Comprehensive structural characterization of the human AAA+ disaggregase CLPB in the apo- and substrate-bound states reveals a unique mode of action driven by oligomerization.

The human AAA+ ATPase CLPB (SKD3) is a protein disaggregase in the mitochondrial intermembrane space (IMS) and functions to promote the solubilization of various mitochondrial proteins. Loss-of-function CLPB mutations are associated with a few human diseases with neutropenia and neurological disorders. Unlike canonical AAA+ proteins, CLPB contains a unique ankyrin repeat domain (ANK) at its N-terminus. How CLPB functions as a disaggregase and the role of its ANK domain are currently unclear. Herein, we report a comprehensive structural characterization of human CLPB in both the apo- and substrate-bound states. CLPB assembles into homo-tetradecamers in apo-state and is remodeled into homo-dodecamers upon substrate binding. Conserved pore-loops (PLs) on the ATPase domains form a spiral staircase to grip and translocate the substrate in a step-size of 2 amino acid residues. The ANK domain is not only responsible for maintaining the higher-order assembly but also essential for the disaggregase activity. Interactome analysis suggests that the ANK domain may directly interact with a variety of mitochondrial substrates. These results reveal unique properties of CLPB as a general disaggregase in mitochondria and highlight its potential as a target for the treatment of various mitochondria-related diseases.

Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression.

In living cells, proteins often exert their functions by interacting with other proteins forming protein complexes. Obtaining homogeneous samples of protein complexes with correct fold and stoichiometry is critical for its biochemical and biophysical characterization as well as functional investigation. Here, we developed a Ribozyme-Assisted Polycistronic co-expression system (pRAP) for heterologous co-production and in vivo assembly of multi-subunit complexes. In the pRAP system, a polycistronic mRNA transcript is co-transcriptionally converted into individual mono-cistrons in vivo. Each cistron can initiate translation with comparable efficiency, resulting in balanced production for all subunits, thus permitting faithful protein complex assembly. With pRAP polycistronic co-expression, we have successfully reconstituted large functional multi-subunit complexes involved in mammalian translation initiation. Our invention provides a valuable tool for studying the molecular mechanisms of biological processes.

DddA homolog search and engineering expand sequence compatibility of mitochondrial base editing.

Expanding mitochondrial base editing tools with broad sequence compatibility is of high need for both research and therapeutic purposes. In this study, we identify a DddA homolog from Simiaoa sunii (Ddd_Ss) which can efficiently deaminate cytosine in DC context in double-stranded DNA (dsDNA). We successfully develop Ddd_Ss-derived cytosine base editors (DdCBE_Ss) and introduce mutations at multiple mitochondrial DNA (mtDNA) loci including disease-associated mtDNA mutations in previously inaccessible GC context. Finally, by introducing a single amino acid substitution from Ddd_Ss, we successfully improve the activity and sequence compatibility of DdCBE derived from DddA of Burkholderia cenocepacia (DdCBE_Bc). Our study expands mtDNA editing tool boxes and provides resources for further screening and engineering dsDNA base editors for biological and therapeutic applications.

Structural dynamics of AAA + ATPase Drg1 and mechanism of benzo-diazaborine inhibition.

The type II AAA + ATPase Drg1 is a ribosome assembly factor, functioning to release Rlp24 from the pre-60S particle just exported from nucleus, and its activity in can be inhibited by a drug molecule diazaborine. However, molecular mechanisms of Drg1-mediated Rlp24 removal and diazaborine-mediated inhibition are not fully understood. Here, we report Drg1 structures in different nucleotide-binding and benzo-diazaborine treated states. Drg1 hexamers transits between two extreme conformations (planar or helical arrangement of protomers). By forming covalent adducts with ATP molecules in both ATPase domain, benzo-diazaborine locks Drg1 hexamers in a symmetric and non-productive conformation to inhibits both inter-protomer and inter-ring communication of Drg1 hexamers. We also obtained a substrate-engaged mutant Drg1 structure, in which conserved pore-loops form a spiral staircase to interact with the polypeptide through a sequence-independent manner. Structure-based mutagenesis data highlight the functional importance of the pore-loop, the D1-D2 linker and the inter-subunit signaling motif of Drg1, which share similar regulatory mechanisms with p97. Our results suggest that Drg1 may function as an unfoldase that threads a substrate protein within the pre-60S particle.