Linear ubiquitin chains are important regulators of cellular signaling pathways that control innate immunity and inflammation through NF-κB activation and protection against TNFα-induced apoptosis1-5. They are synthesized by HOIP, which belongs to the RBR (RING-between-RING) family of E3 ligases and is the catalytic component of LUBAC (linear ubiquitin chain assembly complex), a multi-subunit E3 ligase6. RBR family members act as RING/HECT hybrids, employing RING1 to recognize ubiquitin-loaded E2 while a conserved cysteine in RING2 subsequently forms a thioester intermediate with the transferred or “donor” ubiquitin7. Here we report the crystal structure of the catalytic core of HOIP in its apo form and in complex with ubiquitin. The C-terminal portion of HOIP adopts a novel fold that, together with a zinc finger, forms an ubiquitin-binding platform which orients the acceptor ubiquitin and positions its α-amino group for nucleophilic attack on the E3~ubiquitin thioester. The carboxy-terminal tail of a second ubiquitin molecule is located in close proximity to the catalytic cysteine providing a unique snapshot of the ubiquitin transfer complex containing both donor and acceptor ubiquitin. These interactions are required for activation of the NF-kB pathway in vivo and explain the determinants of linear ubiquitin chain specificity by LUBAC.

We have prepared phosphorylated cyclin-dependent protein kinase 2 (CDK2) for crystallization using the CDK-activating kinase 1 (CAK1) from Saccharomyces cerevisiae and have grown crystals using microseeding techniques. Phosphorylation of monomeric human CDK2 by CAK1 is more efficient than phosphorylation of the binary CDK2-cyclin A complex. Phosphorylated CDK2 exhibits histone H1 kinase activity corresponding to approximately 0.3% of that observed with the fully activated phosphorylated CDK2-cyclin A complex. Fluorescence measurements have shown that Thr 160 phosphorylation increases the affinity of CDK2 for both histone substrate and ATP and decreases its affinity for ADP. By contrast, phosphorylation of CDK2 has a negligible effect on the affinity for cyclin A. The crystal structures of the ATP-bound forms of phosphorylated CDK2 and unphosphorylated CDK2 have been solved at 2.1-Å resolution. The structures are similar, with the major difference occurring in the activation segment, which is disordered in phosphorylated CDK2. The greater mobility of the activation segment in phosphorylated CDK2 and the absence of spontaneous crystallization suggest that phosphorylated CDK2 may adopt several different mobile states. The majority of these states are likely to correspond to inactive conformations, but a small fraction of phosphorylated CDK2 may be in an active conformation and hence explain the basal activity observed.

Staurosporine exhibits nanomolar IC50 values against a wide range of protein kinases. The structure of a CDK2 staurosporine complex explains the tight binding of this inhibitor, and suggests features to be exploited in the design of specific inhibitors of CDKs.

The CD4 (T4) molecule is expressed on a subset of T lymphocytes involved in class II MHC recognition, and is probably the physiological receptor for one or more monomorphic regions of class II MHC (refs 1-3). CD4 also functions as a receptor for the human immunodeficiency virus (HIV) exterior envelope glycoprotein (gp120) (refs 4-9), being essential for virus entry into the host cell and for membrane fusion, which contributes to cell-to-cell transmission of the virus and to its cytopathic effects. We have used a baculovirus expression system to generate mg quantities of a hydrophilic extracellular segment of CD4. Concentrations of soluble CD4 in the nanomolar range, like certain anti-CD4 monoclonal antibodies, inhibit syncytium formation and HIV infection by binding gp120-expressing cells. Perhaps more importantly, class II specific T-cell interactions are uninhibited by soluble CD4 protein, whereas they are virtually abrogated by equivalent amounts of anti-T4 antibody. This may reflect substantial differences in CD4 affinity for gp120 and class II MHC.

CDK1 is the only essential cell cycle CDK in human cells and is required for successful completion of M-phase. It is the founding member of the CDK family and is conserved across all eukaryotes. Here we report the crystal structures of complexes of CDK1–Cks1 and CDK1–cyclin B–Cks2. These structures confirm the conserved nature of the inactive monomeric CDK fold and its ability to be remodeled by cyclin binding. Relative to CDK2–cyclin A, CDK1–cyclin B is less thermally stable, has a smaller interfacial surface, is more susceptible to activation segment dephosphorylation, and shows differences in the substrate sequence features that determine activity. Both CDK1 and CDK2 are potential cancer targets for which selective compounds are required. We also describe the first structure of CDK1 bound to a potent ATP-competitive inhibitor and identify aspects of CDK1 structure and plasticity that might be exploited to develop CDK1-selective inhibitors.

