Nucleosome assembly requires the coordinated deposition of histone complexes H3-H4 and H2A-H2B to form a histone octamer on DNA. In the current paradigm, specific histone chaperones guide the... Show moreNucleosome assembly requires the coordinated deposition of histone complexes H3-H4 and H2A-H2B to form a histone octamer on DNA. In the current paradigm, specific histone chaperones guide the deposition of first H3-H4 and then H2A-H2B. Here, we show that the acidic domain of DNA repair factor APLF (APLF(AD)) can assemble the histone octamer in a single step and deposit it on DNA to form nucleosomes. The crystal structure of the APLF(AD)-histone octamer complex shows that APLF(AD) tethers the histones in their nucleosomal conformation. Mutations of key aromatic anchor residues in APLF(AD) affect chaperone activity in vitro and in cells. Together, we propose that chaperoning of the histone octamer is a mechanism for histone chaperone function at sites where chromatin is temporarily disrupted. Show less
The single-domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, one of the most abundant renewable biomaterials in nature. We demonstrate that... Show moreThe single-domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, attributed to an enzyme induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction. Show less
Elings, W.; Gaur, A.; Blok, A.J.; Timmer, M.; Ingen, H. van; Ubbink, M. 2020
Architectural DNA-binding proteins are key to the organization and compaction of genomic DNA inside cells. The activity of architectural proteins is often subject to further modulation and... Show moreArchitectural DNA-binding proteins are key to the organization and compaction of genomic DNA inside cells. The activity of architectural proteins is often subject to further modulation and regulation through the interaction with a diverse array of other protein factors. Detailed knowledge on the binding modes involved is crucial for our understanding of how these protein-protein and protein-DNA interactions shape the functional landscape of chromatin in all kingdoms of life: bacteria, archaea, and eukarya.Microscale thermophoresis (MST) is a biophysical technique that has seen increasing application in the study of biomolecular interactions thanks to its solution-based nature, its rapid application, modest sample demand, and the sensitivity of the thermophoresis effect to binding events. Here, we describe the use of MST in the study of chromatin interactions, with emphasis on the wide range of ways in which these experiments are set up and the diverse types of information they reveal. These aspects are illustrated with four very different systems: the sequence-dependent DNA compaction by architectural protein HMfB; the sequential binding of core histone complexes to histone chaperone APLF; the impact of the nucleosomal context on the recognition of histone modifications; and the binding of a LANA-derived peptide to nucleosome core. Special emphasis is given to the key steps in the design, execution, and analysis of MST experiments in the context of the provided examples. Show less
