7XQV

The complex of nanobody Rh57 binding to GTP-bound RhoA active form

Summary for 7XQV

• Entry DOI: 10.2210/pdb7xqv/pdb
• Descriptor: RhoA, Rh57, ALANINE, ... (6 entities in total)
• Functional Keywords: protein binding
• Biological source: Rattus norvegicus; More
• Total number of polymer chains: 2
• Total formula weight: 37542.13

Authors

Zhang, Y.R.,Liu, R.,Ding, Y. (deposition date: 2022-05-09, release date: 2022-07-13, Last modification date: 2024-10-30)

Primary citation

Zhang, Y.,Cheng, S.,Zhong, P.,Wang, Z.,Liu, R.,Ding, Y.
Structural insights into the binding of nanobody Rh57 to active RhoA-GTP.
Biochem.Biophys.Res.Commun., 616:122-128, 2022

PubMed Abstract: RhoA protein is a small GTPase that acts as a molecular switch. When bound to guanosine triphosphate (GTP), RhoA can activate several key signal pathways. Recently, nanobody Rh57 specific binding with GTP bound active RhoA was discovered and developed as a BRET biosensor without cytotoxicity. To further clarify the nanobody Rh57's mechanism of action, we co-expressed, purified, and crystallized the RhoA-Rh57 nanobody complex and solved the structure by X-ray diffraction with a resolution of 2.76 Å. The structure showed that the interaction is mainly through hydrogen bonds, salt bridges, aromatic-aromatic interactions, and hydrophobic interactions. The involved regions include CDR3 and non-hypervariable loop of Rh57, and the SWI switch loops of RhoA, respectively. The different SWI conformation of inactivated RhoA-GDP prevented the Rh57's binding. The possible explanation of Rh57 as a non-cytotoxic BRET intracellular tracer is that Rh57's binding did not overlap with downstream PRK1 and thus did not interfere with the downstream signaling pathway. Our research provides an in-depth understanding of how nanobodies recognize activated RhoA-GTP while not binding inactivated RhoA-GDP. This structural information may also provide critical information for further optimization of relevant nanobodies.

PubMed: 35665664
DOI: 10.1016/j.bbrc.2022.05.084

Experimental method

X-RAY DIFFRACTION (2.76 Å)