Structural Basis for the Calmodulin-Mediated Activation of eEF-2K

Translation is a highly energy consumptive process tightly regulated for optimal protein quality and adaptation to energy and nutrient availability. A key facilitator of this process is the α-kinase eEF-2K that specifically phosphorylates the GTP-dependent translocase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis. eEF-2K activation requires calmodulin binding and auto-phosphorylation at the primary stimulatory site, T348. Biochemical studies have predicted that calmodulin activates eEF-2K through a unique allosteric process mechanistically distinct from other calmodulin-dependent kinases. Here we resolve the atomic details of this mechanism through a 2.3 Å crystal structure of the heterodimeric complex of calmodulin with the functional core of eEF-2K (eEF-2KTR). This structure, which represents the activated T348-phosphorylated state of eEF-2KTR, highlights how through an intimate association with the calmodulin C-lobe, the kinase creates a spine that extends from its N-terminal calmodulin-targeting motif through a conserved regulatory element to its active site. Modification of key spine residues has deleterious functional consequences.

Structural Analysis of Retrovirus Assembly and Maturation

Retroviruses have a very complex and tightly controlled life cycle which has been studied intensely for decades. After a virus enters the cell, it reverse-transcribes its genome, which is then integrated into the host genome, and subsequently all structural and regulatory proteins are transcribed and translated. The proteins, along with the viral genome, assemble into a new virion, which buds off the host cell and matures into a newly infectious virion. If any one of these steps are faulty, the virus cannot produce infectious viral progeny. Recent advances in structural and molecular techniques have made it possible to better understand this class of viruses, including details about how they regulate and coordinate the different steps of the virus life cycle. In this review we summarize the molecular analysis of the assembly and maturation steps of the life cycle by providing an overview on structural and biochemical studies to understand these processes. We also outline the differences between various retrovirus families with regards to these processes.

Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9

CRISPR-Cas9 is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system's function.

Sensitivity of Propionibacterium acnes towards Commercial Anti-Acne Formulations

Propionibacterium acnes are aerotolerant anaerobic, gram-positive bacilli that form part of normal flora. They produce several pro-inflammatory substances that can trigger an immune response in the host by an influx of inflammatory leukocytes into the strands, causing inflammatory lesions that leave behind scars. Repeated isolation of Propionibacterium acnes may reduce efficacy among the resistant types, clearly explaining Acne lesions' importance. The Counter acne therapies are often the first treatment choice due to the convenience of cost and time over clinical appointments. However, not all of the commercially available anti-acne formulations are supported by clinical studies. The present study was conducted to test the efficacy of selected commercial anti-acne gel formulations. The microscopic observation and biochemical studies conform to the presence of anti-acne activity. A sensitivity test was performed on all the isolates of Propionibacterium acnes by well diffusion technique. The selected over-the-counter anti-acne gel formulations failed to produce any inhibition zone.

Histone lysine methacrylation is a dynamic post-translational modification regulated by HAT1 and SIRT2

Histone lysine crotonylation is a posttranslational modification with demonstrated functions in transcriptional regulation. Here we report the discovery of a new type of histone posttranslational modification, lysine methacrylation (Kmea), corresponding to a structural isomer of crotonyllysine. We validate the identity of this modification using diverse chemical approaches and further confirm the occurrence of this type of histone mark by pan specific and site-specific anti-methacryllysine antibodies. In total, we identify 27 Kmea modified histone sites in HeLa cells using affinity enrichment with a pan Kmea antibody and mass spectrometry. Subsequent biochemical studies show that histone Kmea is a dynamic mark, which is controlled by HAT1 as a methacryltransferase and SIRT2 as a de-methacrylase. Altogether, these investigations uncover a new type of enzyme-catalyzed histone modification and suggest that methacrylyl-CoA generating metabolism is part of a growing number of epigenome-associated metabolic pathways.

Biochemical Studies in Fibroblasts to Interpret Variants of Unknown Significance in the ABCD1 Gene

Due to newborn screening for X-linked adrenoleukodystrophy (ALD), and the use of exome sequencing in clinical practice, the detection of variants of unknown significance (VUS) in the ABCD1 gene is increasing. In these cases, functional tests in fibroblasts may help to classify a variant as (likely) benign or pathogenic. We sought to establish reference ranges for these tests in ALD patients and control subjects with the aim of helping to determine the pathogenicity of VUS in ABCD1. Fibroblasts from 36 male patients with confirmed ALD, 26 healthy control subjects and 17 individuals without a family history of ALD, all with an uncertain clinical diagnosis and a VUS identified in ABCD1, were included. We performed a combination of tests: (i) a test for very-long-chain fatty acids (VLCFA) levels, (ii) a D3-C22:0 loading test to study the VLCFA metabolism and (iii) immunoblotting for ALD protein. All ALD patient fibroblasts had elevated VLCFA levels and a reduced peroxisomal ß-oxidation capacity (as measured by the D3-C16:0/D3-C22:0 ratio in the D3-C22:0 loading test) compared to the control subjects. Of the VUS cases, the VLCFA metabolism was not significantly impaired (most test results were within the reference range) in 6/17, the VLCFA metabolism was significantly impaired (most test results were within/near the ALD range) in 9/17 and a definite conclusion could not be drawn in 2/17 of the cases. Biochemical studies in fibroblasts provided clearly defined reference and disease ranges for the VLCFA metabolism. In 15/17 (88%) VUS we were able to classify the variant as being likely benign or pathogenic. This is of great clinical importance as new variants will be detected.

Cryo-EM structure of hexameric yeast Lon (PIM1) highlights importance of conserved structural elements

Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphologic perturbations. Although structures of bacterial and human Lon protease reveal a hexameric assembly, PIM1 was speculated to form a heptameric assembly, and is uniquely characterized by a $\sim$50 residue insertion between the ATPase and protease domains. To understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-EM structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrate to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a $\sim$15 residue C-terminal extension. These additional C-terminal residues form an alpha-helix that is located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1 enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.

MICU1 occludes MCU in the mitochondrial calcium uniporter complex

The mitochondrial calcium uniporter imports cytoplasmic Ca2+ into the mitochondrial matrix to regulate cell bioenergetics, Ca2+ signaling, and apoptosis. The uniporter contains the pore-forming MCU subunit, an EMRE protein that binds to MCU, and the regulatory MICU1/MICU2 subunits. Structural and biochemical studies have suggested that MICU1 gates MCU by blocking and unblocking the Ca2+ pore. However, mitoplast patch-clamp experiments argue that MICU1 does not block Ca2+ transport but instead potentiates MCU. To address this direct clash of proposed MICU1 function, we applied purified MICU1 to Ca2+-conducting MCU-EMRE subcomplexes in outside-out patches excised from Xenopus oocytes. MICU1 strongly inhibits Ca2+ currents, and the inhibition is abolished by mutating an MCU-interacting K126 residue in MICU1. Further experiments show that MICU1 block was not observed in mitoplasts because MICU1 dissociates from the uniporter complex. These results firmly establish that MICU1 shuts the uniporter in resting cellular conditions.