16 research outputs found
Disruption of Oligomerization and Dehydroalanine Formation as Mechanisms for ClpP Protease Inhibition
Over 100 protease inhibitors are
currently used in the clinics,
and most of them use blockage of the active site for their mode of
inhibition. Among the protease drug targets are several enzymes for
which the correct multimeric assembly is crucial to their activity,
such as the proteasome and the HIV protease. Here, we present a novel
mechanism of protease inhibition that relies on active-site-directed
small molecules that disassemble the protease complex. We show the
applicability of this mechanism within the ClpP protease family, whose
members are tetradecameric serine proteases and serve as regulators
of several cellular processes, including homeostasis and virulence.
Compound binding to ClpP in a substoichiometric fashion triggers the
formation of completely inactive heptamers. Moreover, we report the
selective β-sultam-induced dehydroalanine formation of the active
site serine. This reaction proceeds through sulfonylation and subsequent
elimination, thereby obliterating the catalytic charge relay system.
The identity of the dehydroalanine was confirmed by mass spectrometry
and crystallography. Activity-based protein profiling experiments
suggest the formation of a dehydroalanine moiety in living S. aureus cells upon β-sultam treatment. Collectively,
these findings extend our view on multicomponent protease inhibition
that until now has mainly relied on blockage of the active site or
occupation of a regulatory allosteric site
Are Free Radicals Involved in IspH Catalysis? An EPR and Crystallographic Investigation
The [4Feâ4S] protein IspH in the methylerythritol
phosphate
isoprenoid biosynthesis pathway is an important anti-infective drug
target, but its mechanism of action is still the subject of debate.
Here, by using electron paramagnetic resonance (EPR) spectroscopy
and <sup>2</sup>H, <sup>17</sup>O, and <sup>57</sup>Fe isotopic labeling,
we have characterized and assigned two key reaction intermediates
in IspH catalysis. The results are consistent with the bioorganometallic
mechanism proposed earlier, and the mechanism is proposed to have
similarities to that of ferredoxin, thioredoxin reductase, in that
one electron is transferred to the [4Feâ4S]<sup>2+</sup> cluster,
which then performs a formal two-electron reduction of its substrate,
generating an oxidized high potential ironâsulfur protein (HiPIP)-like
intermediate. The two paramagnetic reaction intermediates observed
correspond to the two intermediates proposed in the bioorganometallic
mechanism: the early Ď-complex in which the substrateâs
3-CH<sub>2</sub>OH group has rotated away from the reduced ironâsulfur
cluster, and the next, Ρ<sup>3</sup>-allyl complex formed after
dehydroxylation. No free radical intermediates are observed, and the
two paramagnetic intermediates observed do not fit in a Birch reduction-like
or ferraoxetane mechanism. Additionally, we show by using EPR spectroscopy
and X-ray crystallography that two substrate analogues (<b>4</b> and <b>5</b>) follow the same reaction mechanism
Sequential Inactivation of Gliotoxin by the <i>S</i>âMethyltransferase TmtA
The epipolythiodioxopiperazine (ETP)
gliotoxin mediates toxicity
via its reactive thiol groups and thereby contributes to virulence
of the human pathogenic fungus <i>Aspergillus fumigatus</i>. Self-intoxication of the mold is prevented either by reversible
oxidation of reduced gliotoxin or by irreversible conversion to bisÂ(methylthio)Âgliotoxin.
