ClpP protease activation results from the reorganization of the electrostatic interaction networks at the entrance pores

Abstract Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis and Escherichia coli ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation.

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Two distinct mechanisms of transcriptional regulation by the redox sensor YodB

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1604427113 ◽

2016 ◽

Vol 113 (35) ◽

pp. E5202-E5211 ◽

Cited By ~ 8

Author(s):

Sang Jae Lee ◽

In-Gyun Lee ◽

Ki-Young Lee ◽

Dong-Gyun Kim ◽

Hyun-Jong Eun ◽

...

Keyword(s):

Conformational Changes ◽

Disulfide Bonds ◽

Structural Changes ◽

High Specificity ◽

Redox Balance ◽

Structural Basis ◽

X Ray Crystallography ◽

Redox Sensor ◽

Regulatory Systems ◽

Sense And Respond

For bacteria, cysteine thiol groups in proteins are commonly used as thiol-based switches for redox sensing to activate specific detoxification pathways and restore the redox balance. Among the known thiol-based regulatory systems, the MarR/DUF24 family regulators have been reported to sense and respond to reactive electrophilic species, including diamide, quinones, and aldehydes, with high specificity. Here, we report that the prototypical regulator YodB of the MarR/DUF24 family from Bacillus subtilis uses two distinct pathways to regulate transcription in response to two reactive electrophilic species (diamide or methyl-p-benzoquinone), as revealed by X-ray crystallography, NMR spectroscopy, and biochemical experiments. Diamide induces structural changes in the YodB dimer by promoting the formation of disulfide bonds, whereas methyl-p-benzoquinone allows the YodB dimer to be dissociated from DNA, with little effect on the YodB dimer. The results indicate that B. subtilis may discriminate toxic quinones, such as methyl-p-benzoquinone, from diamide to efficiently manage multiple oxidative signals. These results also provide evidence that different thiol-reactive compounds induce dissimilar conformational changes in the regulator to trigger the separate regulation of target DNA. This specific control of YodB is dependent upon the type of thiol-reactive compound present, is linked to its direct transcriptional activity, and is important for the survival of B. subtilis. This study of B. subtilis YodB also provides a structural basis for the relationship that exists between the ligand-induced conformational changes adopted by the protein and its functional switch.

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Selective binding of small molecules to Vibrio cholerae DsbA offers a starting point for the design of novel antibacterials

10.33774/chemrxiv-2021-stw44 ◽

2021 ◽

Author(s):

Geqing Wang ◽

Biswaranjan Mohanty ◽

Martin Williams ◽

Bradley Doak ◽

Rabeb Dhouib ◽

...

Keyword(s):

Vibrio Cholerae ◽

Crystal Structures ◽

Small Molecule ◽

Sodium Taurocholate ◽

Structural Basis ◽

Human Pathogens ◽

X Ray ◽

X Ray Crystallography ◽

Hydrophobic Groove ◽

Starting Point

DsbA enzymes catalyze oxidative folding of proteins that are secreted into the periplasm of Gram-negative bacteria, and they are indispensable for the virulence of human pathogens such as Vibrio cholerae and Escherichia coli. Therefore, targeting DsbA represents an attractive approach to control bacterial virulence. X-ray crystal structures reveal that DsbA enzymes share a similar fold, however, the hydrophobic groove adjacent to the active site, which is implicated in substrate binding, is shorter and flatter in the structure of V. cholerae DsbA (VcDsbA) compared to E. coli DsbA (EcDsbA). The flat and largely featureless nature of this hydrophobic groove is challenging for the development of small molecule inhibitors. Using fragment-based screening approaches, we have identified a novel small molecule, based on the benzimidazole scaffold, that binds to the hydrophobic groove of oxidized VcDsbA with a KD of 446 ± 10 µM. The same benzimidazole compound has ~8-fold selectivity for VcDsbA over EcDsbA and binds to oxidized EcDsbA, with KD > 3.5 mM. We generated a model of the benzimidazole complex with VcDsbA using NMR data but were unable to determine the structure of the benzimidazole bound EcDsbA using either NMR or X-ray crystallography. Therefore, a structural basis for the observed selectivity is unclear. To better understand ligand binding to these two enzymes we crystallized each of them in complex with a known ligand, the bile salt sodium taurocholate. The crystal structures show that taurocholate adopts different binding poses in complex with VcDsbA and EcDsbA, and reveals the protein-ligand interactions that stabilize the different modes of binding. This work highlights the capacity of fragment-based drug discovery to identify inhibitors of challenging protein targets. In addition, it provides a starting point for development of more potent and specific VcDsbA inhibitors that act through a novel anti-virulence mechanism.

