7 research outputs found
Structural Polymorphism of 441-Residue Tau at Single Residue Resolution
Alzheimer disease is characterized by abnormal protein deposits in the brain, such as extracellular amyloid plaques and intracellular neurofibrillary tangles. The tangles are made of a protein called tau comprising 441 residues in its longest isoform. Tau belongs to the class of natively unfolded proteins, binds to and stabilizes microtubules, and partially folds into an ordered β-structure during aggregation to Alzheimer paired helical filaments (PHFs). Here we show that it is possible to overcome the size limitations that have traditionally hampered detailed nuclear magnetic resonance (NMR) spectroscopy studies of such large nonglobular proteins. This is achieved using optimal NMR pulse sequences and matching of chemical shifts from smaller segments in a divide and conquer strategy. The methodology reveals that 441-residue tau is highly dynamic in solution with a distinct domain character and an intricate network of transient long-range contacts important for pathogenic aggregation. Moreover, the single-residue view provided by the NMR analysis reveals unique insights into the interaction of tau with microtubules. Our results establish that NMR spectroscopy can provide detailed insight into the structural polymorphism of very large nonglobular proteins
Assays for Nucleotide Competitive Reversible and Irreversible Inhibitors of Ras GTPases
Although the Ras protein has been
seen as a potential target for
cancer therapy for the past 30 years, there was a tendency to consider
it undruggable until recently. This has changed with the demonstration
that small molecules with a specificity for (disease related mutants
of) Ras can indeed be found, and some of these molecules form covalent
adducts. A subgroup of these molecules can be characterized as competing
with binding of the natural ligands GTP and GDP. Because of the distinct
properties of Ras and related GTPases, in particular the very high
nucleotide affinities and associated very low dissociation rates,
assays for characterizing such molecules are not trivial. This is
compounded by the fact that Ras family GTPases tend to be thermally
unstable in the absence of a bound nucleotide. Here, we show that
instead of using the unstable nucleotide-free Ras, the protein can
be isolated as a 1:1 complex with a modified nucleotide (GDP-β-methyl
ester) with low affinity to Ras. With this nucleotide analogue bound
to the protein, testing of inhibitors is made experimentally more
convenient and we present assays that allow the rapid assessment of
the kinetic constants describing the inhibition process
Protein Minimization of the gp120 Binding Region of Human CD4 \dagger
CD4 is an important component of the immune system and is also the cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. Constructs consisting of all four extracellular domains of human CD4 as well as the first two domains (CD4D12) have previously been expressed and characterized. All of the gp120-binding residues are located within the first N-terminal domain (D1) of CD4. To date, it has not been possible to obtain domain D1 alone in a soluble and active form. Most residues in CD4 that interact with gp120 lie within the region 21-64 of domain D1 of CD4.On the basis of these observations and analysis of the crystal structure of CD4D12, a mutational strategy was designed to express CD4D1 and region 21-64 of CD4 (CD4PEP1) in Escherichia coli. KD values for the binding of CD4 analogues described above to gp120 were measured using a Biacore-based solution-phase competition binding assay. Measured KD values were 15 nM, 40 nM, and 26 \mu M for CD4D12, CD4D1, and CD4PEP1, respectively. All of the proteins interact with gp120 and are able to expose the 17b-binding epitope of gp120. Structural content was determined using CD and proteolysis. Both CD4D1 and CD4PEP1 were partially structured and showed an enhanced structure in the presence of the osmolyte sarcosine. The aggregation behavior of all of the proteins was characterized. While CD4D1 and CD4PEP1 did not aggregate, CD4D12 formed amyloid fibrils at neutral pH within a week at 278 K. These CD4 derivatives should be useful tools in HIV vaccine design and entry inhibition studies
A peptide with fine‐tuned flexibility inhibits NF‐Y transcription factor assembly
Set Me free: A peptide‐derived inhibitor of the NF‐Y transcription factor was developed by constraining the conformation of an epitope through hydrocarbon stapling and then fine‐tuning its flexibility. In the initial set of constrained peptides, a non‐interacting α‐methyl group was observed to have a detrimental effect on complex stability. Adaption of the methylation pattern gave a peptide that inhibits transcription factor assembly and subsequent DNA binding
Linear ubiquitination induces NEMO phase separation to activate NF-B signaling
The NF-B essential modulator NEMO is the core regulatory component of the inhibitor of B kinase complex, which is a critical checkpoint in canonical NF-B signaling downstream of innate and adaptive immune receptors. In response to various stimuli, such as TNF or IL-1, NEMO binds to linear or M1-linked ubiquitin chains generated by LUBAC, promoting its oligomerization and subsequent activation of the associated kinases. Here we show that M1-ubiquitin chains induce phase separation of NEMO and the formation of NEMO assemblies in cells after exposure to IL-1. Phase separation is promoted by both binding of NEMO to linear ubiquitin chains and covalent linkage of M1-ubiquitin to NEMO and is essential but not sufficient for its phase separation. Supporting the functional relevance of NEMO phase separation in signaling, a pathogenic NEMO mutant, which is impaired in both binding and linkage to linear ubiquitin chains, does not undergo phase separation and is defective in mediating IL-1–induced NF-B activation