Gao Ning's research group in the School of Life Sciences revealed the functional molecular mechanism of the ribosome assembly factor Drg1

On November 9, 2022, Professor Gao Ning's research group from the School of Life Sciences of Peking University published an online report entitled " Structural dynamics of AAA?+? ATPase Drg1 and mechanism of benzo-diazaborine inhibition"
.
In this study, cryo-EM was used to analyze the structure of Drg1 in the state of multiple nucleotides and the structure of hexamer in the substrate bound state, and the molecular mechanism
of Drg1 function was elucidated in detail based on high-resolution structure and biochemical data analysis.
At the same time, this study also analyzed the high-resolution structure of Drg1 hexamer in the bound state of the drug Benzo-diazaborine (B-Dia), revealing the molecular mechanism
of action of the drug.

Ribosomes are the site of protein translation, the processing plants
that transmit genetic information from mRNA into protein amino acid sequences.
Eukaryotic ribosome assembly is a highly complex, delicate and energy-intensive process involving transcription, processing and maturation of rRNA.
translation and transport of ribosomal proteins; Processes such as binding and dissociation of multiple ribosome assembly factors
.
The initial assembly of ribosomes takes place in the nucleoli, after which ribosome precursors are transported to the nucleoplasm and cytoplasm, where final maturation
is completed.
In eukaryotic cells, more than 200 ribosome assembly factors are involved in this process, including three AAA+ ATPases associated with a variety of cellular activities in the nucleoli, nuclear stroma, and cellular matrix—Rix7, Real, and Drg1
.
Among them, Drg1 is an essential gene in yeast, and its homologous protein SPATA5 in human is closely related
to spermatogenesis.
Mutations in SPATA5 can lead to disorders such as epilepsy, hearing impairment or intellectual disability
.
After the prososome 60S ribosome (pre-60S) is transported from the nucleus to the cytoplasm via the nuclear pore complex, Drg1 binds to pre-60S, initiating the ripening process of ribosomes in the cytoplasm and releasing ribosome assembly factors
such as Nog1, Rlp24, Tif6 and Mrt4.
It has been reported that the C-terminal domain of Rlp24 increases its ATPase activity by interacting with Drg1.
And Drg1 releases assembly factors, a process that may require the involvement
of a helper protein (Adaptor/co-factor).
The biological activity of Drg1 can be inhibited by the boron heterocyclic compound diazaborine (Benzo-diazaborine), resulting in the accumulation of pre-60S in the cytoplasm, causing cell dysfunction
.
However, so far, the complete and high-resolution structure of Drg1 is unknown, the detailed molecular mechanism of its transport substrate is unknown, and the binding site and mechanism of action of the inhibitor are unclear
.

Based on the high-resolution structure, the high-resolution structure
of Drg1 was analyzed.
The study found that similar to other classical type II AAA+ ATPase, a three-layer structure is formed by NTD, D1 and D2, and a homohexamer is formed to function
.
Interestingly, although the NTD of Drg1 has low homology with Cdc48/p97 in sequence, its three-dimensional structure is highly similar.
This also suggests structurally that Drg1 may also need to completely unfold the substrate protein into a polypeptide
when it functions.
In the Drg1 hexamer structure in the substrate-bound state, the conserved domains of Drg1 protein, Walker A, Walker B, Sensor I, Sensor II, arginine finger (AF), pore loops I and II (PL-I and PL-II), ISS, etc.
are clearly visible
。 D1 and D2 of Drg1 surround the substrate through conservative Pore-loop (PL) aromatic amino acid residues and rise in spiral steps, with each PL acting like a hand grasping the substrate and transporting
the substrate in a "Hand-over-hand" manner.
Of the six subunits (P:protomer) of Drg1, P1-5 interacts with the peptide substrate via PLs, while P6 is far from the substrate and the nucleotide-binding pocket of D2 is empty
.
Based on the analysis of the structure, it is speculated that the mechanism of action of Drg1 is as follows: when P6 binds to ATP, it will rebind the substrate and then change to the state of P1; At the same time, P5-bound ATP will be hydrolyzed into ADP, transformed into the state of P6, and away from the substrate, and Drg1 will repeatedly fold and transport
the substrate in this mode.
The structure shows that Drg1's conserved ISS (Inter-subunit signaling) motif participates in synergistic interactions between adjacent subunits and regulates ATPase activity
through its conformational changes.
After binding to ATP, the ISS adjacent to protomer will change from a helical conformation to a triangular loop and reach into a nucleotide-binding pocket; At the same time, the conformational changes of ISS and the binding and dissociation of PLs to substrates are coupled to each other
.
Genetic data also suggest that mutations in ISS, linker and PLs between ISS and D1-D2 affect ribosome assembly efficiency and cell growth
.

Figure 1.
Drg1 structure in the substrate-bound state

Diazaborine was originally discovered as an antibacterial compound that hinders the biosynthesis of fatty acids and phospholipids, thereby preventing bacterial proliferation
.
Diazaborine is able to inhibit the ATPase activity of Drg1, which leads to the accumulation of shuttle protein on pre-60S, affecting the maturation
of pre-60S.
In the resolved B-Dia-bound Drg1 hexameric structure, 12 ATP/B-dia binding in the nucleotide-binding pockets of D1 and D2 can be clearly seen
.
B-Dia forms a covalent bond with the 2'-OH of ribose in the ATP molecule, inhibits the hydrolysis of ATP and locks Drg1 in a flatter, symmetrical structure, thereby blocking the conformational changes and functional execution
of D1 and D2.

Figure 2.
Drg1 cryo-EM structure in the B-Dia bound state

In summary, this work analyzes the structure of Drg1 hexamers in different nucleotide states, and elucidates the conformational changes
of Drg1 hexamers in different nucleotide states.
The structure of Drg1 hexamer in substrate treatment was analyzed, and the detailed molecular mechanism
of Drg1 substrate transport was elaborated.
It is of great significance
for understanding the role played by Drg1 in the maturation of large ribosome subunits.
The structural and functional data of the paper suggest that Drg1 is likely involved in the assembly
of large subunits of ribosomes by exerting the activity of protein unfoldase, rather than disassemblase.

Gao Ning is the corresponding author of the paper, Dr.
Ma Chengying (currently an associate researcher in Changping Laboratory) and Dr.
Wu Damu (2017 PTN) are the co-first authors of this paper, and intern Chen Qian (currently studying at Zhejiang University) also contributed
to the work.
The research was supported
by the National Natural Science Foundation of China, the National Key R&D Program of China, the Qidong-SLS Innovation Fund, the Peking University-Tsinghua Joint Center for Life Sciences, and the State Key Laboratory of Membrane Biology.
Peking University cryo-EM platform, electron microscopy laboratory, high-performance computing platform, School of Life Sciences instrument center and Phoenix Engineering and other instrument platforms have provided important technical support
for this project.