Open Access

Show Abstract ▼

Abstract: Meiosis protein TEX12 is an essential component of the synaptonemal complex (SC), which mediates homologous chromosome synapsis. It is also recruited to centrosomes in meiosis, and aberrantly in certain cancers, leading to centrosome dysfunction. Within the SC, TEX12 forms an intertwined complex with SYCE2 that undergoes fibrous assembly, driven by TEX12’s C-terminal tip. However, we hitherto lack structural information regarding SYCE2-independent functions of TEX12. Here, we report X-ray crystal structures of TEX12 mutants in three distinct conformations, and utilise solution light and X-ray scattering to determine its wild-type dimeric four-helical coiled-coil structure. TEX12 undergoes conformational change upon C-terminal tip mutations, indicating that the sequence responsible for driving SYCE2-TEX12 assembly within the SC also controls the oligomeric state and conformation of isolated TEX12. Our findings provide the structural basis for SYCE2-independent roles of TEX12, including the possible regulation of SC assembly, and its known functions in meiotic centrosomes and cancer.

Show Abstract ▼

Abstract: Acoustic levitation may utilise standing waves at ultrasonic frequencies to manipulate suspended substances and small objects in a contactless manner. These materials may be levitated in the positions in which the nodes are located, corresponding to positions of low acoustic pressure. In recent years, off the shelf transducer based acoustic levitators have been used for contactless manipulation of liquids. These systems benefit from requiring low power and low-cost components making acoustic levitation more accessible to the masses. Such a system was investigated in this work for presenting protein crystals, within their mother liquor, to the I24 beamline at Diamond Light Source for x-ray diffraction experiments. It was found that the crystals tended to sediment toward the bottom of the droplets, which were oblate in shape. The droplets which were levitated often became unstable and fell from their suspended position, or they would not detach from the pipette tip when they were being injected. To rectify this, a coating of silicone oil was added allowing the droplets to remain stable as well as limit the evaporation of the droplet whilst it was manually inserted and the area cleared of personnel before the x-ray beam was engaged. This silicone oil coating is non-crystalline and thus did not invalidate the results collected which showed the lysozyme crystal structure with a resolution of 1.69 A, confirming acoustic levitation as a good sample presentation method for these types of experiments. To remove the requirement for the silicone oil, a bespoke system was created named the DLS-Lev that allowed top-loading of the sample. The droplets of mother liquor which contained protein crystals were easily detached from the pipette tip into the traps within the DLS-Lev system owing to the increased strength of the traps in the modified design. This system, paired with an automated pipette, facilitated sample mixing experiments whilst the x-ray beam was engaged. The further development of the pipetting system was halted due to the COVID-19 pandemic. However, future work should see the permanent installation of these systems at the I24 beamline at Diamond Light Source, as well as additional bespoke acoustic levitators designed for the other beamlines specialising in the research of protein structure via x-ray scattering techniques.

Open Access

Show Abstract ▼

Abstract: The multifunctional human Parkinson’s disease protein 7 (PARK7/DJ1) is an attractive therapeutic target due to its link with early-onset Parkinson’s disease, upregulation in various cancers, and contribution to chemoresistance. However, only a few compounds have been identified to bind PARK7 due to the lack of a dedicated chemical toolbox. We report the creation of such a toolbox and showcase the application of each of its components. The selective PARK7 submicromolar inhibitor with a cyanimide reactive group covalently modifies the active site Cys106. Installment of different dyes onto the inhibitor delivered two PARK7 probes. The Rhodamine110 probe provides a high-throughput screening compatible FP assay, showcased by screening a compound library (8000 molecules). The SulfoCy5-equipped probe is a valuable tool to assess the effect of PARK7 inhibitors in a cell lysate. Our work creates new possibilities to explore PARK7 function in a physiologically relevant setting and develop new and improved PARK7 inhibitors.

Open Access

Show Abstract ▼

Abstract: CK2 is a ubiquitous protein kinase with an anti-apoptotic role and is found to be overexpressed in multiple cancer types. To this end, the inhibition of CK2 is of great interest with regard to the development of novel anti-cancer therapeutics. ATP-site inhibition of CK2 is possible; however, this typically results in poor selectivity due to the highly conserved nature of the catalytic site amongst kinases. An alternative methodology for the modulation of CK2 activity is through allosteric inhibition. The recently identified αD site represents a promising binding site for allosteric inhibition of CK2α. The work presented herein describes the development of a series of CK2α allosteric inhibitors through iterative cycles of X-ray crystallography and enzymatic assays, in addition to both fragment growing and fragment merging design strategies. The lead fragment developed, fragment 8, exhibits a high ligand efficiency, displays no drop off in activity between enzymatic and cellular assays, and successfully engages CK2α in cells. Furthermore, X-ray crystallographic analysis provided indications towards a novel mechanism of allosteric inhibition through αD site binding. Fragments described in this paper therefore represent promising starting points for the development of highly selective allosteric CK2 inhibitors.

Open Access

Show Abstract ▼

Abstract: A 1494 Dalton hemoglycin space polymer of Glycine18 Hydroxy-glycine4 Fe2O4 termed the “core unit” is part of a polymer of Glycine, Si, Fe and O that forms tubes, vesicles and a lattice structure. It has been isolated from four different CV3 meteorites and characterized by mass spectrometry, FIB/SIMS and X-ray analysis. In quantum calculations (HF and DF wB97X-D 6-31G) the polymer has an absorption at 480 nm that is dependent on rectus “R” (= dextro D) chirality in a hydroxy glycine residue whose C-terminus is bonded to an iron atom. The absorption originates in the Fe II state as a consequence of chiral symmetry breaking. In confirmation of theory, measurements at Diamond Light Source UK, on crystals of hemoglycin derived from Acfer-086 and Sutter’s Mill meteorites have shown a strong 483 ± 3 nm absorption that confirms the proposed location of hydroxy glycine residues within the polymer. A high 483 nm to 580 nm absorption ratio points to an “R” chirality excess in hemoglycin, suggesting that 480 nm photons could have provided the energy for its replication in the protoplanetary disc.