11 research outputs found
Crystal Structure of the FeS Cluster–Containing Nucleotide Excision Repair Helicase XPD
DNA damage recognition by the nucleotide excision repair pathway requires an initial step identifying helical distortions in the DNA and a proofreading step verifying the presence of a lesion. This proofreading step is accomplished in eukaryotes by the TFIIH complex. The critical damage recognition component of TFIIH is the XPD protein, a DNA helicase that unwinds DNA and identifies the damage. Here, we describe the crystal structure of an archaeal XPD protein with high sequence identity to the human XPD protein that reveals how the structural helicase framework is combined with additional elements for strand separation and DNA scanning. Two RecA-like helicase domains are complemented by a 4Fe4S cluster domain, which has been implicated in damage recognition, and an α-helical domain. The first helicase domain together with the helical and 4Fe4S-cluster–containing domains form a central hole with a diameter sufficient in size to allow passage of a single stranded DNA. Based on our results, we suggest a model of how DNA is bound to the XPD protein, and can rationalize several of the mutations in the human XPD gene that lead to one of three severe diseases, xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy
Mechanism of Substrate and Inhibitor Binding of Rhodobacter capsulatus Xanthine Dehydrogenase*
Rhodobacter capsulatus xanthine dehydrogenase (XDH) is an
(αβ)2 heterotetrameric cytoplasmic enzyme that resembles
eukaryotic xanthine oxidoreductases in respect to both amino acid sequence and
structural fold. To obtain a detailed understanding of the mechanism of
substrate and inhibitor binding at the active site, we solved crystal
structures of R. capsulatus XDH in the presence of its substrates
hypoxanthine, xanthine, and the inhibitor pterin-6-aldehyde using either the
inactive desulfo form of the enzyme or an active site mutant
(EB232Q) to prevent substrate turnover. The hypoxanthine- and
xanthine-bound structures reveal the orientation of both substrates at the
active site and show the importance of residue GluB-232 for
substrate positioning. The oxygen atom at the C-6 position of both substrates
is oriented toward ArgB-310 in the active site. Thus the substrates
bind in an orientation opposite to the one seen in the structure of the
reduced enzyme with the inhibitor oxypurinol. The tightness of the substrates
in the active site suggests that the intermediate products must exit the
binding pocket to allow first the attack of the C-2, followed by oxidation of
the C-8 atom to form the final product uric acid. Structural studies of
pterin-6-aldehyde, a potent inhibitor of R. capsulatus XDH,
contribute further to the understanding of the relative positioning of
inhibitors and substrates in the binding pocket. Steady state kinetics reveal
a competitive inhibition pattern with a Ki of 103.57
± 18.96 nm for pterin-6-aldehyde
Structure of the C-terminal half of UvrC reveals an RNase H endonuclease domain with an Argonaute-like catalytic triad
Removal and repair of DNA damage by the nucleotide excision repair pathway requires two sequential incision reactions, which are achieved by the endonuclease UvrC in eubacteria. Here, we describe the crystal structure of the C-terminal half of UvrC, which contains the catalytic domain responsible for 5′ incision and a helix–hairpin–helix–domain that is implicated in DNA binding. Surprisingly, the 5′ catalytic domain shares structural homology with RNase H despite the lack of sequence homology and contains an uncommon DDH triad. The structure also reveals two highly conserved patches on the surface of the protein, which are not related to the active site. Mutations of residues in one of these patches led to the inability of the enzyme to bind DNA and severely compromised both incision reactions. Based on our results, we suggest a model of how UvrC forms a productive protein–DNA complex to excise the damage from DNA
Evaluating the Contribution of Transition-State Destabilization to Changes in the Residence Time of Triazole-Based InhA Inhibitors
Mechanism of the AppA<sub>BLUF</sub> Photocycle Probed by Site-Specific Incorporation of Fluorotyrosine Residues: Effect of the Y21 p<i>K</i><sub>a</sub> on the Forward and Reverse Ground-State Reactions
The
transcriptional antirepressor AppA is a blue light using flavin
(BLUF) photoreceptor that releases the transcriptional repressor PpsR
upon photoexcitation. Light activation of AppA involves changes in
a hydrogen-bonding network that surrounds the flavin chromophore on
the nanosecond time scale, while the dark state of AppA is then recovered
in a light-independent reaction with a dramatically longer half-life
of 15 min. Residue Y21, a component of the hydrogen-bonding network,
is known to be essential for photoactivity. Here, we directly explore
the effect of the Y21 p<i>K</i><sub>a</sub> on dark state
recovery by replacing Y21 with fluorotyrosine analogues that increase
the acidity of Y21 by 3.5 pH units. Ultrafast transient infrared measurements
confirm that the structure of AppA is unperturbed by fluorotyrosine
substitution, and that there is a small (3-fold) change in the photokinetics
of the forward reaction over the fluorotyrosine series. However, reduction
of 3.5 pH units in the p<i>K</i><sub>a</sub> of Y21 increases
the rate of dark state recovery by 4000-fold with a Brønsted
coefficient of ∼1, indicating that the Y21 proton is completely
transferred in the transition state leading from light to dark adapted
AppA. A large solvent isotope effect of ∼6–8 is also
observed on the rate of dark state recovery. These data establish
that the acidity of Y21 is a crucial factor for stabilizing the light
activated form of the protein, and have been used to propose a model
for dark state recovery that will ultimately prove useful for tuning
the properties of BLUF photosensors for optogenetic applications