Molecular clockwork from cyanobacteria: The cyanobacterial circadian clock oscillator can be reconstituted in a test tube from just three proteins—KaiA, KaiB, and KaiC—and adenosine triphosphate (ATP). Tseng et al. studied crystal and nuclear magnetic resonance structures of complexes of the oscillator proteins and their signaling output proteins and tested the in vivo effects of structure-based mutants. Large conformational changes in KaiB and ATP hydrolysis by KaiC are coordinated with binding to output protein, which couples signaling and the day-night transitions of the clock. Snijder et al. provide complementary analysis of the oscillator proteins by mass spectrometry and cryo–electron microscopy. Their results help to explain the structural basis for the dynamic assembly of the oscillator complexes. Science , this issue p. 1174 , p. 1181

A biological clock in a test tube: The biological clock of cyanobacteria, which remarkably requires just three proteins, has been reconstituted in vitro in a system that allows detailed study of its inputs and outputs, bringing new understanding of how environmental signals can influence a biological oscillator and how the clock controls cellular events such as gene transcription. Chavan et al . extended the known in vitro function of the core clock components to include output signals to transcriptional regulation and allow monitoring through fluorescence measurements in real time. The authors combined crystallography, mutagenesis, and quantitative modeling to further explore the clock mechanism, which may enable future synthetic biology applications. —LBR

Uniquely, the circadian clock of cyanobacteria can be reconstructed outside the complex milieu of live cells, greatly simplifying the investigation of a functioning biological chronometer. The core oscillator component is composed of only three proteins, KaiA, KaiB, and KaiC, and together with ATP they undergo waves of assembly and disassembly that drive phosphorylation rhythms in KaiC. Typically, the time points of these reactions are analyzed ex post facto by denaturing polyacrylamide gel electrophoresis, because this technique resolves the different states of phosphorylation of KaiC. Here, we describe a more sensitive method that allows real-time monitoring of the clock reaction. By labeling one of the clock proteins with a fluorophore, in this case KaiB, the in vitro clock reaction can be monitored by fluorescence anisotropy on the minutes time scale for weeks.

The self-association of therapeutic antibodies can result in elevated viscosity and create problems in manufacturing and formulation, as well as limit delivery by subcutaneous injection. The high concentration viscosity of some antibodies has been reduced by variable domain mutations or by the addition of formulation excipients. In contrast, the impact of Fc mutations on antibody viscosity has been minimally explored. Here, we studied the effect of a panel of common and clinically validated Fc mutations on the viscosity of two closely related humanized IgG_1, κ antibodies, omalizumab (anti-IgE) and trastuzumab (anti-HER2). Data presented here suggest that both Fab-Fab and Fab-Fc interactions contribute to the high viscosity of omalizumab, in a four-contact model of self-association. Most strikingly, the high viscosity of omalizumab (176 cP) was reduced 10.7- and 2.2-fold by Fc modifications for half-life extension (M252Y:S254T:T256E) and aglycosylation (N297G), respectively. Related single mutations (S254T and T256E) each reduced the viscosity of omalizumab by ~6-fold. An alternative half-life extension Fc mutant (M428L:N434S) had the opposite effect in increasing the viscosity of omalizumab by 1.5-fold. The low viscosity of trastuzumab (8.6 cP) was unchanged or increased by ≤2-fold by the different Fc variants. Molecular dynamics simulations provided mechanistic insight into the impact of Fc mutations in modulating electrostatic and hydrophobic surface properties as well as conformational stability of the Fc. This study demonstrates that high viscosity of some IgG₁ antibodies can be mitigated by Fc mutations, and thereby offers an additional tool to help design future antibody therapeutics potentially suitable for subcutaneous delivery.

The histidine kinase SasA enhances robustness of circadian rhythms in the cyanobacterium S. elongatus by temporally controlling expression of the core clock components, kaiB and kaiC . Here we show that SasA also engages directly with KaiB and KaiC proteins to regulate the period and enhance robustness of the reconstituted circadian oscillator in vitro , particularly under limiting concentrations of KaiB. In contrast to its role regulating gene expression, oscillator function does not require SasA kinase activity; rather, SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex. Cooperativity gives way to competition with increasing concentrations of SasA to define a dynamic window by which SasA directly modulates clock robustness. One Sentence Summary: SasA controls the assembly of clock protein complexes through a balance of cooperative and competitive interactions.

The variable domains in Fab regions are well-established contributors to high concentration viscosity of IgG, primarily through charge and hydrophobic interactions. In contrast, the roles of the Fc and the number of self-interacting sites (valency) are less well understood. Here, we investigate the relative contributions of Fab–Fab and Fab–Fc interactions to high concentration viscosity for a diverse panel of 20 IgG ₁ antibodies, by rheometry, coarse-grained simulations, and molecular surface property analysis. Strikingly, fragmentation of IgG ₁ into F(ab′) ₂ plus Fc reduced viscosity (−11 to −93%) for all antibodies tested, demonstrating prevalent contributions of Fc to viscosity. Coarse-grained simulations with one site per Fab and two sites per Fc qualitatively tracked trends in experimental rheometry data for 20 parental antibodies and their fragments. In these simulations Fab–Fab and Fab–Fc interaction strengths were independently varied to capture possible interaction differences arising from parental sequences or any mutations. These coarse-grained simulations suggest that Fab–Fc attractions generate branched IgG ₁ networks and disproportionately larger clusters relative to Fab–Fab interactions of comparable strength. This study suggests that a four-site self-interaction model, previously proposed for a single antibody (omalizumab), is broadly applicable to diverse IgG ₁ . Beyond well-established variable domain engineering, this self-interaction model predicts that Fc engineering may reduce IgG ₁ viscosity, a much sought after goal to enable subcutaneous delivery. Clinically validated Fc mutations are demonstrated here to substantially reduce the viscosity for multiple IgG ₁ (−33 to −91% reduction, n = 6), supporting this emerging antibody design concept.