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Photocage-initiated time-resolved solution X-ray scattering investigation of protein dimerization

IUCrJ ◽

10.1107/s2052252518012149 ◽

2018 ◽

Vol 5 (6) ◽

pp. 667-672 ◽

Cited By ~ 6

Author(s):

Inokentijs Josts ◽

Stephan Niebling ◽

Yunyun Gao ◽

Matteo Levantino ◽

Henning Tidow ◽

...

Keyword(s):

Small Molecule ◽

Conformational Changes ◽

Structural Changes ◽

Time Resolved ◽

Protein Dimerization ◽

X Ray ◽

Single Turnover ◽

X Ray Scattering ◽

Photoactivatable Proteins ◽

Ray Scattering

This work demonstrates a new method for investigating time-resolved structural changes in protein conformation and oligomerization via photocage-initiated time-resolved X-ray solution scattering by observing the ATP-driven dimerization of the MsbA nucleotide-binding domain. Photocaged small molecules allow the observation of single-turnover reactions of non-naturally photoactivatable proteins. The kinetics of the reaction can be derived from changes in X-ray scattering associated with ATP-binding and subsequent dimerization. This method can be expanded to any small-molecule-driven protein reaction with conformational changes traceable by X-ray scattering where the small molecule can be photocaged.

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Structural Insights of SARS-CoV-2 Spike Protein from Delta and Omicron Variants

10.1101/2021.12.08.471777 ◽

2021 ◽

Author(s):

Ali Sadek ◽

David Zaha ◽

Mahmoud Salama Ahmed

Keyword(s):

Viral Entry ◽

Structural Changes ◽

Structural Basis ◽

S Protein ◽

Angiotensin Converting Enzyme 2 ◽

X Ray ◽

X Ray Crystallography ◽

Binding Domains ◽

Structural Insights ◽

Sheer Number

Given the continuing heavy toll of the COVID-19 pandemic and the emergence of the Delta (B.1.617.2) and Omicron (B.1.1.529) variants, the WHO declared both as variants of concern (VOC). There are valid concerns that the latest Omicron variant might have increased infectivity and pathogenicity. In addition, the sheer number of S protein mutations in the Omicron variant raise concerns of potential immune evasion and resistance to therapeutics such as monoclonal antibodies. However, structural insights that underpin the potential increased pathogenicity are unknown. Here we adopted an artificial intelligence (AI)-based approach to predict the structural changes induced by mutations of the Delta and Omicron variants in the spike (S) protein using Alphafold. This was followed by docking the human angiotensin-converting enzyme 2 (ACE2) with the predicted S proteins for Wuhan-Hu-1, Delta, and Omicron variants. Our in-silico structural analysis indicates that S protein for Omicron variant has a higher binding affinity to ACE-2 receptor, compared to Wuhan-Hu-1 and Delta variants. In addition, the recognition sites of the receptor binding domains for Delta and Omicron variants showed lower electronegativity compared to Wuhan-Hu-1. Importantly, further molecular insights revealed significant changes induced at fusion protein (FP) site, which may mediate enhanced viral entry. These results represent the first computational analysis of structural changes associated with Omicron variant using Alphafold, Collectively, our results highlight potential structural basis for enhanced pathogenicity of the Omicron variant, however further validation using X-ray crystallography and cryo-EM are warranted.

