12 research outputs found

    The Elastase-PK101 Structure: Mechanism of an Ultrasensitive Activity-based Probe Revealed

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    Human neutrophil elastase (HNE) plays a central role in neutrophil host defense, but its broad specificity makes HNE a difficult target for both inhibitor and probe development. Recently, we identified the unnatural amino acid containing activity-based probe PK101, which exhibits astounding sensitivity and selectivity for HNE, yet completely lacks mechanistic explanation for its unique characteristics. Here, we present the crystal structure of the HNE-PK101 complex which not only reveals the basis for PK101 ultrasensitivity but also uncovers so far unrecognized HNE features. Strikingly, the Nle­(<i>O</i>-Bzl) function in the P4 position of PK101 reveals and leverages an “exo-pocket” on HNE as a critical factor for selectivity. Furthermore, the PK101 P3 position harbors a methionine dioxide function, which mimics a post-translationally oxidized methionine residue and forms a critical hydrogen bond to the backbone amide of Gly219 of HNE. Gly219 resides in a Gly–Gly motif that is unique to HNE, yet compulsory for this interaction. Consequently, this feature enables HNE to accommodate substrates that have undergone methionine oxidation, which constitutes a hallmark post-translational modification of neutrophil signaling

    Figure 5

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    <p>A Multiple sequence alignment of NLR and Apaf-1 CARD domains. Acidic key residues participating in the CARD-CARD interface are indicated by red borders. Residues that belong to the basic patch of the CARD-CARD interface are indicated by blue borders. Nod2.1 and Nod2.2 refer to NOD2 CARD domain 1 and 2, respectively. B Multiple sequence alignment of NLR PYRIN domains. Patch of negatively charged residues from ASC2 in helices 1 and 4 and their corresponding residues in the PYRIN domain containing NLR proteins (red box). Patch of positively charged residues from ASC2 in helices 2 and 3 and their corresponding residues in the PYRIN containing NLR proteins (blue box).</p

    Figure 4

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    <p>A NLR oligomerization interface: Apaf-1 oligomer modeled on the basis of the NtrC1 heptamer crystal structure. For clarity only three NACHT domains are shown in ribbon representation with an ADP molecule depicted in sticks to highlight the nucleotide-binding site in each domain. Side-chains of residues in the oligomerization interfaces are shown in sticks color-coded according to the alignment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002119#pone-0002119-g001" target="_blank">Figure 1</a>. The two interfaces form across the nucleotide-binding site of the NACHT domain including the GxP domain. B Model of NLR activation and inflammasome formation based on the Apaf-1 apoptosome.</p

    Key elements of Apaf-1 and NLR NACHT-WH-SH domains extracted from the multiple sequence alignment in Figure 1.

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    <p>Sequence identities shown are derived from pairwise alignments using FFAS between Apaf-1 and individual NLRs. WH His and WH cons show the conserved histidine and the conserved METEEV sequence patch, respectively which are located in the winged helix (WH) domain.</p

    Overview of NLR family members according to their domain organization.

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    <p>Protein name and synonyms (new synonyms in bold), accession number, and chromosomal location according to <a href="http://www.genenames.org/genefamily/nlr.php" target="_blank">http://www.genenames.org/genefamily/nlr.php</a> followed by domains as defined by FFAS. CARD, caspase activation and recruitment domain; PYD, pyrin; NACHT, domain present in NAIP, CIITA, HET-E, TP-1; NALP, NACHT-LRR-PYD-containing protein; WH, winged helix domain; SH, superhelical domain, LRR, leucine-rich repeats.</p

    Model of the NOD2 nucleotide-binding site with an ADP molecule and conserved sequence motifs Walker A, Walker B, Sensor 1, GxP, and WH-His shown in sticks.

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    <p>Model of the NOD2 nucleotide-binding site with an ADP molecule and conserved sequence motifs Walker A, Walker B, Sensor 1, GxP, and WH-His shown in sticks.</p

    Multiple sequence alignment of NLR NACHT-WH-SH domains and the Apaf-1 NACHT-WH-SH domain.

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    <p>Degree of conservation is shown as blue shading. The secondary structure of Apaf-1 is shown above the Apaf-1 sequence. Arrows underneath the alignment indicate domain boundaries. Conserved sequence features important for catalytic activity are shown in black boxes. Orange and magenta boxes depict interfaces residues while the orange ones contribute to interactions with the left partner and the magenta residues are thought to interact with the right partner in the oligomer. Green boxes indicate additional motifs as described in the text.</p

    Development and Structural Analysis of a Nanomolar Cyclic Peptide Antagonist for the EphA4 Receptor

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    The EphA4 receptor is highly expressed in the nervous system, and recent findings suggest that its signaling activity hinders neural repair and exacerbates certain neurodegenerative processes. EphA4 has also been implicated in cancer progression. Thus, EphA4 inhibitors represent potential therapeutic leads and useful research tools to elucidate the role of EphA4 in physiology and disease. Here, we report the structure of a cyclic peptide antagonist, APY, in complex with the EphA4 ligand-binding domain (LBD), which represents the first structure of a cyclic peptide bound to a receptor tyrosine kinase. The structure shows that the dodecameric APY efficiently occupies the ephrin ligand-binding pocket of EphA4 and promotes a “closed” conformation of the surrounding loops. Structure-guided relaxation of the strained APY β-turn and amidation of the C terminus to allow an additional intrapeptide hydrogen bond yielded APY-βAla8.am, an improved APY derivative that binds to EphA4 with nanomolar affinity. APY-βAla8.am potently inhibits ephrin-induced EphA4 activation in cells and EphA4-dependent neuronal growth cone collapse, while retaining high selectivity for EphA4. The two crystal structures of APY and APY-βAla8.am bound to EphA4, in conjunction with secondary phage display screens, highlighted peptide residues that are essential for EphA4 binding as well as residues that can be modified. Thus, the APY scaffold represents an exciting prototype, particularly since cyclic peptides have potentially favorable metabolic stability and are emerging as an important class of molecules for disruption of protein–protein interactions

    Modifications of a Nanomolar Cyclic Peptide Antagonist for the EphA4 Receptor To Achieve High Plasma Stability

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    EphA4 is a receptor tyrosine kinase with a critical role in repulsive axon guidance and synaptic function. However, aberrant EphA4 activity can inhibit neural repair after injury and exacerbate neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer’s. We previously identified the cyclic peptide <b>APY-d2</b> (APYCVYRβASWSC-nh<sub>2</sub>, containing a disulfide bond) as a potent and selective EphA4 antagonist. However, <b>APY-d2</b> lacks sufficient plasma stability to be useful for EphA4 inhibition <i>in vivo</i> through peripheral administration. Using structure–activity relationship studies, we show that protecting the peptide N-terminus from proteolytic degradation dramatically increases the persistence of the active peptide in plasma and that a positively charged peptide N-terminus is essential for high EphA4 binding affinity. Among several improved <b>APY-d2</b> derivatives, the cyclic peptides <b>APY-d3</b> (<u>βA</u>PYCVYRβASWSC-nh<sub>2</sub>) and <b>APY-d4</b> (<u>βA</u>PYCVYRβA<u>E</u>W<u>E</u>C-nh<sub>2</sub>) combine high stability in plasma and cerebrospinal fluid with slightly enhanced potency. These properties make them valuable research tools and leads toward development of therapeutics for neurological diseases
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