Functional roles of the pre‐sensor I insertion sequence in an AAA+ bacterial enhancer binding protein

Patricia C Burrows,Tamaswati Ghosh,Timothy A Burgis,Martin Buck,Xiaodong Zhang,B Tracy Nixon,Jörg Schumacher,Samuel Amartey

doi:10.1111/j.1365-2958.2009.06744.x

Abstract

Molecular machines belonging to the AAA+ superfamily of ATPases use NTP hydrolysis to remodel their versatile substrates. The presence of an insertion sequence defines the major phylogenetic pre-sensor I insertion (pre-SIi) AAA+ superclade. In the bacterial σ54-dependent enhancer binding protein phage shock protein F (PspF) the pre-SIi loop adopts different conformations depending on the nucleotide-bound state. Single amino acid substitutions within the dynamic pre-SIi loop of PspF drastically change the ATP hydrolysis parameters, indicating a structural link to the distant hydrolysis site. We used a site-specific protein–DNA proximity assay to measure the contribution of the pre-SIi loop in σ54-dependent transcription and demonstrate that the pre-SIi loop is a major structural feature mediating nucleotide state-dependent differential engagement with Eσ54. We suggest that much, if not all, of the action of the pre-SIi loop is mediated through the L1 loop and relies on a conserved molecular switch, identified in a crystal structure of one pre-SIi variant and in accordance with the high covariance between some pre-SIi residues and distinct residues outside the pre-SIi sequence.

Highlights

The large AAA+ (ATPases associated with various cellular activities) protein family are responsible for a wide range of ATP-dependent molecular transformations that involve the disassembly and remodelling of protein and nucleic acid complexes (Thomsen and Berger, 2008)
If not all, of the action of the presensor I insertion (pre-SIi) loop is mediated through the L1 loop and relies on a conserved molecular switch, identified in a crystal structure of one pre-SIi variant and in accordance with the high covariance between some pre-SIi residues and distinct residues outside the pre-SIi sequence
To study the function of the pre-SIi loop in phage shock protein F (PspF), single amino acid substitutions to alanine were made across residues 131–139

Summary

Introduction

The large AAA+ (ATPases associated with various cellular activities) protein family are responsible for a wide range of ATP-dependent molecular transformations that involve the disassembly and remodelling of protein and nucleic acid complexes (Thomsen and Berger, 2008). All AAA+ members have the ability to self-assemble into higher-order oligomeric structures, usually as hexamers arranged in a ring (Hanson and Whiteheart, 2005) They contain the highly conserved Walker A and Walker B motifs, involved in ATP binding and ATP hydrolysis, the sensor I sequence and the second region of homology that comprises at least one intersubunit catalytic R-finger (Zhang et al, 2002). AAA+ members belonging to this superclade include HslU, ClpX, Lon, MCM, RuvB, the Ltag helicase and bacterial enhancer binding proteins (bEBPs; Schumacher et al, 2006) These sequence insertions (typically 15–25 amino acids in length) are not highly conserved among different AAA+ members, they are generally located in, or at the edge of the inner pore defined by the common hexameric AAA+ ring assembly (Iyer et al, 2004)

Results

Discussion

Conclusion