Glutamine amidotransferases are multienzyme machineries in which reactive ammonia is generated by a glutaminase and then transferred through a sequestered protein tunnel to a synthase active site for incorporation into diverse metabolites. To avoid wasteful metabolite consumption, there is a requirement for synchronized catalysis, but any generally applicable mechanistic insight is still lacking. As synthase activity depends on glutamine turnover, we investigated possible mechanisms controlling glutaminase catalysis using aminodeoxychorismate synthase involved in folate biosynthesis as a model. By analyzing this system in distinct states of catalysis, we found that incubation with glutamine leads to a subunit interface expansion by one-third of its original area. These changes completely enclose the glutaminase active site for sequestered catalysis and the subsequent transport of volatile ammonia to the synthase active site. In view of similar rearrangements in other glutamine amidotransferases, our observations may provide a general mechanism for the catalysis synchronization of this multienzyme family.

There has been a surge of interest in covalent inhibitors for protein kinases in recent years. Despite success in oncology, the off-target reactivity of these molecules is still hampering the use of covalent warhead-based strategies. Herein, we disclose the development of precision-guided warheads to mitigate the off-target challenge. These reversible warheads have a complex and cyclic structure with optional chirality center and tailored steric and electronic properties. To validate our proof-of-concept, we modified acrylamide-based covalent inhibitors of c-Jun N-terminal kinases (JNKs). We show that the cyclic warheads have high resilience against off-target thiols. Additionally, the binding affinity, residence time, and even JNK isoform specificity can be fine-tuned by adjusting the substitution pattern or using divergent and orthogonal synthetic elaboration of the warhead. Taken together, the cyclic warheads presented in this study will be a useful tool for medicinal chemists for the deliberate design of safer and functionally fine-tuned covalent inhibitors.

Clathrin forms a triskelion, or three-legged, network that regulates cellular processes by facilitating cargo internalization and trafficking in eukaryotes. Its N-terminal domain is crucial for interacting with adaptor proteins, which link clathrin to the membrane and engage with specific cargo. The N-terminal domain contains up to four adaptor-binding sites, though their role in preferential occupancy by adaptor proteins remains unclear. In this study, we examine the binding hierarchy of adaptors for clathrin, using integrative biophysical and structural approaches, along with in vivo functional experiments. We find that yeast epsin Ent5 has the highest affinity for clathrin, highlighting its key role in cellular trafficking. Epsins Ent1 and Ent2, crucial for endocytosis but thought to have redundant functions, show distinct binding patterns. Ent1 exhibits stronger interactions with clathrin than Ent2, suggesting a functional divergence toward actin binding. These results offer molecular insights into adaptor protein selectivity, suggesting they competitively bind clathrin while also targeting three different clathrin sites. Clathrin helps cells internalize and transport cargo by interacting with membrane adaptor proteins. Here, the authors reveal that adaptor proteins bind selectively, hierarchically and simultaneously to three sites of clathrin's N-terminal domain.

Granulicella tundricola hydroxynitrilelyase (GtHNL) is a manganese dependent cupin thatcatalyzes theenantioselective synthesis of cyanohydrins. The analysis of its activesite shows high similarity with the active site of cupin Tm1459 from Thermotoga maritima, an enzyme that catalyzesthe oxidative cleavage of styrene derivatives. GtHNL (GtHNL-WT) was found to catalyze the oxidativecleavage of & alpha;-methyl styrene, too. The conversion of & alpha;-methylstyrene yielded 23.6 & PLUSMN; 0.8% of acetophenone after 20 h. On theother hand, Tm1459 was not able to catalyze the synthesisof cyanohydrins efficiently. A low yield of rac-mandelonitrilewas obtained from benzaldehyde and HCN using either Tm1459-WT or Tm1459-C106L, a variant more active inoxidative catalysis. On the basis of the molecular analysis of GtHNL and Tm1459 active sites, the variants GtHNL-H96A, GtHNL-H96F, and GtHNL-A40H/V42T/H96A/Q110H were produced and evaluated for improvedcatalytic activity toward oxidative cleavage of styrenes. The aminoacid substitution H96A liberates an additional manganese coordinationposition and enlarges the GtHNL-WT active site cavity.Similarly, the amino acid substitution H96F liberates a coordinationsite as described for the GtHNL-H96A variant butwithout enlarging the active site space. All variants were able tocatalyze the oxidative cleavage of styrene derivatives. The best resultswere observed using GtHNL-H96A as catalyst. It displayeda higher yield of acetophenone (42%) as compared to GtHNL-A40H/V42T/H96A/Q110H (12%) and GtHNL-H96F (11%)after 20 h of reaction time. No oxidation of Mn(II) to Mn(III) couldbe detected by electron paramagnetic resonance (EPR), whereas evidencefor a radical mechanism is presented. Control reactions using 0.1and 0.5 mM of MnCl2 in the absence of enzyme showed nosignificant oxidation reaction.

