XFELs
to reveal the heterogeneity in M.
tuberculosis β-lactamase inhibition by Sulbactam. This work builds on possibilities
unleashed by mix-and-inject serial crystallography at XFELs. We have
triggered an enzymatic reaction by mixing an inhibitor with enzyme
microcrystals to report, in atomic detail and at room temperature, how
the Mycobacterium
tuberculosis enzyme BlaC is inhibited by sulbactam. Our
results reveal ligand binding heterogeneity, ligand gating,
cooperativity, induced fit, and conformational selection, detailing how
the inhibitor approaches the catalytic clefts and binds to the enzyme
noncovalently before reacting to a trans-enamine.
Nature Communications (2023)
(doi:
10.1038/s41467-023-41246-1)
Catalytic
process of anhydro-N-acetylmuramic acid kinase (AnmK) from Pseudomonas
aeruginosa.
Peptidoglycan is a rigid envelope surrounding the cytoplasmic membrane
of most bacterial species. The enzymatic processes that build, remodel,
and recycle the chemical components of this cross-linked polymer are
preeminent targets of antibiotics. In a collaborative effort with the
group of Shahriar Mobashery and Amr M. El-Araby (Univ. Notre Dame), and
Juan A. Hermoso (IQF-CSIC) we report a comprehensive kinetic and
structural analysis for one such enzyme, the Pseudomonas aeruginosa
anhydro-N- acetylmuramic acid (anhNAM) kinase (AnmK). AnmK follows a
random-sequential kinetic mechanism with respect to its anhNAM and ATP
substrates. Crystallographic analyses of four distinct structures
demonstrate that both substrates enter the active site independently in
an ungated conformation of the substrate subsites, with protein loops
acting as gates for anhNAM binding. A remarkable X-ray structure for
dimeric AnmK sheds light on the pre-catalytic and post-catalytic
ternary complexes, one in each subunit. Computational simulations in
conjunction with the four high-resolution X-ray structures reveal the
full catalytic cycle.
Journal of Biological Chemistry (2023)
( doi:
10.1016/j.jbc.2023.105198)
Decoding
the Molecular Basis of the Cell Division Final Step in Streptococcus pneumoniae.
Bacterial cell-wall hydrolases must be tightly regulated during
bacterial cell division to prevent aberrant cell lysis and to allow
final separation of viable daughter cells. In a multidisciplinary work,
we disclose the molecular dialogue between the cell-wall hydrolase
LytB, wall teichoic acids, and the eukaryotic-like protein kinase StkP
in Streptococcus
pneumoniae. After characterizing the peptidoglycan
recognition mode by the catalytic domain of LytB, we further
demonstrate that LytB possesses a modular organization allowing the
specific binding to wall teichoic acids and to the protein kinase StkP.
Structural and cellular studies notably reveal that the temporal and
spatial localization of LytB is governed by the interaction between
specific modules of LytB and the final PASTA domain of StkP. Our data
collectively provide a comprehensive understanding of how LytB performs
final separation of daughter cells and highlights the regulatory role
of eukaryotic-like kinases on lytic machineries in the last step of
cell division in streptococci. Considering that LytB is recognized as a
virulence factor involved in different aspects of host infection and
that the pneumococcus is on the WHO list of priority pathogens for
research and development of new antibiotics, our work holds the promise
of providing a structural basis for the rational design of new drugs to
combat pneumococcal infections.
Cell
Reports (2023) (doi:
10.1016/j.celrep.2023.112756)
Structure-guided
engineering of a receptor-agonist pair for inducible activation of the
ABA adaptive response to drought.
Abscisic acid (ABA) is a
plant hormone that naturally controls the
response of plants in drought situations. Based on the atomic structure
of ABA receptor proteins, we have designed a synthetic ABA receptor and
a small chemical compound that acting together in plants are capable of
activating ABA signaling in plants and very efficiently improving their
tolerance to drought.
Science
Advances (2023) 9(10) (doi:
10.1126/sciadv.ade9948) (see video
1) (see video 2)
Structural
Basis for Cyclosporin Isoform-Specific Inhibition of Cyclophilins from Toxoplasma
gondii.
Cyclosporin
(CsA) has antiparasite activity against the human pathogen Toxoplasma
gondii. In a collaborative effort between University of Verona and the
IQFR we characterized the functional and structural properties of two
cyclophilins from T.
gondii,
TgCyp23 and TgCyp18.4. While TgCyp23 is a highly active
cis−trans-prolyl isomerase (PPIase) and binds CsA with
nanomolar
affinity, TgCyp18.4 shows low PPIase activity and is significantly less
sensitive to CsA inhibition. The crystal structure of the TgCyp23:CsA
complex was solved at 1.1 Å resolution showing the molecular
details of CsA recognition by the protein, and revealing relevant
differences at the CsA-binding site compared to TgCyp18.4. The
biochemical and structural data presented herein represents a relevant
step toward understanding the molecular mechanisms of the
anti-Toxoplasma action of CsA and may be instrumental in the rational
design of new therapeutic drugs modulating TgCyp activity
ACS
Infectious Diseases
(2023)
(doi:
10.1021/acsinfecdis.2c00566)
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The
neuronal calcium sensor NCS-1 regulates the phosphorylation state and
activity of the Gα chaperone and GEF Ric-8A.The Neuronal Calcium Sensor 1, an
EF-hand Ca2+ binding protein, and Ric-8A
coregulate synapse number and probability of neurotransmitter release.
