A motor protein moves on a large scale at molecular level. We have used site-directed spin-labeling electron spin resonance (ESR) to detect molecular orientation, residual side-chain mobility and inter-residual distances. Especially, the distances of 8-80 Å can be measured by continuous-wave and recently developed pulse ESR. We applied these techniques to the studies on conformational dynamics of motor proteins, myosin and kinesin, and muscular switch proteins troponin-tropomyosin. In these systems, the flexible elements undergo thermal motion and fluctuate on large scale between distinct structural states during activity.
Post-translational modifications of proteins change dramatically and dynamically protein structures and functions. Tubulin, which forms microtubules, undergoes highly unique post-translational modifications, polyglutamylation and polyglycylation. These modifications, in particular, accumulate in the axoneme of cilia or flagella. Recently, we and others have identified enzymes that perform or reverse those modifications. The discovery of enzymes enables to investigate the physiological roles of the modifications by means of model organisms with knockout or knockdown techniques. We here review recent knowledge, focusing on our own findings, about the roles of polyglutamylation and polyglycylation in ciliary or flagellar structure and motility.
No-go decay (NGD) and Nonstop decay (NSD) are mRNA surveillance pathways that detect the stalled ribosome containing the defective mRNA. In archaea, archaeal Pelota (aPelota) associates with archaeal elongation factor 1α (aEF1α) to act in the mRNA surveillance pathway. Here, we present the complex structure of aPelota and GTP-bound aEF1α determined at 2.3 Å resolution. Notably, the aPelota·aEF1α·GTP complex structurally resembles the tRNA·EF-Tu complex bound to the ribosome. Combined with the functional analysis in yeast, our findings provide structural insights into how aPelota·aEF1α·GTP complex detects the stalled ribosome and triggers NGD and NSD.
Nitric oxide reductase (NOR) is a transmembrane enzyme that catalyzes reductive coupling of two molecules of nitric oxide NO to form nitrous oxide N2O in microbial denitrification process. Recently, we solved the crystal structures of two types of bacterial NORs, cNOR and qNOR. As has been proposed, their overall structures are similar to those of aerobic and microaerobic respiratory enzymes, cytochrome c oxidases, supporting the idea that NORs are classified as the divergent members of respiratory Heme-Cupper oxidase superfamily. By comparing these structural data in detail, we can briefly describe functional conversion of the respiratory enzyme in their molecular evolution.
Flagella and cilia are bending organelles, which generate cellular movements or extracellular fluid. Flagella and cilia from various organisms share the common “9 + 2” structure in which nine microtubule doublets surround two singlet microtubules and are linked by dynein motor proteins. However the mechanism to integrate the sliding motion of dynein into well-orchestrated bending was unknown. We have been working on structural analysis of flagella/cilia by electron cryo-tomography to elucidate the mechanism of bending motion. In this article we overview recent progress of understanding flagellar/ciliary bending motion, especially by our analysis of dynein arrangement and nucleotide-induced conformational change of dynein molecules in situ.