Postsynaptic scaffold proteins such as Shank, PSD-95, Homer and SAPAP/GKAP family members establish the postsynaptic density of glutamatergic synapses through a dense network of molecular interactions. Mutations in SHANK genes are associated with neurodevelopmental disorders including autism and intellectual disability. However, no SHANK missense mutations have been described which interfere with the key functions of Shank proteins believed to be central for synapse formation, such as GKAP binding via the PDZ domain, or Zn2+-dependent multimerization of the SAM domain. We identify two individuals with a neurodevelopmental disorder carrying de novo missense mutations in SHANK2. The p.G643R variant distorts the binding pocket for GKAP in the Shank2 PDZ domain and prevents interaction with Thr(-2) in the canonical PDZ ligand motif of GKAP. The p.L1800W variant severely delays the kinetics of Zn2+-dependent polymerization of the Shank2-SAM domain. Structural analysis shows that Trp1800 dislodges one histidine crucial for Zn2+ binding. The resulting conformational changes block the stacking of helical polymers of SAM domains into sheets through side-by-side contacts, which is a hallmark of Shank proteins, thereby disrupting the highly cooperative assembly process induced by Zn2+. Both variants reduce the postsynaptic targeting of Shank2 in primary cultured neurons and alter glutamatergic synaptic transmission. Super-resolution microscopy shows that both mutants interfere with the formation of postsynaptic nanoclusters. Our data indicate that both the PDZ- and the SAM-mediated interactions of Shank2 contribute to the compaction of postsynaptic protein complexes into nanoclusters, and that deficiencies in this process interfere with normal brain development in humans.

Computer-aided drug discovery methods play a major role in the development of therapeutically important small molecules, but their performance needs to be improved. Molecular dynamics simulations in mixed solvents are useful in understanding protein-ligand recognition and improving molecular docking predictions. In this work, we used ethanol as a cosolvent to find relevant interactions for ligands toward protein kinase G, an essential protein of Mycobacterium tuberculosis (Mtb). We validated the hot spots by screening a database of fragment-like compounds and another one of known kinase inhibitors. Next, we performed a pharmacophore-guided docking simulation and found three low micromolar inhibitors, including one with a novel chemical scaffold that we expanded to four derivative compounds. Binding affinities were characterized by intrinsic fluorescence quenching assays, isothermal titration calorimetry, and the analysis of melting curves. The predicted binding mode was confirmed by X-ray crystallography. Finally, the compounds significantly inhibited the viability of Mtb in infected THP-1 macrophages.

The Kaposi's sarcoma associated herpesvirus protein ORF45 binds the extracellular signal-regulated kinase (ERK) and the p90 Ribosomal S6 kinase (RSK). ORF45 was shown to be a kinase activator in cells but a kinase inhibitor in vitro, and its effects on the ERK-RSK complex are unknown. Here, we demonstrate that ORF45 binds ERK and RSK using optimized linear binding motifs. The crystal structure of the ORF45-ERK2 complex shows how kinase docking motifs recognize the activated form of ERK. The crystal structure of the ORF45-RSK2 complex reveals an AGC kinase docking system, for which we provide evidence that it is functional in the host. We find that ORF45 manipulates ERK-RSK signaling by favoring the formation of a complex, in which activated kinases are better protected from phosphatases and docking motif-independent RSK substrate phosphorylation is selectively up-regulated. As such, our data suggest that ORF45 interferes with the natural design of kinase docking systems in the host.

Regulation of enzyme activity is vital for living organisms. In metalloenzymes, far-reaching rearrangements of the protein scaffold are generally required to tune the metal cofactor's properties by allosteric regulation. Here structural analysis of hydroxyketoacid aldolase from Sphingomonas wittichii RW1 ( Sw HKA) revealed a dynamic movement of the metal cofactor between two coordination spheres without protein scaffold rearrangements. In its resting state configuration (M 2+ R ), the metal constitutes an integral part of the dimer interface within the overall hexameric assembly, but sterical constraints do not allow for substrate binding. Conversely, a second coordination sphere constitutes the catalytically active state (M 2+ A ) at 2.4 Å distance. Bidentate coordination of a ketoacid substrate to M 2+ A affords the overall lowest energy complex, which drives the transition from M 2+ R to M 2+ A . While not described earlier, this type of regulation may be widespread and largely overlooked due to low occupancy of some of its states in protein crystal structures.