Biology subjects

Rao, Q.

Publications and source records attributed to Rao, Q..

5 recordsLinked to original sources

bioRxiv2024

Molecular basis for the assembly of the dynein transport machinery on microtubules

Cytoplasmic dynein-1, a microtubule-based motor protein, requires dynactin and an adaptor to form the processive dynein-dynactin-adaptor (DDA) complex. The role of microtubules in DDA assembly has been elusive. Here, we reveal detailed structural insights into microtubule-mediated DDA assembly using cryo-electron microscopy. We find that an adaptor-independent dynein- dynactin complex (DD) predominantly forms on microtubules in an intrinsic 2:1 stoichiometry, induced by spontaneous parallelization of dynein upon microtubule binding. Adaptors can squeeze in and exchange within the assembled microtubule-bound DD complex, which is enabled by relative rotations between dynein and dynactin, and further facilitated by dynein light intermediate chains that assist in an adaptor search mechanism. Our findings elucidate the dynamic adaptability of the dynein transport machinery, and reveal a new mode for assembly of the motile complex.

biochemistry↗

bioRxiv2022

Cryo-EM structure of an active central apparatus

Accurately regulated ciliary beating in time and space is critical for diverse cellular activities, which impact the survival and development of nearly all eukaryotic species. An essential beating regulator is the conserved central apparatus (CA) of motile cilia, composed of a pair of microtubules (C1 and C2) associated with hundreds of protein subunits per repeating unit. It is largely unclear how CA plays its regulatory roles in ciliary motility. Here, we present high-resolution structures of Chlamydomonas reinhardtii CA by cryo-electron microscopy (cryo-EM) and its dynamic conformational behavior in multiple scales. The structures show how functionally related projection proteins of CA are clustered onto a spring-shaped scaffold of armadillo-repeat proteins, facilitated by elongated rachis-like proteins. The two halves of CA are brought together by elastic chain-like bridge proteins to achieve coordinated activities. We captured an array of kinesin-like protein (KLP1) in two different stepping states, which are actively correlated with beating wave propagation of cilia. These findings establish a structural framework for understanding the role of CA in cilia.

cell biology↗

bioRxiv2022

Multi-curve fitting and tubulin-lattice signal removal for structure determination of large microtubule-based motors

Revealing high-resolution structures of microtubule-associated proteins (MAPs) is critical for understanding their fundamental roles in various cellular activities, such as cell motility and intracellular cargo transport. Nevertheless, large molecular motors that dynamically bind and release microtubule networks are challenging for cryo-electron microscopy (cryo-EM). Traditional structure determination of MAPs bound to microtubules needs alignment information from the reconstruction of microtubules, which cannot be readily applied to large MAPs without a fixed binding pattern. Here, we developed a comprehensive approach to estimate the microtubule networks (multicurve fitting), model the tubulin-lattice signals, and remove them (tubulin-lattice subtraction) from the raw cryo-EM micrographs. The approach does not require an ordered binding pattern of MAPs on microtubules, nor does it need a reconstruction of the microtubules. We demonstrated the capability of our approach using the reconstituted outer-arm dynein bound to microtubule doublets. In addition, we applied our multi-curve fitting approach to other biological filaments and achieved accurate estimations. Our work provides a new tool to determine high-resolution structures of large MAPs bound to curved microtubule networks.

biophysics↗

bioRxiv2021

Structural insights into hormone recognition by the human glucose-dependent insulinotropic polypeptide receptor

Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone that exerts crucial metabolic functions by binding and activating its cognate receptor, GIPR. As an important therapeutic target, GIPR has been subjected to intensive structural studies without success. Here, we report the cryo-EM structure of the human GIPR in complex with GIP and a Gs heterotrimer at a global resolution of 2.9 [A]. GIP adopts a single straight helix with its N terminus dipped into the receptor transmembrane domain (TMD), while the C-terminus is closely associated with the extracellular domain and extracellular loop 1. GIPR employs conserved residues in the lower half of the TMD pocket to recognize the common segments shared by GIP homologous peptides, while uses non-conserved residues in the upper half of the TMD pocket to interact with residues specific for GIP. These results provide a structural framework of hormone recognition and GIPR activation.

molecular biology↗

bioRxiv2020

Cryo-EM structures of outer-arm dynein array bound to microtubule doublet reveal a mechanism for motor coordination

Thousands of outer-arm dyneins (OADs) are arrayed in the axoneme to drive a rhythmic ciliary beat. Coordination among multiple OADs is essential for generating mechanical forces to bend microtubule doublets (MTDs). Using electron microscopy, we determined high-resolution structures of OAD arrays bound to MTD in two different states. OAD preferentially binds to MTD protofilaments with a pattern resembling the native tracks for its distinct microtubule-binding domains. Upon MTD binding, free OADs are induced to adopt a stable parallel conformation, primed for array formation. Extensive tail-to-head (TTH) interactions between OADs are observed, which need to be broken for ATP turnover by the dynein motor. We propose that OADs in an array sequentially hydrolyze ATP to slide MTDs. ATP-hydrolysis in turn relaxes the TTH interfaces to effectuate free nucleotide cycles of downstream OADs. These findings lead to a model explaining how conformational changes in the axoneme produce coordinated action of dyneins.

biophysics↗