Allosteric mechanism of MntR transcription factor from alkalophilic bacterium Halalkalibacterium halodurans.

Bacterium Halalkalibacterium halodurans is an industrially important alkalophilic bacteria. It is recognized as a producer of enzymes such as ß-galactosidase, xylanase, amylase and protease which are able to function at higher pH values and thus can be used in textile, food, paper industry and more. This bacterium, as any other bacterium, requires a sensitive mechanism for regulation of homeostasis of manganese ions (Mn2+) in order to survive. The key protein regulating this mechanism in H. halodurans is MntR - a transcriptional factor that binds to DNA and regulates the transcription of genes for proteins involved in manganese homeostasis. Long range all-atom molecular dynamics (MD) simulations, from 500 ns up to 1.25 µs, were used to study different forms of H. halodurans MntR in order to investigate the differences in the protein's structural and dynamical properties upon Mn2+ binding. Simulations revealed an allosteric mechanism which is activated by Mn2+ binding. The results of simulations show that Mn2+ binding alters the non-covalent interaction network of the protein structure which leads to a conformational change that primarily affects the positions of the DNA binding domains and, consequently, the DNA binding affinity of H. halodurans MntR. The key amino acid residues of the proposed mechanism were identified and their role in the proposed mechanism was computationally confirmed by MD simulations of in silico mutants.Communicated by Ramaswamy H. Sarma.

Augmin prevents merotelic attachments by promoting proper arrangement of bridging and kinetochore fibers.

The human mitotic spindle is made of microtubules nucleated at centrosomes, at kinetochores, and from pre-existing microtubules by the augmin complex. However, it is unknown how the augmin-mediated nucleation affects distinct microtubule classes and thereby mitotic fidelity. Here, we use superresolution microscopy to analyze the previously indistinguishable microtubule arrangements within the crowded metaphase plate area and demonstrate that augmin is vital for the formation of uniformly arranged parallel units consisting of sister kinetochore fibers connected by a bridging fiber. This ordered geometry helps both prevent and resolve merotelic attachments. Whereas augmin-nucleated bridging fibers prevent merotelic attachments by creating a nearly parallel and highly bundled microtubule arrangement unfavorable for creating additional attachments, augmin-nucleated k-fibers produce robust force required to resolve errors during anaphase. STED microscopy revealed that bridging fibers were impaired twice as much as k-fibers following augmin depletion. The complete absence of bridging fibers from a significant portion of kinetochore pairs, especially in the inner part of the spindle, resulted in the specific reduction of the interkinetochore distance. Taken together, we propose a model where augmin promotes mitotic fidelity by generating assemblies consisting of bridging and kinetochore fibers that align sister kinetochores to face opposite poles, thereby preventing erroneous attachments.