The latter is produced by the <i>S</i>-methyltransferase
TmtA and attenuates ETP biosynthesis. Here, we report the crystal
structure of TmtA in complex with <i>S</i>-(5â˛-adenosyl)-l-homocysteine. TmtA features one substrate and one cofactor
binding pocket per protein, and thus, bis-thiomethylation of gliotoxin
occurs sequentially. Molecular docking of substrates and products
into the active site of TmtA reveals that gliotoxin forms specific
interactions with the protein surroundings, and free energy calculations
indicate that methylation of the C10a-SH group precedes alkylation
of the C3-SH site. Altogether, TmtA is well suited to selectively
convert gliotoxin and to control its biosynthesis, suggesting that
homologous enzymes serve to regulate the production of their toxic
natural sulfur compounds in a similar manner
Systematic Analyses of Substrate Preferences of 20S Proteasomes Using Peptidic Epoxyketone Inhibitors
Cleavage
analyses of 20S proteasomes with natural or synthetic
substrates allowed to infer the substrate specificities of the active
sites and paved the way for the rational design of high-affinity proteasome
inhibitors. However, details of cleavage preferences remained enigmatic
due to the lack of appropriate structural data. In a unique approach,
we here systematically examined substrate specificities of yeast and
human proteasomes using irreversibly acting Îąâ˛,βâ˛epoxyketone
(ep) inhibitors. Biochemical and structural analyses provide unique
insights into the substrate preferences of the distinct active sites
and highlight differences between proteasome types that may be considered
in future inhibitor design efforts. (1) For steric reasons, epoxyketones
with Val or Ile at the P1 position are weak inhibitors of all active
sites. (2) Identification of the β2c selective compound Ac-LAE-ep
represents a promising starting point for the development of compounds
that discriminate between β2c and β2i. (3) The compound
Ac-LAA-ep was found to favor subunit β5c over β5i by three
orders of magnitude. (4) Yeast β1 and human β1c subunits
preferentially bind Asp and Leu in their S1 pockets, while Glu and
large hydrophobic residues are not accepted. (5) Exceptional structural
features in the β1/2 substrate binding channel give rise to
the β1 selectivity of compounds featuring Pro at the P3 site.
Altogether, 23 different epoxyketone inhibitors, five proteasome mutants,
and 43 crystal structures served to delineate a detailed picture of
the substrate and ligand specificities of proteasomes and will further
guide drug development efforts toward subunit-specific proteasome
inhibitors for applications as diverse as cancer and autoimmune disorders
A Predictive Approach for the Optical Control of Carbonic Anhydrase II Activity
Optogenetics
and photopharmacology are powerful approaches to investigating
biochemical systems. While the former is based on genetically encoded
photoreceptors that utilize abundant chromophores, the latter relies
on synthetic photoswitches that are either freely diffusible or covalently
attached to specific bioconjugation sites, which are often native
or engineered cysteines. The identification of suitable cysteine sites
and appropriate linkers for attachment is generally a lengthy and
cumbersome process. Herein, we describe an <i>in silico</i> screening approach that is designed to propose a small number of
optimal combinations. By applying this computational approach to human
carbonic anhydrase and a set of three photochromic tethered ligands,
the number of potential site-ligand combinations was narrowed from
over 750 down to 6, which we then evaluated experimentally. Two of
these six combinations resulted in light-responsive human Carbonic
Anhydrases (LihCAs), which were characterized with enzymatic activity
assays, mass spectrometry, and X-ray crystallography. Our study also
provides insights into the reactivity of cysteines toward maleimides
and the hydrolytic stability of the adducts obtained
Mechanism of Allosteric Inhibition of the Enzyme IspD by Three Different Classes of Ligands
Enzymes
of the nonmevalonate pathway of isoprenoid biosynthesis
are attractive targets for the development of herbicides and drugs
against infectious diseases. While this pathway is essential for many
pathogens and plants, mammals do not depend on it for the synthesis
of isoprenoids. IspD, the third enzyme of the nonmevalonate pathway,
is unique in that it has an allosteric regulatory site. We elucidated
the binding mode of phenylisoxazoles, a new class of allosteric inhibitors.
Allosteric inhibition is effected by large conformational changes
of a loop region proximal to the active site. We investigated the
different roles of residues in this loop by mutation studies and identified
repulsive interactions with Asp291 and Asp292 to be responsible for
inhibition. Crystallographic data and the response of mutant enzymes
to three different classes of allosteric inhibitors provide an in-depth
understanding of the allosteric mechanism. The obtained mutant enzymes
show selective resistance to allosteric inhibitors and provide conceptually
valuable information for future engineering of herbicide-resistant
crops. We found that the isoprenoid precursors IPP and DMAPP are natural
inhibitors of <i>Arabidopsis thaliana</i> IspD; however,
they do not seem to bind to the allosteric site
Mechanism of Allosteric Inhibition of the Enzyme IspD by Three Different Classes of Ligands
Enzymes
of the nonmevalonate pathway of isoprenoid biosynthesis
are attractive targets for the development of herbicides and drugs
against infectious diseases. While this pathway is essential for many
pathogens and plants, mammals do not depend on it for the synthesis
of isoprenoids. IspD, the third enzyme of the nonmevalonate pathway,
is unique in that it has an allosteric regulatory site. We elucidated
the binding mode of phenylisoxazoles, a new class of allosteric inhibitors.
