Glycogen is the primary energy reserve in mammals, and dysregulation of glycogen metabolism can result in glycogen storage diseases (GSDs). In muscle, glycogen synthesis is initiated by the enzymes ...glycogenin-1 (GYG1), which seeds the molecule by autoglucosylation, and glycogen synthase-1 (GYS1), which extends the glycogen chain. Although both enzymes are required for proper glycogen production, the nature of their interaction has been enigmatic. Here, we present the human GYS1:GYG1 complex in multiple conformations representing different functional states. We observe an asymmetric conformation of GYS1 that exposes an interface for close GYG1 association, and propose this state facilitates handoff of the GYG1-associated glycogen chain to a GYS1 subunit for elongation. Full activation of GYS1 widens the GYG1-binding groove, enabling GYG1 release concomitant with glycogen chain growth. This structural mechanism connecting chain nucleation and extension explains the apparent stepwise nature of glycogen synthesis and suggests distinct states to target for GSD-modifying therapeutics.
ALK (anaplastic lymphoma kinase) is an RTK (receptor tyrosine kinase) of the IRK (insulin receptor kinase) superfamily, which share an YXXXYY autophosphorylation motif within their A-loops ...(activation loops). A common activation and regulatory mechanism is believed to exist for members of this superfamily typified by IRK and IGF1RK (insulin-like growth factor receptor kinase-1). Chromosomal translocations involving ALK were first identified in anaplastic large-cell lymphoma, a subtype of non-Hodgkin's lymphoma, where aberrant fusion of the ALK kinase domain with the NPM (nucleophosmin) dimerization domain results in autophosphosphorylation and ligand-independent activation. Activating mutations within the full-length ALK kinase domain, most commonly R1275Q and F1174L, which play a major role in neuroblastoma, were recently identified. To provide a structural framework for understanding these mutations and to guide structure-assisted drug discovery efforts, the X-ray crystal structure of the unphosphorylated ALK catalytic domain was determined in the apo, ADP- and staurosporine-bound forms. The structures reveal a partially inactive protein kinase conformation distinct from, and lacking, many of the negative regulatory features observed in inactive IGF1RK/IRK structures in their unphosphorylated forms. The A-loop adopts an inhibitory pose where a short proximal A-loop helix (alphaAL) packs against the alphaC helix and a novel N-terminal beta-turn motif, whereas the distal portion obstructs part of the predicted peptide-binding region. The structure helps explain the reported unique peptide substrate specificity and the importance of phosphorylation of the first A-loop Tyr1278 for kinase activity and NPM-ALK transforming potential. A single amino acid difference in the ALK substrate peptide binding P-1 site (where the P-site is the phosphoacceptor site) was identified that, in conjunction with A-loop sequence variation including the RAS (Arg-Ala-Ser)-motif, rationalizes the difference in the A-loop tyrosine autophosphorylation preference between ALK and IGF1RK/IRK. Enzymatic analysis of recombinant R1275Q and F1174L ALK mutant catalytic domains confirms the enhanced activity and transforming potential of these mutants. The transforming ability of the full-length ALK mutants in soft agar colony growth assays corroborates these findings. The availability of a three-dimensional structure for ALK will facilitate future structure-function and rational drug design efforts targeting this receptor tyrosine kinase.
•NRX-0492 induced rapid and sustained degradation of wild-type and C481 mutant BTK at subnanomolar concentrations in primary CLL samples.•Orally administered NRX-0492 abrogated BTK-dependent ...signaling and demonstrated anti-CLL activity in patient-derived xenografts in vivo.
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Bruton tyrosine kinase (BTK) is essential for B-cell receptor (BCR) signaling, a driver of chronic lymphocytic leukemia (CLL). Covalent inhibitors bind C481 in the active site of BTK and have become a preferred CLL therapy. Disease progression on covalent BTK inhibitors is commonly associated with C481 mutations. Here, we investigated a targeted protein degrader, NRX-0492, that links a noncovalent BTK-binding domain to cereblon, an adaptor protein of the E3 ubiquitin ligase complex. NRX-0492 selectively catalyzes ubiquitylation and proteasomal degradation of BTK. In primary CLL cells, NRX-0492 induced rapid and sustained degradation of both wild-type and C481 mutant BTK at half maximal degradation concentration (DC50) of ≤0.2 nM and DC90 of ≤0.5 nM, respectively. Sustained degrader activity was maintained for at least 24 hours after washout and was equally observed in high-risk (deletion 17p) and standard-risk (deletion 13q only) CLL subtypes. In in vitro testing against treatment-naïve CLL samples, NRX-0492 was as effective as ibrutinib at inhibiting BCR-mediated signaling, transcriptional programs, and chemokine secretion. In patient-derived xenografts, orally administered NRX-0492 induced BTK degradation and inhibited activation and proliferation of CLL cells in blood and spleen and remained efficacious against primary C481S mutant CLL cells collected from a patient progressing on ibrutinib. Oral bioavailability, >90% degradation of BTK at subnanomolar concentrations, and sustained pharmacodynamic effects after drug clearance make this class of targeted protein degraders uniquely suitable for clinical translation, in particular as a strategy to overcome BTK inhibitor resistance. Clinical studies testing this approach have been initiated (NCT04830137, NCT05131022).
Bruton tyrosine kinase (BTK) is a driver of chronic lymphocytic leukemia (CLL), and covalent inhibitors of BTK like ibrutinib have become central to CLL therapy. Resistance to ibrutinib is often associated with mutations that block binding. Zhang and colleagues report on excellent activity in patient-derived xenografts of a targeted protein degrader that links a noncovalent BTK-binding domain to cereblon, catalyzing ubiquitylation of BTK and targeting it for proteosomal degradation. Ongoing clinical studies may establish this as a strategy for overcoming BTK-inhibitor resistance.
A novel aminoacyl‐tRNA synthetase that contains an iron–sulfur cluster in the tRNA anticodon‐binding region and efficiently charges tRNA with tryptophan has been found in Thermotoga maritima. The ...crystal structure of TmTrpRS (tryptophanyl‐tRNA synthetase; TrpRS; EC 6.1.1.2) reveals an iron–sulfur 4Fe–4S cluster bound to the tRNA anticodon‐binding (TAB) domain and an l‐tryptophan ligand in the active site. None of the other T. maritima aminoacyl‐tRNA synthetases (AARSs) contain this 4Fe–4S cluster‐binding motif (C‐x22‐C‐x6‐C‐x2‐C). It is speculated that the iron–sulfur cluster contributes to the stability of TmTrpRS and could play a role in the recognition of the anticodon.
The crystal structures of two homologous endopeptidases from cyanobacteria Anabaena variabilis and Nostoc punctiforme were determined at 1.05 and 1.60 Å resolution, respectively, and contain a ...bacterial SH3-like domain (SH3b) and a ubiquitous cell-wall-associated NlpC/P60 (or CHAP) cysteine peptidase domain. The NlpC/P60 domain is a primitive, papain-like peptidase in the CA clan of cysteine peptidases with a Cys126/His176/His188 catalytic triad and a conserved catalytic core. We deduced from structure and sequence analysis, and then experimentally, that these two proteins act as γ-D-glutamyl-L-diamino acid endopeptidases (EC 3.4.22.-). The active site is located near the interface between the SH3b and NlpC/P60 domains, where the SH3b domain may help define substrate specificity, instead of functioning as a targeting domain, so that only muropeptides with an N-terminal L-alanine can bind to the active site.
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