Modulating
disease-relevant protein–protein interactions
(PPIs) using pharmacological tools is a critical step toward the design
of novel therapeutic strategies. Over the years, however, targeting
PPIs has proven a very challenging task owing to the large interfacial
areas. Our recent efforts identified possible novel routes for the
design of potent and selective inhibitors of PPIs using a structure-based
design of covalent inhibitors targeting Lys residues. In this present
study, we report on the design, synthesis, and characterizations of
the first Lys-covalent BH3 peptide that has a remarkable affinity
and selectivity for hMcl-1 over the closely related hBfl-1 protein.
Our structural studies, aided by X-ray crystallography, provide atomic-level
details of the inhibitor interactions that can be used to further
translate these discoveries into novel generation, Lys-covalent pro-apoptotic
agents.
The hydroxyornithine transformylase from Pseudomonas aeruginosa is known by the gene name pvdF, and has been hypothesized to use N 10 -formyltetrahydrofolate (N 10 -fTHF) as a co-substrate formyl donor to convert N 5 -hydroxyornithine (OHOrn) to N 5 -formyl-N 5 -hydroxyornithine (fOHOrn). PvdF is in the biosynthetic pathway for pyoverdin biosynthesis, a siderophore generated under iron-limiting conditions that has been linked to virulence, quorum sensing and biofilm formation. The structure of PvdF was determined by X-ray crystallography to 2.3 Å, revealing a formyltransferase fold consistent with N 10 -formyltetrahydrofolate dependent enzymes, such as the glycinamide ribonucleotide transformylases, N-sugar transformylases and methionyl-tRNA transformylases. Whereas the core structure, including the catalytic triad, is conserved, PvdF has three insertions of 18 or more amino acids, which we hypothesize are key to binding the OHOrn substrate. Steady state kinetics revealed a non-hyperbolic rate curve, promoting the hypothesis that PvdF uses a random-sequential mechanism, and favors folate binding over OHOrn.
RAD52 is a structurally and functionally conserved component of the DNA double-strand break (DSB) repair apparatus from budding yeast to humans. We recently showed that expressing the human gene, HsRAD52 in rad52 mutant budding yeast cells can suppress both their ionizing radiation (IR) sensitivity and homologous recombination repair (HRR) defects. Intriguingly, we observed that HsRAD52 supports DSB repair by a mechanism of HRR that conserves genome structure and is independent of the canonical HR machinery. In this study we report that naturally occurring variants of HsRAD52, one of which suppresses the pathogenicity of BRCA2 mutations, were unable to suppress the IR sensitivity and HRR defects of rad52 mutant yeast cells, but fully suppressed a defect in DSB repair by single-strand annealing (SSA). This failure to suppress both IR sensitivity and the HRR defect correlated with an inability of HsRAD52 protein to associate with and drive an interaction between genomic sequences during DSB repair by HRR. These results suggest that HsRAD52 supports multiple, distinct DSB repair apparatuses in budding yeast cells and help further define its mechanism of action in HRR. They also imply that disruption of HsRAD52-dependent HRR in BRCA2defective human cells may contribute to protection against tumorigenesis and provide a target for killing BRCA2-defective cancers.
RibB (3,4-dihydroxy-2-butanone 4-phosphate synthase)
is a magnesium-dependent
enzyme that excises the C4 of d-ribulose-5-phosphate (d-Ru5P) as formate. RibB generates the four-carbon substrate
for lumazine synthase that is incorporated into the xylene moiety
of lumazine and ultimately the riboflavin isoalloxazine. The reaction
was first identified by Bacher and co-workers in the 1990s, and their
chemical mechanism hypothesis became canonical despite minimal direct
evidence. X-ray crystal structures of RibB typically show two metal
ions when solved in the presence of non-native metals and/or liganding
non-substrate analogues, and the consensus hypothetical mechanism
has incorporated this cofactor set. We have used a variety of biochemical
approaches to further characterize the chemistry catalyzed by RibB
from Vibrio cholera (VcRibB). We show
that full activity is achieved at metal ion concentrations equal to
the enzyme concentration. This was confirmed by electron paramagnetic
resonance of the enzyme reconstituted with manganese and crystal structures
liganded with Mn2+ and a variety of sugar phosphates. Two
transient species prior to the formation of products were identified
using acid quench of single turnover reactions in combination with
NMR for singly and fully 13C-labeled d-Ru5P. These
data indicate that dehydration of C1 forms the first transient species,
which undergoes rearrangement by a 1,2 migration, fusing C5 to C3
and generating a hydrated C4 that is poised for elimination as formate.
Structures determined from time-dependent Mn2+ soaks of
VcRibB-d-Ru5P crystals show accumulation in crystallo of
the same intermediates. Collectively, these data reveal for the first
time crucial transient chemical states in the mechanism of RibB.
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