Understanding the structural diversity of honeybee-infecting viruses is critical to maintain pollinator health and manage the spread of diseases in ecology and agriculture. We determine cryo-EM structures of T = 4 and T = 3 capsids of virus-like particles (VLPs) of Lake Sinai virus (LSV) 2 and delta-N48 LSV1, belonging to tetraviruses, at resolutions of 2.3–2.6 Å in various pH environments. Structural analysis shows that the LSV2 capsid protein (CP) structural features, particularly the protruding domain and C-arm, differ from those of other tetraviruses. The anchor loop on the central β-barrel domain interacts with the neighboring subunit to stabilize homo-trimeric capsomeres during assembly. Delta-N48 LSV1 CP interacts with ssRNA via the rigid helix α1’, α1’–α1 loop, β-barrel domain, and C-arm. Cryo-EM reconstructions, combined with X-ray crystallographic and small-angle scattering analyses, indicate that pH affects capsid conformations by regulating reversible dynamic particle motions and sizes of LSV2 VLPs. C-arms exist in all LSV2 and delta-N48 LSV1 VLPs across varied pH conditions, indicating that autoproteolysis cleavage is not required for LSV maturation. The observed linear domino-scaffold structures of various lengths, made up of trapezoid-shape capsomeres, provide a basis for icosahedral T = 4 and T = 3 architecture assemblies. These findings advance understanding of honeybee-infecting viruses that can cause Colony Collapse Disorder.

Regulation of CO2 fixation in cyanobacteria is important both for the organism and global carbon balance. Here we show that phosphoketolase in Synechococcus elongatus PCC7942 (SeXPK) possesses a distinct ATP-sensing mechanism, where a drop in ATP level allows SeXPK to divert precursors of the RuBisCO substrate away from the Calvin–Benson–Bassham cycle. Deleting the SeXPK gene increased CO2 fixation particularly during light–dark transitions. In high-density cultures, the Δxpk strain showed a 60% increase in carbon fixation and unexpectedly resulted in sucrose secretion without any pathway engineering. Using cryo-EM analysis, we discovered that these functions were enabled by a unique allosteric regulatory site involving two subunits jointly binding two ATP, which constantly suppresses the activity of SeXPK until the ATP level drops. This magnesium-independent ATP allosteric site is present in many species across all three domains of life, where it may also play important regulatory functions.

Regulation of CO2 fixation in cyanobacteria is important both for the organism and the global carbon balance. Here we show that phosphoketolase in Synechococcus elongatus PCC7942 (SeXPK) possesses a distinct ATP sensing mechanism, which upon ATP drops, allows SeXPK to divert precursors of the RuBisCO substrate away from the Calvin-Benson-Bassham (CBB) cycle. Deleting the SeXPK gene increased CO2 fixation particularly during light-dark transitions. In high-density cultures, the Dxpk strain showed a 60% increase in carbon fixation, and unexpectedly resulted in sucrose secretion without any pathway engineering. Using cryo-EM analysis, we discovered that these functions were enabled by a unique allosteric regulatory site involving two subunits jointly binding two ATP, which constantly suppresses the activity of SeXPK until the ATP level drops. This magnesium-independent ATP allosteric site is present in many species across all three domains of life, where it may also play important regulatory functions.

Human mitochondrial NAD(P)+-dependent malic enzyme (ME2) is well-known for its role in cell metabolism, which may be involved in cancer or epilepsy. We present potent ME2 inhibitors based on cyro-EM structures that target ME2 enzyme activity. Two structures of ME2-inhibitor complexes demonstrate that 5,5’-Methylenedisalicylic acid (MDSA) and embonic acid (EA) bind allosterically to ME2’s fumarate-binding site. Mutagenesis studies demonstrate that Asn35 and the Gln64-Tyr562 network are required for both inhibitors’ binding. ME2 overexpression increases pyruvate and NADH production while decreasing the cell’s NAD+/NADH ratio; however, ME2 knockdown has the opposite effect. MDSA and EA inhibit pyruvate synthesis and thus increase the NAD+/NADH ratio, implying that these two inhibitors interfere with metabolic changes by inhibiting cellular ME2 activity. ME2 silence or inhibiting ME2 activity with MDSA or EA decreases cellular respiration and ATP synthesis. Our findings suggest that ME2 is crucial for mitochondrial pyruvate and energy metabolism, as well as cellular respiration, and that ME2 inhibitors could be useful in the treatment of cancer or other diseases that involve these processes.