4-Anilinoquinazoline Derivatives as the First Potent NOD1-RIPK2 Signaling Pathway Inhibitors at the Nanomolar Range.

Inflammation is a defense mechanism that restores tissue damage and eliminates pathogens. Among the pattern recognition receptors that recognize danger or pathogenic signals, nucleotide oligomerization domains 1 and 2 (NOD1/2) have been identified to play an important role in innate immunity responses, and inhibition of NOD1 could be interesting to treat severe infections and inflammatory diseases. In this work, we identified the first selective NOD1 versus NOD2 pathway inhibitors at the nanomolar range based on a 4-anilinoquinazoline scaffold.

Metal fluorides-multi-functional tools for the study of phosphoryl transfer enzymes, a practical guide.

Enzymes facilitating the transfer of phosphate groups constitute the most extensive protein families across all kingdoms of life. They make up approximately 10% of the proteins found in the human genome. Understanding the mechanisms by which enzymes catalyze these reactions is essential in characterizing the processes they regulate.

Structural basis for competitive binding of productive and degradative co-transcriptional effectors to the nuclear cap-binding complex.

The nuclear cap-binding complex (CBC) coordinates co-transcriptional maturation, transport, or degradation of nascent RNA polymerase II (Pol II) transcripts. CBC with its partner ARS2 forms mutually exclusive complexes with diverse "effectors" that promote either productive or destructive outcomes. Combining AlphaFold predictions with structural and biochemical validation, we show how effectors NCBP3, NELF-E, ARS2, PHAX, and ZC3H18 form competing binary complexes with CBC and how PHAX, NCBP3, ZC3H18, and other effectors compete for binding to ARS2.

Architecture of the MKK6-p38α complex defines the basis of MAPK specificity and activation.

The mitogen-activated protein kinase (MAPK) p38α is a central component of signaling in inflammation and the immune response and is, therefore, an important drug target. Little is known about the molecular mechanism of its activation by double phosphorylation from MAPK kinases (MAP2Ks), because of the challenge of trapping a transient and dynamic heterokinase complex. We applied a multidisciplinary approach to generate a structural model of p38α in complex with its MAP2K, MKK6, and to understand the activation mechanism.

Structure shows that the BIR2 domain of E3 ligase XIAP binds across the RIPK2 kinase dimer interface.

RIPK2 is an essential adaptor for NOD signalling and its kinase domain is a drug target for NOD-related diseases, such as inflammatory bowel disease. However, recent work indicates that the phosphorylation activity of RIPK2 is dispensable for signalling and that inhibitors of both RIPK2 activity and RIPK2 ubiquitination prevent the essential interaction between RIPK2 and the BIR2 domain of XIAP, the key RIPK2 ubiquitin E3 ligase. Moreover, XIAP BIR2 antagonists also block this interaction.

Burden and resources in caregivers of people with multiple sclerosis: A qualitative study.

Background: Caregivers of people with Multiple Sclerosis are required to provide ongoing assistance especially during the advanced stages of the disease. They have to manage interventions and assume responsibilities which significantly impact both their personal quality of life and family's dynamics.

Objective: A qualitative phenomenological study was carried out to understand the experience of burden in caregivers and their resources to manage it.

A GAP-GTPase-GDP-P Intermediate Crystal Structure Analyzed by DFT Shows GTP Hydrolysis Involves Serial Proton Transfers.

Cell signaling by small G proteins uses an ON to OFF signal based on conformational changes following the hydrolysis of GTP to GDP and release of dihydrogen phosphate (P ). The catalytic mechanism of GTP hydrolysis by RhoA is strongly accelerated by a GAP protein and is now well defined, but timing of inorganic phosphate release and signal change remains unresolved. We have generated a quaternary complex for RhoA-GAP-GDP-P .

RIP2 filament formation is required for NOD2 dependent NF-κB signalling.

Activation of the innate immune pattern recognition receptor NOD2 by the bacterial muramyl-dipeptide peptidoglycan fragment triggers recruitment of the downstream adaptor kinase RIP2, eventually leading to NF-κB activation and proinflammatory cytokine production. Here we show that full-length RIP2 can form long filaments mediated by its caspase recruitment domain (CARD), in common with other innate immune adaptor proteins. We further show that the NOD2 tandem CARDs bind to one end of the RIP2 CARD filament, suggesting a mechanism for polar filament nucleation by activated NOD2.

Structures of the inactive and active states of RIP2 kinase inform on the mechanism of activation.

Innate immune receptors NOD1 and NOD2 are activated by bacterial peptidoglycans leading to recruitment of adaptor kinase RIP2, which, upon phosphorylation and ubiquitination, becomes a scaffold for downstream effectors. The kinase domain (RIP2K) is a pharmaceutical target for inflammatory diseases caused by aberrant NOD2-RIP2 signalling. Although structures of active RIP2K in complex with inhibitors have been reported, the mechanism of RIP2K activation remains to be elucidated.

Assessing the Influence of Mutation on GTPase Transition States by Using X-ray Crystallography, F NMR, and DFT Approaches.

We report X-ray crystallographic and F NMR studies of the G-protein RhoA complexed with MgF , GDP, and RhoGAP, which has the mutation Arg85'Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X-ray data show how Tyr34 maintains solvent exclusion and the core H-bond network in the active site by relocating to replace the missing Arg85' sidechain.

Structural Basis for the Subversion of MAP Kinase Signaling by an Intrinsically Disordered Parasite Secreted Agonist.

The causative agent of toxoplasmosis, the intracellular parasite Toxoplasma gondii, delivers a protein, GRA24, into the cells it infects that interacts with the mitogen-activated protein (MAP) kinase p38α (MAPK14), leading to activation and nuclear translocation of the host kinase and a subsequent inflammatory response that controls the progress of the parasite. The purification of a recombinant complex of GRA24 and human p38α has allowed the molecular basis of this activation to be determined. GRA24 is shown to be intrinsically disordered, binding two kinases that act independently, and is the only factor required to bypass the canonical mitogen-activated protein kinase activation pathway.

α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction.

β-Phosphoglucomutase (βPGM) catalyzes isomerization of β-D-glucose 1-phosphate (βG1P) into D-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a β-D-glucose 1,6-bisphosphate (βG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of βG1P deliver novel step 1 transition state analog (TSA) complexes for βPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the β-D-glucopyranose ring in the βG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2).

Direct cryocooling of naked crystals: are cryoprotection agents always necessary?

Over the last 20 years cryocrystallography has revolutionized the field of macromolecular crystallography, greatly reducing radiation damage and allowing the collection of complete data sets at synchrotron sources. However, in order to cool crystals to 100 K cryoprotective agents must usually be added to prevent the formation of crystalline ice, which disrupts the macromolecular crystal lattice and often results in a degradation of diffraction quality. This process can involve the extensive testing of solution compositions and soaking protocols to find suitable conditions that maintain diffraction quality.

Inducing phase changes in crystals of macromolecules: status and perspectives for controlled crystal dehydration.

The increase in the number of large multi-component complexes and membrane protein crystal structures determined over the last few years can be ascribed to a number of factors such as better protein expression and purification systems, the emergence of high-throughput crystallization techniques and the advent of 3rd generation synchrotron sources. However, many systems tend to produce crystals that can be extremely heterogeneous in their diffraction properties. This prevents, in many cases, the collection of diffraction data of sufficient quality to yield useful biological or phase information.