Structural studies on AAA+ chaperone complexes
EMSL Project ID
51615
Abstract
Cells have evolved various mechanisms for harnessing the energy from nucleotide hydrolysis and directing it towards useful work. One class of proteins using such a mechanism is the P-loop NTPases, which play critical roles in a vast array of cellular functions. P-loop NTPases are defined by the presence of the nominal P-loop, a conserved nucleotide phosphate-binding motif, also referred to as the Walker A motif, and a second, more variable region, called the Walker B motif. Both the Walker A and Walker B motifs are important for nucleotide binding and hydrolysis. One of the major P-loop lineages are proteins of the AAA+ superfamily. AAA stands for ‘ATPases Associated with diverse cellular Activities’, and, as the name implies, was first used to describe a class of ATP hydrolyzing enzymes with a range of functional roles. Typically, AAA+ proteins function as oligomeric rings, with a hexameric arrangement being the most common. Our proposed structural studies will concentrate on three types of AAA+ proteins: (1) a chaperone that we discovered and named R2TP, (2) an ATP-dependent protease named ClpXP, and (3) a protein disaggregase named ClpB.
We recently identified a novel conserved complex in eukaryotes, which we named the R2TP complex, that has essential and novel chaperone activity dedicated to protein complex assembly. In mammalian cells, R2TP consists of four subunits: RUVBL1, RUVBL2, RPAP3, and PIH1D1. RUVBL1 and RUVBL2 are highly conserved AAA+ ATPases, while RPAP3 and PIH1D1 are newly characterized proteins. R2TP was found to be involved in the assembly of other critical complexes such as snoRNPs, several chromatin remodeling complexes, RNA polymerase II, and PIKK signaling complexes. Recently, we identified a novel adaptor of R2TP that, based on preliminary data, we propose to target the chaperone to ciliogenesis. Our goal is to determine the high resolution structure of R2TP in complex with the adaptor.
ClpXP is a complex composed of the AAA+ ATPase ClpX and the serine protease ClpP. It is highly conserved in bacteria and the mitochondria of mammalian cells. Structurally, ClpP forms a cylindrical tetradecameric oligomer that encloses a proteolytic chamber containing 14 Ser-His-Asp active sites. The cylindrical ClpP is capped at one or both ends by ClpX hexamers, which actively unfold a bound substrate protein and feed it through narrow axial pores into ClpP’s proteolytic chamber for degradation. Hence, ClpX is essential for substrate recognition and unfolding. The cryoEM structure of ClpXP has been determined from multiple bacterial species. In this aim, we will determine the structure of ClpXP in the presence of substrates, adaptors, and compounds with the goal of elucidating the molecular basis of function of this critical ATP-dependent protease.
In our third aim we will work towards determining the structure of a unique AAA+ chaperone, termed ClpB, that is involved in the disaggregation of protein aggregates. More specifically, we will determine the structure of this protein from Plasmodium falciparum, the causative agent of malaria.
In conclusion, our efforts are expected to shed important insights into the structure and mechanism of function of diverse members of the AAA+ superfamily of ATPases.
Project Details
Start Date
2020-09-15
End Date
2021-03-17
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members