Allosteric inhibition is effected by large conformational changes
of a loop region proximal to the active site. We investigated the
different roles of residues in this loop by mutation studies and identified
repulsive interactions with Asp291 and Asp292 to be responsible for
inhibition. Crystallographic data and the response of mutant enzymes
to three different classes of allosteric inhibitors provide an in-depth
understanding of the allosteric mechanism. The obtained mutant enzymes
show selective resistance to allosteric inhibitors and provide conceptually
valuable information for future engineering of herbicide-resistant
crops. We found that the isoprenoid precursors IPP and DMAPP are natural
inhibitors of <i>Arabidopsis thaliana</i> IspD; however,
they do not seem to bind to the allosteric site
Dimerized Linear Mimics of a Natural Cyclopeptide (TMC-95A) Are Potent Noncovalent Inhibitors of the Eukaryotic 20S Proteasome
Noncovalent
proteasome inhibitors introduce an alternative mechanism
of inhibition to that of covalent inhibitors used in cancer therapy.
Starting from a noncovalent linear mimic of TMC-95A, a series of dimerized
inhibitors using polyaminohexanoic acid spacers has been designed
and optimized to target simultaneously two of the six active sites
of the eukaryotic 20S proteasome. The homodimerized compounds actively
inhibited chymotrypsin-like (<i>K</i><sub>i</sub> = 6â11
nM) and trypsin-like activities, whereas postacid activity was poorly
modified. The noncovalent binding mode was ascertained by X-ray crystallography
of the inhibitors complexed with the yeast 20S proteasome. The inhibition
of proteasomal activities in human cells was evaluated. The use of
the multivalency inhibitor concept has produced highly efficient and
selective noncovalent compounds (no inhibition of calpain and cathepsin)
that have potential therapeutic advantages compared to covalent binders
such as bortezomib and carfilzomib
Potent Proteasome Inhibitors Derived from the Unnatural <i>cis</i>-Cyclopropane Isomer of Belactosin A: Synthesis, Biological Activity, and Mode of Action
The
natural product belactosin A (<b>1</b>) with a <i>trans</i>-cyclopropane structure is a useful prototype compound
for developing potent proteasome (core particle, CP) inhibitors. To
date, <b>1</b> and its analogues are the only CP ligands that
bind to both the nonprimed S1 pocket as well as the primed substrate
binding channel; however, these molecules harbor a high IC<sub>50</sub> value of more than 1 ÎźM. We have performed structureâactivity
relationship studies, thereby elucidating unnatural <i>cis</i>-cyclopropane derivatives of <b>1</b> that exhibit high potency
to primarily block the chymotrypsin-like active site of the human
constitutive (cCP) and immunoproteasome (iCP). The most active compound <b>3e</b> reversibly inhibits cCP and iCP similarly with an IC<sub>50</sub> of 5.7 nM. X-ray crystallographic analysis of the yeast
proteasome in complex with <b>3e</b> revealed that the ligand
is accommodated predominantly into the primed substrate binding channel
and covalently binds to the active site threonine residue via its
β-lactone ring-opening
Gliotoxin Biosynthesis: Structure, Mechanism, and Metal Promiscuity of Carboxypeptidase GliJ
The
formation of glutathione (GSH) conjugates, best known from
the detoxification of xenobiotics, is a widespread strategy to incorporate
sulfur into biomolecules. The biosynthesis of gliotoxin, a virulence
factor of the human pathogenic fungus <i>Aspergillus fumigatus</i>, involves attachment of two GSH molecules and their sequential decomposition
to yield two reactive thiol groups. The degradation of the GSH moieties
requires the activity of the CysâGly carboxypeptidase GliJ,
for which we describe the X-ray structure here. The enzyme forms a
homodimer with each monomer comprising one active site. Two metal
ions are present per proteolytic center, thus assigning GliJ to the
diverse family of dinuclear metallohydrolases. Depending on availability,
Zn<sup>2+</sup>, Fe<sup>2+</sup>, Fe<sup>3+</sup>, Mn<sup>2+</sup>, Cu<sup>2+</sup>, Co<sup>2+</sup>, or Ni<sup>2+</sup> ions are accepted
as cofactors. Despite this high metal promiscuity, a preference for
zinc versus iron and manganese was noted. Mutagenesis experiments
revealed details of metal coordination, and molecular modeling delivered
insights into substrate recognition and processing by GliJ. The latter
results suggest a reaction mechanism in which the two scissile peptide
bonds of one gliotoxin precursor molecule are hydrolyzed sequentially
and in a given order