23 research outputs found
Kinetic gating mechanism of DNA damage recognition by Rad4/XPC
The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites
Crystal Structure of Thioesterase SgcE10 Supporting Common Polyene Intermediates in 9- and 10-Membered Enediyne Core Biosynthesis
Enediynes are potent natural product
anticancer antibiotics, and
are classified as 9- or 10-membered according to the size of their
enediyne core carbon skeleton. Both 9- and 10-membered enediyne cores
are biosynthesized by the enediyne polyketide synthase (PKSE), thioesterase
(TE), and PKSE-associated enzymes. Although the divergence between
9- and 10-membered enediyne core biosynthesis remains unclear, it
has been observed that nascent polyketide intermediates, tethered
to the acyl carrier protein (ACP) domain of PKSE, could be released
by TE in the absence of the PKSE-associated enzymes. In this study,
we determined the crystal structure of SgcE10, the TE that participates
in the biosynthesis of the 9-membered enediyne C-1027. Structural
comparison of SgcE10 with CalE7 and DynE7, two TEs that participate
in the biosynthesis of the 10-membered enediynes calicheamicin and
dynemicin, respectively, revealed that they share a common α/ÎČ
hot-dog fold. The amino acids involved in both substrate binding and
catalysis are conserved among SgcE10, CalE7, and DynE7. The volume
and the shape of the substrate-binding channel and active site in
SgcE10, CalE7, and DynE7 confirm that TEs from both 9- and 10-membered
enediyne biosynthetic machineries bind the linear form of similar
ACP-tethered polyene intermediates. Taken together, these findings
further support the proposal that the divergence between 9- and 10-membered
enediyne core biosynthesis occurs beyond PKSE and TE catalysis
Optimization of Benzoxazole-Based Inhibitors of <i>Cryptosporidium parvum</i> Inosine 5âČ-Monophosphate Dehydrogenase
<i>Cryptosporidium parvum</i> is an enteric protozoan parasite
that has emerged as a major cause of diarrhea, malnutrition, and gastroenteritis
and poses a potential bioterrorism threat. <i>C. parvum</i> synthesizes guanine nucleotides from host adenosine in a streamlined
pathway that relies on inosine 5âČ-monophosphate dehydrogenase
(IMPDH). We have previously identified several parasite-selective <i>C. parvum</i> IMPDH (<i>Cp</i>IMPDH) inhibitors by
high-throughput screening. In this paper, we report the structureâactivity
relationship (SAR) for a series of benzoxazole derivatives with many
compounds demonstrating <i>Cp</i>IMPDH IC<sub>50</sub> values
in the nanomolar range and >500-fold selectivity over human IMPDH
(hIMPDH). Unlike previously reported <i>Cp</i>IMPDH inhibitors,
these compounds are competitive inhibitors versus NAD<sup>+</sup>.
The SAR study reveals that pyridine and other small heteroaromatic
substituents are required at the 2-position of the benzoxazole for
potent inhibitory activity. In addition, several other SAR conclusions
are highlighted with regard to the benzoxazole and the amide portion
of the inhibitor, including preferred stereochemistry. An X-ray crystal
structure of a representative E·IMP·inhibitor complex is
also presented. Overall, the secondary amine derivative <b>15a</b> demonstrated excellent <i>Cp</i>IMPDH
inhibitory activity (IC<sub>50</sub> = 0.5 ± 0.1 nM) and moderate
stability (<i>t</i><sub>1/2</sub> = 44 min) in mouse liver
microsomes. Compound <b>73</b>, the racemic version of <b>15a</b>, also displayed superb antiparasitic activity in a <i>Toxoplasma gondii</i> strain that relies on <i>Cp</i>IMPDH (EC<sub>50</sub> = 20 ± 20 nM), and selectivity versus
a wild-type <i>T. gondii</i> strain (200-fold). No toxicity
was observed (LD<sub>50</sub> > 50 ÎŒM) against a panel of
four mammalian cells lines
Interaction between KDs of oligomeric hIKK2.
<p>(A) Within the crystal, neighboring tetrameric assemblies interact symmetrically such that they contact one another through their V-shaped interfaces and two KDs are positioned within close proximity to one another (dashed box). (B) The close packed KDs are positioned so that their activation loops (dashed box) rest directly over the active site of a neighbor. Orange spheres mark the Cα positions on V229 and H232. (C) Close-up view of the kinase activation loops (yellow and blue) with glutamic acid residues 177 and 181 mimicking activation loop serines and the catalytic base D145 labeled. (D) In vitro kinase assay on immunoprecipitated hIKK2 with mutations at key residues that mediate KDâKD interactions in the crystal (lanes 3,4) reveals their involvement in catalytic activity. (E) Mutation of activation loop serines 177 and 181 to glutamates restores activity of immunoprecipitated IKK2 in vitro. (F) Immunoblotting with anti-phospho-Ser177,181 antibody reveals that the decrease in catalytic activity observed in the KDâKD interface mutants correlates with decreased activation loop phosphorylation.</p
A Structural Basis for IÎșB Kinase 2 Activation Via Oligomerization-Dependent <i>Trans</i> Auto-Phosphorylation
<div><p>Activation of the IÎșB kinase (IKK) is central to NF-ÎșB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-ÎșB transcriptional activity is not well understood. Here we report a 4 Ă
x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of <i>Xenopus</i> IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote <i>trans</i> auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via <i>trans</i> auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent <i>trans</i> auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase.</p></div
IKK2 oligomerization activation model.
