Oshri Afanzar

All Publications

• CryoEM structures reveal how the bacterial flagellum rotates and switches direction. Nature microbiology Singh, P. K., Sharma, P., Afanzar, O., Goldfarb, M. H., Maklashina, E., Eisenbach, M., Cecchini, G., Iverson, T. M. 2024

  Abstract

  Bacterial chemotaxis requires bidirectional flagellar rotation at different rates. Rotation is driven by a flagellar motor, which is a supercomplex containing multiple rings. Architectural uncertainty regarding the cytoplasmic C-ring, or 'switch', limits our understanding of how the motor transmits torque and direction to the flagellar rod. Here we report cryogenic electron microscopy structures for Salmonella enterica serovar typhimurium inner membrane MS-ring and C-ring in a counterclockwise pose (4.0A) and isolated C-ring in a clockwise pose alone (4.6A) and bound to a regulator (5.9A). Conformational differences between rotational poses include a 180° shift in FliF/FliG domains that rotates the outward-facing MotA/B binding site to inward facing. The regulator has specificity for the clockwise pose by bridging elements unique to this conformation. We used these structures to propose how the switch reverses rotation and transmits torque to the flagellum, which advances the understanding of bacterial chemotaxis and bidirectional motor rotation.

  View details for DOI 10.1038/s41564-024-01674-1

  View details for PubMedID 38632342

• The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility EMBO JOURNAL Afanzar, O., Di Paolo, D., Eisenstein, M., Levi, K., Plochowietz, A., Kapanidis, A. N., Berry, R., Eisenbach, M. 2021: e104683

  Abstract

  Regulatory switches are wide spread in many biological systems. Uniquely among them, the switch of the bacterial flagellar motor is not an on/off switch but rather controls the motor's direction of rotation in response to binding of the signaling protein CheY. Despite its extensive study, the molecular mechanism underlying this switch has remained largely unclear. Here, we resolved the functions of each of the three CheY-binding sites at the switch in E. coli, as well as their different dependencies on phosphorylation and acetylation of CheY. Based on this, we propose that CheY motor switching activity is potentiated upon binding to the first site. Binding of potentiated CheY to the second site produces unstable switching and at the same time enables CheY binding to the third site, an event that stabilizes the switched state. Thereby, this mechanism exemplifies a unique combination of tight motor regulation with inherent switching flexibility.

  View details for DOI 10.15252/embj.2020104683

  View details for Web of Science ID 000620692300001

  View details for PubMedID 33620739

• The nucleus serves as the pacemaker for the cell cycle. eLife Afanzar, O., Buss, G. K., Stearns, T., Ferrell, J. E. 2020; 9

  Abstract

  Mitosis is a dramatic process that affects all parts of the cell. It is driven by an oscillator whose various components are localized in the nucleus, centrosome, and cytoplasm. In principle, the cellular location with the fastest intrinsic rhythm should act as a pacemaker for the process. Here we traced the waves of tubulin polymerization and depolymerization that occur at mitotic entry and exit in Xenopus egg extracts back to their origins. We found that mitosis was commonly initiated at sperm-derived nuclei and their accompanying centrosomes. The cell cycle was ~20% faster at these initiation points than in the slowest regions of the extract. Nuclei produced from phage DNA, which did not possess centrosomes, also acted as trigger wave sources, but purified centrosomes in the absence of nuclei did not. We conclude that the nucleus accelerates mitotic entry and propose that it acts as a pacemaker for cell cycle.

  View details for DOI 10.7554/eLife.59989

  View details for PubMedID 33284106