate immune signaling pathways. Consequently, viruses have evolved effector proteins to modify PML-NBs; however, little is known concerning structure-function relationships of viral antagonists. The herpesvirus human cytomegalovirus (HCMV) expresses the abundant immediate-early protein IE1 that colocalizes with PML-NBs and induces their dispersal, which correlates with the antagonization of NB-mediated intrinsic immunity. Here, we delineate the molecular basis for this antagonization by presenting the first crystal structure for the evolutionary conserved primate cytomegalovirus IE1 proteins. We show that IE1 consists of a globular core (IE1CORE) flanked by intrinsically disordered regions. The 2.3 Å crystal structure of IE1CORE displays an all α-helical, femur-shaped fold, which lacks overall fold similarity with known protein structures, but shares secondary structure features recently observed in the coiled-coil domain of TRIM proteins. Yeast two-hybrid and coimmunoprecipitation experiments demonstrate that IE1CORE binds efficiently to the TRIM family member PML, and is able to induce PML deSUMOylation. Intriguingly, this results in the release of NB-associated proteins into the nucleoplasm, but not of PML itself. Importantly, we show that PML deSUMOylation by IE1CORE is sufficient to antagonize PML-NB-instituted intrinsic immunity. Moreover, co-immunoprecipitation experiments demonstrate that IE1CORE binds via the coiled-coil domain to PML and also interacts with TRIM5α We propose that IE1CORE sequesters PML and possibly other TRIM family members via structural mimicry using an extended binding surface formed by the coiled-coil region. This mode of interaction might render the antagonizing activity less susceptible to mutational escape.
AmtR belongs to the TetR family of transcription regulators and is a global nitrogen regulator that is induced under nitrogen‐starvation conditions in Corynebacterium glutamicum. AmtR regulates the ...expression of transporters and enzymes for the assimilation of ammonium and alternative nitrogen sources, for example urea, amino acids etc. The recognition of operator DNA by homodimeric AmtR is not regulated by small‐molecule effectors as in other TetR‐family members but by a trimeric adenylylated PII‐type signal transduction protein named GlnK. The crystal structure of ligand‐free AmtR (AmtRorth) has been solved at a resolution of 2.1 Å in space group P21212. Comparison of its quaternary assembly with the previously solved native AmtR structure (PDB entry 5dy1) in a trigonal crystal system (AmtRtri) not only shows how a solvent‐content reduction triggers a space‐group switch but also suggests a model for how dimeric AmtR might stoichiometrically interact with trimeric adenylylated GlnK.
The structure of ligand‐free and DNA‐unbound AmtR, a TetR‐type global nitrogen regulator from C. glutamicum, was solved at 2.1 Å resolution in space group P21212 with six molecules in the asymmetric unit. A structure comparison with the previously solved structure of AmtR in a trigonal crystal system suggests a 6:6 stoichiometric association of three AmtR dimers with two trimeric GlnK complexes that prevents AmtR from binding to DNA and induces gene transcription.
Crystals of the cytotoxic thionin proteins viscotoxins A1 and B2 extracted from mistletoe diffracted to high resolution (1.25 and 1.05 Å, respectively) and are excellent candidates for testing ...crystallographic methods. Ab initio direct methods were only successful in solving the viscotoxin B2 structure, which with 861 unique non‐H atoms is one of the largest unknown structures without an atom heavier than sulfur to be solved in this way, but sulfur‐SAD phasing provided a convincing solution for viscotoxin A1. Both proteins form dimers in the crystal and viscotoxin B2 (net charge +4 per monomer), but not viscotoxin A1 (net charge +6), is coordinated by sulfate or phosphate anions. The viscotoxin A1 crystal has a higher solvent content than the viscotoxin B2 crystal (49% as opposed to 28%) with solvent channels along the crystallographic 43 axes.
Mouse apolipoprotein M (m-apoM) displays a 79% sequence identity to human apolipoprotein M (h-apoM). Both proteins are apolipoproteins associated with high-density lipoproteins, with similar ...anticipated biological functions. The structure of h-apoM has recently been determined by X-ray crystallography, which revealed that h-apoM displays, as expected, a lipocalin-like fold characterized by an eight-stranded β‑barrel that encloses an internal fatty-acid-binding site. Surprisingly, this is not true for m-apoM. After refolding from inclusion bodies, the crystal structure of m-apoM (reported here at 2.5 Å resolution) displays a novel yet unprecedented seven-stranded β-barrel structure. The fold difference is not caused by a mere deletion of a single β-strand; instead, β-strands E and F are removed and replaced by a single β-strand A′ formed from residues from the N-terminus. Molecular dynamics simulations suggest that m-apoM is able to adopt both a seven-stranded barrel structure and an eight-stranded barrel structure in solution, and that both folds are comparably stable. Thermal unfolding simulations identify the position where β-strand exchange occurs as the weak point of the β-barrel. We wonder whether the switch in topology could have a biological function and could facilitate ligand release, since it goes hand in hand with a narrowing of the barrel diameter. Possibly also, the observed conformation represents an on-pathway or off-pathway folding intermediate of apoM. The difference in fold topology is quite remarkable, and the fold promiscuity observed for m-apoM might possibly provide a glimpse at potential cross-points during the evolution of β-barrels.
An intriguing catch: Gutingimycin (1) from the marine streptomycete isolate B8652 is a conjugate of guanine and trioxacarcin A, as extensive NMR measurements and a crystal structure analysis show. ...The natural product is formed most likely by the selective attack of trioxacarcin A on the bacterial DNA, which results in strand breakage and may explain the high cytotoxicity of the latter.
AmtR, a member of the TetR family of transcription regulators, is a global regulator of nitrogen control in Corynebacterium glutamicum. Unlike other TetR‐family members, which are regulated by ...small‐molecule effectors, AmtR is regulated by a protein called GlnK. It has been shown that a GlnK trimer has to become adenylylated prior to formation of a complex with AmtR. The physiological function of AmtR has been very well studied, but structural characterization of the mechanistic aspects of AmtR‐regulated transcription has yet to be accomplished. AmtR has successfully been crystallized in space group P21212, with six molecules in the asymmetric unit and unit‐cell parameters a = 153.34, b = 163.10, c = 51.93 Å. Preliminary phases were obtained using Se‐SAD.
Steroidal aryliminium salts were prepared from
d-
seco-pregnene aldehyde
2b, and their BF
3·OEt
2-catalyzed reactions were studied. The nature of the substituent R
1 in the anilines
3–
6 essentially ...influenced the chemoselectivity. Using unsubstituted
3, 4-methoxy- (
4) or 4-bromoaniline (
5), different tetrahydroquinoline derivatives
7a–
13a via intramolecular hetero
Diels-Alder reaction were formed. In the case of 4-nitroaniline (
6) the
N-arylamino-
d-homopregnane (
14a) were also obtained. We assume, that an intramolecular
Prins reaction led to this type of fluoro-
d-homosteroid. The main products represent a new class of tetrahydroquinolino-androstenes.
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