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Discovery of dual-activity small-molecule ligands of Pseudomonas aeruginosa LpxA and LpxD using SPR and X-ray crystallography

Scientific Reports ◽

10.1038/s41598-019-51844-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Kyle G. Kroeck ◽

Michael D. Sacco ◽

Emmanuel W. Smith ◽

Xiujun Zhang ◽

Daniel Shoun ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Small Molecule ◽

Conformational Changes ◽

Lipid A ◽

Acyl Carrier Protein ◽

Structural Features ◽

X Ray ◽

X Ray Crystallography ◽

Small Molecule Ligands ◽

Lipid A Biosynthesis

Abstract The lipid A biosynthesis pathway is essential in Pseudomonas aeruginosa. LpxA and LpxD are the first and third enzymes in this pathway respectively, and are regarded as promising antibiotic targets. The unique structural similarities between these two enzymes make them suitable targets for dual-binding inhibitors, a characteristic that would decrease the likelihood of mutational resistance and increase cell-based activity. We report the discovery of multiple small molecule ligands that bind to P. aeruginosa LpxA and LpxD, including dual-binding ligands. Binding poses were determined for select compounds by X-ray crystallography. The new structures reveal a previously uncharacterized magnesium ion residing at the core of the LpxD trimer. In addition, ligand binding in the LpxD active site resulted in conformational changes in the distal C-terminal helix-bundle, which forms extensive contacts with acyl carrier protein (ACP) during catalysis. These ligand-dependent conformational changes suggest a potential allosteric influence of reaction intermediates on ACP binding, and vice versa. Taken together, the novel small molecule ligands and their crystal structures provide new chemical scaffolds for ligand discovery targeting lipid A biosynthesis, while revealing structural features of interest for future investigation of LpxD function.

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Integrative x-ray structure and molecular modeling for the rationalization of procaspase-8 inhibitor potency and selectivity

10.1101/721951 ◽

2019 ◽

Author(s):

Janice H. Xu ◽

Jerome Eberhardt ◽

Brianna Hill-Payne ◽

Gonzalo E. González-Páez ◽

José Omar Castellón ◽

...

Keyword(s):

Molecular Modeling ◽

Small Molecules ◽

Small Molecule ◽

Conformational Changes ◽

Active Sites ◽

Structural Similarity ◽

X Ray ◽

X Ray Crystallography ◽

Molecule Inhibitor ◽

High Structural Similarity

AbstractCaspases are a critical class of proteases involved in regulating programmed cell death and other biological processes. Selective inhibitors of individual caspases, however, are lacking, due in large part to the high structural similarity found in the active sites of these enzymes. We recently discovered a small-molecule inhibitor, 63-R, that covalently binds the zymogen, or inactive precursor (pro-form), of caspase-8, but not other caspases, pointing to an untapped potential of procaspases as targets for chemical probes. Realizing this goal would benefit from a structural understanding of how small molecules bind to and inhibit caspase zymogens. There have, however, been very few reported procaspase structures. Here, we employ x-ray crystallography to elucidate a procaspase-8 crystal structure in complex with 63-R, which reveals large conformational changes in active-site loops that accommodate the intramolecular cleavage events required for protease activation. Combining these structural insights with molecular modeling and mutagenesis-based biochemical assays, we elucidate key interactions required for 63-R inhibition of procaspase-8. Our findings inform the mechanism of caspase activation and its disruption by small molecules, and, more generally, have implications for the development of small molecule inhibitors and/or activators that target alternative (e.g., inactive precursor) protein states to ultimately expand the druggable proteome.