BackgroundDespite the rapid adoption of immunotherapies in advanced non–small cell lung cancer (advNSCLC), knowledge gaps remain about their real‐world (rw) performance.MethodsThis retrospective, observational, multicenter analysis used the Flatiron Health deidentified electronic health record‐derived database of rw patients with advNSCLC who received treatment with PD‐1 and/or PD‐L1 (PD‐[L]1) inhibitors before July 1, 2017 (N = 5257) and had ≥6 months of follow‐up. The authors investigated PD‐(L)1 line of treatment and PD‐L1 testing rates and the relationship between overall survival (OS) and rw intermediate endpoints: progression‐free survival (rwPFS), rw time to progression (rwTTP), rw time to next treatment (rwTTNT), and rw time to discontinuation (rwTTD).ResultsFirst‐line PD‐(L)1 inhibitor use increased from 0% (in the third quarter of 2014 [Q3 2014]) to 42% (Q2 2017) over the study period. PD‐L1 testing also increased (from 3% in Q3 2015 to 70% in Q2 2017). The estimated median OS was 9.3 months (95% CI, 8.9‐9.8 months), and the estimated rwPFS was 3.2 months (95% CI, 3.1‐3.3 months). Longer OS and rwPFS were associated with ≥50% PD‐L1 percentage staining results. Correlations (⍴) between OS and intermediate endpoints were ⍴ = 0.75 (95% CI, 0.73‐0.76) for rwPFS and ⍴ = 0.60 (95% CI, 0.57‐0.63) for rwTTP, and, for treatment‐based intermediate endpoints, correlations were ⍴ = 0.60 (95% CI, 0.56‐0.64) for rwTTNT (N = 856) and ⍴ = 0.81 (95% CI, 0.80‐0.82) for rwTTD.ConclusionsThe use of first‐line PD‐(L)1 inhibitors and PD‐L1 testing has substantially increased, with better outcomes for patients who have ≥50% PD‐L1 percentage staining. Intermediate rw tumor‐dynamics estimates were moderately correlated with OS in patients with advNSCLC who received immunotherapy, highlighting the need for optimizing and standardizing rw endpoints to enhance the understanding of patient outcomes outside clinical trials.

The CDK-interacting protein phosphatase KAP dephosphorylates phosphoThr-160 (pThr-160) of the CDK2 activation segment, the site of regulatory phosphorylation that is essential for kinase activity. Here we describe the crystal structure of KAP in association with pThr-160-CDK2, representing an example of a protein phosphatase in complex with its intact protein substrate. The major protein interface between the two molecules is formed by the C-terminal lobe of CDK2 and the C-terminal helix of KAP, regions remote from the kinase-activation segment and the KAP catalytic site. The kinase-activation segment interacts with the catalytic site of KAP almost entirely via the phosphate group of pThr-160. This interaction requires that the activation segment is unfolded and drawn away from the kinase molecule, inducing a conformation of CDK2 similar to the activated state observed in the CDK2/cyclin A complex.

Heterochromatin represents a cytologically visible state of heritable gene repression. In the yeast, Schizosaccharomyces pombe, the swi6 gene encodes a heterochromatin protein 1 (HP1)-like chromodomain protein that localizes to heterochromatin domains, including the centromeres, telomeres, and the donor mating-type loci, and is involved in silencing at these loci. We identify here the functional domains of swi6p and demonstrate that the chromodomain from a mammalian HP1-like protein, M31, can functionally replace that of swi6p, showing that chromodomain function is conserved from yeasts to humans. Site-directed mutagenesis, based on a modeled three-dimensional structure of the swi6p chromodomain, shows that the hydrophobic amino acids which lie in the core of the structure are critical for biological function. Gel filtration, gel overlay experiments, and mass spectroscopy show that HP1 proteins can self-associate, and we suggest that it is as oligomers that HP1 proteins are incorporated into heterochromatin complexes that silence gene activity.The highly conserved heterochromatin protein 1 (HP1) class of chromobox genes (HP1) encode structural adapters whose probable role is to assemble a variety of macromolecular complexes in chromatin (30). The possible functions of these complexes are wide-ranging and include roles in transcriptional repression (12,36,54,55), transgene silencing (17, 26), chromosome segregation (14, 31), recruitment of silent genes to heterochromatin (7, 54), localization of heterochromatin to the nuclear periphery (67), and sex chromosome inactivation during mammalian spermatogenesis (44).The swi6 gene in Schizosaccharomyces pombe is a nonessential gene that is required for the recombination-suppression and silencing which encompasses the mat2-K-mat3 region (33). Cloning of the gene showed that swi6 is a member of the HP1 class of chromobox genes (38), suggesting that the recombination-suppression and silencing are due to the packaging of the mat2-K-mat3 region into a heterochromatin-like complex that renders the region inaccessible to the transcriptional and recombination machinery (38,63). Other trans-acting factors that are required for repression at the silent loci include rik1, clr1, clr2, clr3, clr4, and clr6 (34, 64). rik1, clr1, and clr4 are thought to encode structural components of the heterochromatin-like complex, while clr3 and clr6 share considerable homology with histone deacetylases (20). Along with the silent mating-type loci, swi6p is also involved in silencing at the fission yeast centromeres and telomeres (14, 47) and plays a role in chromosome segregation at anaphase (14).HP1 proteins are characterized by the possession of both a classical chromodomain (CD) and a chromo shadow domain (CSD) (2) linked by a variable intervening region (IVR) or "hinge" (16). In addition, a stretch of acidic amino acids immediately precedes the CD of HP1 proteins (see Fig. 1A). The solution structure of the CD from the murine HP1-like heterochromatin-associated protein, M31 (also known as mH...