Motivation

In order to keep alive and grow, the cell has to fuel a multitude of transport, recycling and communication processes using the energy gained from breaking down organic matter. Under the competitive environmental pressure, cellular machines evolved that can be more energy efficient than man made motors.

Educated as an engineer and biochemist, I am fascinated by molecular motor proteins that convert chemical energy into motion or mechanical work in the cell. By definition these motors transfer physical energy onto a substrate molecule by exerting force along a path, causing a displacement of the motor or the substrate. In the cell we find many different motor proteins working on a variety of substrates.

A heterogenous group of molecular motors is formed by AAA+ enzymes (ATPases associated with various cellular activities). AAA+ proteins generate mechanical work from binding and decomposing ATP (adenosine triphosphate), the universal energy source in the cell. They usually act as oligomers and are often found at the core of essential multi protein assemblies involved in re-organisation and recycling processes of proteins, membranes or DNA in the cell. Despite sharing a strongly conserved nucleotide binding domain (see Figure), each AAA+ protein exhibits unique functional specificity and substrate selectivity. It is believed that accessory domains and proteins facilitate fine tuning of the AAA+ motor activity and thus confer specificity. However, the nature of these interactions as well as their impact on the motor activity of the AAA+ domains remains to be established.

So far less than 50 crystal structures and cryo electron microscopy maps of different AAA+ proteins from diverse species have been deposited in PDB and electron microscopy databases. These numbers compare to ~30,000 proteins with AAA+ domains identified over the past 20 years in all species examined and 50-100 different AAA+ proteins typically encoded in eukaryotic genomes. In budding yeast, the AAA+ superfamily outnumbers ATPases of the myosin and kinesin superfamilies by a factor of five. Despite the available structural information very little is known about the conformational changes during the ATPase cycle and how these changes relate to substrate and co-factor binding. The major difficulty in obtaining structural information to address these questions is the dynamic nature of the motor proteins and their functional form, the oligomeric complex. X-ray structures solved in the hexameric assembly show a very similar packing independent of the nucleotide bound, revealing little information about the nucleotide dependent structural changes. In particular, double tier AAA+ proteins rarely crystallize as hexameric complexes, which makes it difficult to deduce the native conformation of the machine. Because of their complexity, size and dynamic properties, cryo electron microscopy is an essential tool for visualizing the AAA+ multi-protein complexes in their native state. However, so far the AAA+ assemblies reconstructed using single particle cryo EM techniques only resolved to 12- 20 Å, presumably due to heterogeneity within the complex. Recent work on ClpX and Hsp104 suggests that the active complexes are intrinsically asymmetric and that the movements of individual AAA+ domains are much larger than anticipated from crystal structures.