Crystallization chaperones have been used to facilitate the crystallization of challenging proteins. Even though the maltose-binding protein (MBP) is one of the most commonly used crystallization chaperones, the design of optimal expression constructs for crystallization of MBP fusion proteins remains a challenge. To increase the success rate of MBP-facilitated crystallization, a series of expression vectors have been designed with either a short flexible linker or a set of rigid helical linkers. Seven death domain superfamily members were tested for crystallization with this set of vectors, six of which had never been crystallized before. All of the seven targets were crystallized, and their structures were determined using at least one of the vectors. Our successful crystallization of all of the targets demonstrates the validity of our approach and expands the arsenal of the crystallization chaperone toolkit, which may be applicable to crystallization of other difficult protein targets, as well as to other crystallization chaperones.

Preclinical data demonstrated that combining an anti-programmed cell death 1 (PD-1) inhibitor with a CDK9 inhibitor provided enhanced antitumor activity with no significant toxicities, suggesting this combination may be a potential therapeutic option. The multicohort, phase 1 KEYNOTE-155 study evaluated the safety and antitumor activity of the PD-1 inhibitor pembrolizumab plus the CDK9 inhibitor dinaciclib in patients with relapsed or refractory (rr) chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM). Patients enrolled were ≥18 years of age with a confirmed diagnosis of CLL, DLBCL, or MM. The study included 2 phases: a dose-evaluation phase to determine dose-limiting toxicities and a signal-detection phase. Patients received pembrolizumab 200 mg every 3 weeks plus dinaciclib 7 mg/m2 on day 1 and 10 mg/m2 on day 8 of cycle 1 and 14 mg/m2 on days 1 and 8 of cycles 2 and later. Primary endpoint was safety, and a key secondary endpoint was objective response rate (ORR), Seventy-two patients were enrolled and received ≥1 dose of study treatment (CLL, n = 17; DLBCL, n = 38; MM, n = 17). Pembrolizumab plus dinaciclib was generally well tolerated and produced no unexpected toxicities. The ORRs were 29.4% (5/17, rrCLL), 21.1% (8/38, rrDLBCL), and 0% (0/17, rrMM), respectively. At data cutoff, all 72 patients had discontinued treatment, 38 (52.8%) because of progressive disease. These findings demonstrate activity with combination pembrolizumab plus dinaciclib and suggest that a careful and comprehensive approach to explore anti-PD-1 and CDK9 inhibitor combinations is warranted. Clinical trial registration: ClinicalTrials.gov, NCT02684617

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CARD8 plays crucial roles in regulating apoptotic and inflammatory signaling pathways through the association of its caspase‐recruitment domain (CARD) with those of procaspase‐9 and procaspase‐1. The CARD8 CARD has also been predicted to form an intramolecular complex with its FIIND domain. Here, the first crystal structure of the CARD8 CARD is reported; it adopts a six‐helix bundle fold with a unique conformation of the α6 helix that is described here for the first time. The surface of the CARD8 CARD displays a prominent acidic patch at its α2, α3 and α5 helices that may interact with the procaspase‐9 CARD, whereas an adjacent charged surface at its α3 and α4 helices may associate with the CARD8 FIIND domain without interfering with the CARD–CARD interaction. This suggests that the function of CARD8 may be regulated by both intramolecular and intermolecular interactions involving electrostatic attractions.