<p>(A) The hIKK2 X-ray crystal structure in space filling representation viewed from three different angles. The four surfaces that mediate oligomerization in the X-ray crystal structure are colored purple (dimer interface), blue (antiparallel interface), orange (V-shaped interface), and green (KDâKD interface). (B) A structure-based model for IKK2 activation via <i>trans</i> auto-phosphorylation. IKK2 interconverts between its open and closed dimeric forms. The open dimer can further associate to form transient homooligomers, such as observed in the hIKK2 X-ray crystal structure. Phosphorylation of one IKK2 subunit by an upstream kinase activates the kinase activity of that subunit and, as a consequence of its propensity to assemble into higher order oligomers through it V-shaped and KD-KD interfaces, is rapidly amplified via <i>trans</i> auto-phosphorylation.</p
Oligomerization of hIKK2 dimers.
<p>(A) Ribbon diagram of the interaction between two neighboring hIKK2 dimers in the crystal. Their asymmetric association gives rise to two unique intersubunit interfaces. (B) Close-up view of residues that interact between the KDs at the V-shaped interface. (C) Additional residues that mediate V-shaped interface interactions between the ULD an SDD. (D) Close-up view of interacting residues within the anti-parallel interface. (E) In vitro kinase assay reveals that catalytic activity of hIKK2 with mutations that disrupt the V-shaped interface (lanes 3â5) is drastically reduced compared to wild-type protein (WT-lane 2). (F) In vitro kinase assays with the same WT mutant proteins in which activation loop serines are mutated to glutamate. (G) Immunoblotting with anti-phospho-Ser177,181 antibody reveals that the decrease in catalytic activity observed in the V-shaped interface mutants correlates with activation loop phosphorylation status. (H) In vitro kinase assays reveal the modest effects on hIKK2 catalytic activity of mutation at the antiparallel interface.</p
Oligomerization of hIKK2 dimers in the crystal.
<p>(A) Three successive asymmetric units, each composed of six protomers, are taken from the hIKK2 X-ray crystal structure and depicted with the center asymmetric unit (labeled AâF) and colored with surface rendering as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001581#pbio-1001581-g003" target="_blank">Figure 3A</a>. Within this arrangement can be found four unique but closely related versions of the same dimer of dimers. Beginning from the left, there are four protomers (D, E, and another DâČ and EâČ from the symmetry-related asymmetric unit) that assemble into a tetramer. These four polypeptide chains are depicted as opaque, while the remaining protomers are rendered as semitransparent. In the second depiction of the same assembly of three asymmetric units, chains B, C, D, and E are rendered opaque and reveal themselves to assemble with a similar tetrameric arrangement. Likewise, protomers A, B, C, and F in the third panel and A, F, and the symmetry-related Aâł and Fâł chains assemble into renditions of the same tetramer. (B) Close-up views of the four unique assemblies viewed perpendicular to their 2-fold rotation axes reveal their close similarity.</p
Data collection and refinement statistics
a<p>Data in parentheses are for highest resolution shell.</p>b<p>Reflections with |<i>F</i><sub>O</sub>|/Ï<1.0 rejected.</p>c<p>Calculated against a cross-validation set of 3.8% of data selected at random prior to refinement.</p>d<p>Combines clashscore, rotamer, and Ramachandran evaluations in to a single score, normalized to the same scale as x-ray resolution <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001581#pbio.1001581-Chen1" target="_blank">[40]</a>.</p
The hIKK2 X-ray crystal structure.
<p>(A) Domain organization schematics of full-length hIKK2 (above) and crystallized protein construct (below). The KD, ubiquitin-like domain (ULD), scaffold dimerization domain (SDD), and NEMO-binding region (N) are indicated and the domain borders are numbered. âEEâ represents mutation of two active site serines 177 and 181 to glutamic acid residues in the crystallized protein. (B) Ribbon diagram representation of the hIKK2 X-ray crystal structure. Coloring and labels correspond to part A. Individual helices of the SDD are labeled as are the N and C termini (N and C, respectively). (C) Ribbon diagram representation of the hIKK2 KD with secondary structure elements and key amino acid side chain positions labeled. (D) Close-up view of the structural elements and amino acid residues immediately surrounding the activation loop (blue). (E) Superposition of hIKK2 (green) and xIKK2 (brown) KDs depicted in Cα-trace representation. Several amino acid residues that adopt significantly different positions in the two structures are rendered as sticks (yellow for hIKK2; orange for xIKK2) and labeled.</p