Cryo-EM structures of PP2A:B55-FAM122A and PP2A:B55-ARPP19.

Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation1. Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases2, whereas mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B553. Although the role of kinases in mitotic entry is well established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited4. Inhibition of PP2A:B55 is achieved by the intrinsically disordered proteins ARPP195,6 and FAM122A7. Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the single-particle cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies, both intrinsically disordered proteins bind PP2A:B55, but do so in highly distinct manners, leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provide a molecular roadmap for the development of therapeutic interventions for PP2A:B55-related diseases.

Cryo-EM structures of PP2A:B55-FAM122A and PP2A:B55-ARPP19.

Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation.1 Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases,2 while mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B55.3 While the role of kinases in mitotic entry is well-established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited.4 For PP2A:B55, inhibition is achieved by the two intrinsically disordered proteins (IDPs), ARPP19 (phosphorylation-dependent)6,7 and FAM122A5 (inhibition is phosphorylation-independent). Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies both IDPs bind PP2A:B55, but do so in highly distinct manners, unexpectedly leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provides a molecular roadmap for the development of therapeutic interventions for PP2A:B55 related diseases.

Degradation of the E. coli antitoxin MqsA by the proteolytic complex ClpXP is regulated by zinc occupancy and oxidation.

It is well established that the antitoxins of toxin-antitoxin (TA) systems are selectively degraded by bacterial proteases in response to stress. However, how distinct stressors result in the selective degradation of specific antitoxins remain unanswered. MqsRA is a TA system activated by various stresses, including oxidation. Here, we reconstituted the Escherichia coli ClpXP proteolytic machinery in vitro to monitor degradation of MqsRA TA components. We show that the MqsA antitoxin is a ClpXP proteolysis substrate, and that its degradation is regulated by both zinc occupancy in MqsA and MqsR toxin binding. Using NMR chemical shift perturbation mapping, we show that MqsA is targeted directly to ClpXP via the ClpX substrate targeting N-domain, and ClpX mutations that disrupt N-domain binding inhibit ClpXP-mediated degradation in vitro. Finally, we discovered that MqsA contains a cryptic N-domain recognition sequence that is accessible only in the absence of zinc and MqsR toxin, both of which stabilize the MqsA fold. This recognition sequence is transplantable and sufficient to target a fusion protein for degradation in vitro and in vivo. Based on these results, we propose a model in which stress selectively targets nascent and zinc-free MqsA, resulting in exposure of the ClpX recognition motif for ClpXP-mediated degradation.

Analysis of Student Attitudes of a Neurobiology Themed Inquiry Based Research Experience in First Year Biology Labs.

Inquiry based research experiences are thought to increase learning gains in biology, STEM retention, and confidence in students of diverse backgrounds. Furthermore, such research experiences within the first year of college may foster increased student retention and interest in biology. However, providing first year students in biology labs with inquiry-based experiences is challenging given demands of large student enrollments, restricted lab space, and instructor time. Thus, we aimed to integrate a small neurobiology themed research experience within a three-week modular, first-year biology laboratory setting. For this, students first performed a whole class lab examining the effects of ethanol on movement and associative learning. Using skills they acquired, the students devised, executed, and presented their self-designed experiments and results. Using pre-and post-course surveys, we analyzed student attitudes on their experiences, including technical skills, inquiry-based learning styles in which experimental outcomes are often unknown, and research in their first year of biology. Analyzing data collected for three years, we found that students self-reported gains in technical skills and positive attitudes toward inquiry-based learning. In contrast, we found that students did not self-report increased interest in research experiences in general.