The protein-protein interaction interface constitutes a pharmacological
target under brain pathological conditions. Previous structural studies
of Ric-8A bound to Gα have revealed how Ric-8A
phosphorylation promotes
Gα recognition and activity as a chaperone and guanine
nucleotide
exchange factor. However, the molecular mechanism by which NCS-1
regulates Ric-8A activity and its interaction with Gα
subunits was not
well understood. Here, we have conducted a multimodal approach to show
that NCS-1 and Ric-8A constitute a hub that integrates Ca2+,
phosphorylation and G-protein signaling. The emergent picture indicates
that at Ca2+ resting state, Ric-8A activity is
under NCS-1 control and
the Ca2+ sensor traps Ric-8A in a conformational
state that hinders
phosphorylation and Gα recognition. However, a specific Ca2+
signal
triggers the disassembly of the NCS-1/Ric-8A complex, which in turn
allows phosphorylation of Ric-8A, formation of the Ric-8A/Gα
complex
and activation of Gα nucleotide exchange. Strikingly, we
found that
NCS-1 binds Na+ in its regulatory Ca2+
site, decreasing the affinity of
NCS-1 for Ca2+. Furthermore, we show that
different Ca2+ signals
promote the recognition of Ric-8A and dopamine D2 receptor. Finally,
the high-resolution crystallographic data reported here define the
NCS-1/Ric-8A interface and will allow the development of therapeutic
synapse function regulators with improved activity and selectivity.
eLife (2023)
12:e86151 (doi:
10.7554/eLife.86151)
New
structural insight into the conformational heterogeneity of NQO1 enzyme
with XFELs. NQO1
is a flavoenzyme essential for the antioxidant defense system,
stabilization of tumor suppressors, and the NAD(P)H-dependent
two-electron reduction of a wide variety of substrates, including the
activation of quinone-based chemotherapeutics. In addition, alterations
in NQO1 function are associated with cancer, Alzheimer's and
Parkinson's disease, which makes this enzyme an attractive target for
drug discovery. The results reported in this article demonstrate the
power of the SFX technique with XFELs to describe the
structure-function relationship in NQO1. We provide important insight
into the conformational heterogeneity of the human NQO1, highlighting
the high plasticity of this enzyme in the catalytic site and hence shed
light on the molecular basis of NQO1 functional cooperativity.
Lab on a Chip (2023)
(doi:
10.1039/D3LC00176H)
DipM
controls multiple autolysins and mediates a regulatory feedback loop
promoting cell constriction in Caulobacter crescentus. A multidimensional investigation led by
Martin Thanbichler from the University of Marburg and in collaboration
with Juan A. Hermoso (Institute of Physical Chemistry “Blas
Cabrera”), has identified two critical regulatory
hubs that control cell division in Caulobacter
crescentus, a widely studied bacterium. Published in
Nature Communications, the research reveals the pivotal role of DipM
and LdpF in coordinating peptidoglycan remodeling pathways, ensuring
proper cell constriction and daughter cell separation. The findings
offer valuable insights into bacterial growth and potential targets for
the development of effective antibacterial drugs.
Nature
Communications (2023) 14, Article number: 4095 (doi:
10.1038/s41467-023-39783-w)
Proceedings
of the National Academy of Sciences (2023)
(doi:
10.1073/pnas.2301897120)
See also two
short
movies: ATP
binding leading to PLD restraining and EnvC
activation caused by the restraining of PLD upon ATP binding
Deciphering
the Second Messenger Processing Mechanism by Standalone CRISPR-Cas Ring
Nucleases.
CRISPR-Cas systems comprise
an adaptive immune system in bacteria and archaea against foreign
mobile genetic elements, such as plasmids and phages, which has
constituted a revolution in life sciences. Their discovery and
straightforward development into versatile nucleases by guide RNA
exchange paved the way for gene modifications à la carte
that can be employed in biomedicine and biotechnology.
Type III CRISPR-Cas
effector systems detect foreign RNA triggering DNA and RNA cleavage and
synthesizing cyclic oligoadenylate molecules (cA) in their Cas10
subunit. cAs act as a second messenger activating auxiliary nucleases,
leading to an indiscriminate RNA degradation that can end in cell
dormancy or death. Standalone ring nucleases are CRISPR ancillary
proteins which downregulate the strong immune response of Type III
systems by degrading cA. Two genes with this function (Sis0811 and
Sis0455) have been found within the Sulfolobus islandicus
(Sis) genome. They code for a long polypeptide composed by a CARF
domain fused to an HTH domain (Sis0811 described in Molina et al.,
Nucleic Acids Research, 2021) and a short polypeptide constituted by a
CARF domain with a 40 residue C-terminal insertion (Sis0455). Here, we
determine the structure of the apo and substrate bound states of the
Sis0455 enzyme, revealing an insertion at the C-terminal region of the
CARF domain, which plays a key role closing the catalytic site upon
substrate binding. Our analysis reveals the key residues of Sis0455
during cleavage and the coupling of the active site closing with their
positioning to proceed with cA4 phosphodiester hydrolysis. A time
course comparison of cA4 cleavage between the short, Sis0455, and long
ring nucleases, Sis0811, shows the slower cleavage kinetics of the
former, suggesting that the combination of these two types of enzymes
with the same function in a genome could be an evolutionary strategy to
regulate the levels of the second messenger in different infection
scenarios.
Nucleic
Acids Research (2022)
(doi: 10.1093/nar/gkac923)
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