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New insights into the structural basis of integrin activation

Blood ◽

10.1182/blood-2003-01-0334 ◽

2003 ◽

Vol 102 (4) ◽

pp. 1155-1159 ◽

Cited By ~ 133

Author(s):

Jian-Ping Xiong ◽

Thilo Stehle ◽

Simon L. Goodman ◽

M. Amin Arnaout

Keyword(s):

Conformational Changes ◽

Structural Changes ◽

Structural Basis ◽

Integrin Activation ◽

Cellular Functions ◽

Electron Microscopic ◽

The Past ◽

Intracellular Signals ◽

Bidirectional Signaling ◽

New Hypothesis

Abstract Integrins are cell adhesion receptors that communicate biochemical and mechanical signals in a bidirectional manner across the plasma membrane and thus influence most cellular functions. Intracellular signals switch integrins into a ligand-competent state as a result of elicited conformational changes in the integrin ectodomain. Binding of extracellular ligands induces, in turn, structural changes that convey distinct signals to the cell interior. The structural basis of this bidirectional signaling has been the focus of intensive study for the past 3 decades. In this perspective, we develop a new hypothesis for integrin activation based on recent crystallographic, electron microscopic, and biochemical studies.

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Unusual Conformational Changes in 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase as Revealed by X-ray Crystallography and NMR

Journal of Biological Chemistry ◽

10.1074/jbc.m103837200 ◽

2001 ◽

Vol 276 (43) ◽

pp. 40274-40281 ◽

Cited By ~ 26

Author(s):

Bing Xiao ◽

Genbin Shi ◽

Jinhai Gao ◽

Jaroslaw Blaszczyk ◽

Qin Liu ◽

...

Keyword(s):

Conformational Changes ◽

X Ray ◽

X Ray Crystallography

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Structural Basis of the Sec Translocon and YidC Revealed Through X-ray Crystallography

The Protein Journal ◽

10.1007/s10930-019-09830-x ◽

2019 ◽

Vol 38 (3) ◽

pp. 249-261 ◽

Cited By ~ 4

Author(s):

Tomoya Tsukazaki

Keyword(s):

Structural Basis ◽

X Ray ◽

X Ray Crystallography ◽

Sec Translocon

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Structural basis of the transactivation deficiency of the human PPARγ F360L mutant associated with familial partial lipodystrophy

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714009638 ◽

2014 ◽

Vol 70 (7) ◽

pp. 1965-1976 ◽

Cited By ~ 9

Author(s):

Clorinda Lori ◽

Alessandra Pasquo ◽

Roberta Montanari ◽

Davide Capelli ◽

Valerio Consalvi ◽

...

Keyword(s):

Crystal Structure ◽

Conformational Changes ◽

Partial Agonist ◽

Salt Bridge ◽

Van Der Waals Interactions ◽

Structural Basis ◽

Wild Type ◽

Partial Lipodystrophy ◽

X Ray ◽

Familial Partial Lipodystrophy

The peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate glucose and lipid metabolism. The role of PPARs in several chronic diseases such as type 2 diabetes, obesity and atherosclerosis is well known and, for this reason, they are the targets of antidiabetic and hypolipidaemic drugs. In the last decade, some rare mutations in human PPARγ that might be associated with partial lipodystrophy, dyslipidaemia, insulin resistance and colon cancer have emerged. In particular, the F360L mutant of PPARγ (PPARγ2 residue 388), which is associated with familial partial lipodystrophy, significantly decreases basal transcriptional activity and impairs stimulation by synthetic ligands. To date, the structural reason for this defective behaviour is unclear. Therefore, the crystal structure of PPARγ F360L together with the partial agonist LT175 has been solved and the mutant has been characterized by circular-dichroism spectroscopy (CD) in order to compare its thermal stability with that of the wild-type receptor. The X-ray analysis showed that the mutation induces dramatic conformational changes in the C-terminal part of the receptor ligand-binding domain (LBD) owing to the loss of van der Waals interactions made by the Phe360 residue in the wild type and an important salt bridge made by Arg357, with consequent rearrangement of loop 11/12 and the activation function helix 12 (H12). The increased mobility of H12 makes the binding of co-activators in the hydrophobic cleft less efficient, thereby markedly lowering the transactivation activity. The spectroscopic analysis in solution and molecular-dynamics (MD) simulations provided results which were in agreement and consistent with the mutant conformational changes observed by X-ray analysis. Moreover, to evaluate the importance of the salt bridge made by Arg357, the crystal structure of the PPARγ R357A mutant in complex with the agonist rosiglitazone has been solved.

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