Trigger Factor (TF)

Trigger factor is the only known ribosome-associated chaperone in bacteria (Lill et al, 1988) and was first discovered in E. coli as a cytosolic protein involved in translocation of the outer membrane protein pro-OmpA across the plasma membrane (Crooke & Wickner, 1987). It is a modular protein of 48 kDa (432 aa) consisting of an N-terminal ribosome binding domain (N-domain), a central peptidyl-propyl isomerase (PPIase) domain, and a C-terminal domain (C-domain) (Figure 9A) (Stoller et al, 1996). Remarkably, TF adopts an extended three-dimensional conformation with an unusual domain arrangement (Ferbitz et al, 2004). The C-domain forms arm-like protrusions and is located in the center of the molecule between the N-domain and the PPIase domain (Figure 9B). TF binds to ribosomes via a conserved signature motif (42-GFRxGxxP-51) within a loop-region in the N-domain, which contacts the ribosomal protein L23 (Kramer et al, 2002). Mutation of either the TF signature motif (TF-FRK/AAA), or the conserved surface-exposed residue E18 in the ribosomal protein L23, strongly impairs binding of TF to ribosomes. The PPIase domain catalyzes propyl cis/trans isomerization in vitro (Hesterkamp et al, 1996) but is not essential for the folding of cytosolic proteins in vivo (Kramer et al, 2004). The C-terminus together with the N-domain forms a bent cavity with a hydrophobic inner surface that is assumed to provide interaction sites for unfolded substrate proteins. Although TF is an ATP-independent chaperone, it promotes the refolding of chemically denatured proteins in vitro (Huang et al, 2000). This activity of TF depends primarily on its C-domain (Merz et al, 2006). Nevertheless, the in vivo function of TF is not yet understood completely. TF is not essential for viability of E. coli and deletion of the TF encoding gene (tig) does not influence cell growth (Deuerling et al, 1999). However, the simultaneous deletion of TF together with the cytosolic Hsp70 chaperone DnaK (ΔtigΔdnaK) is synthetically lethal at temperatures above 30 C and leads to severe aggregation of more

protein homeostasis network and cooperates with other chaperone systems to promote de novo protein folding. In agreement with this hypothesis, both, TF and DnaK, were shown to interact preferentially with peptides enriched in hydrophobic and basic amino acids, suggesting that they have overlapping substrate pools (Patzelt et al, 2001; Rudiger et al, 1997). A further indication for the plasticity of the chaperone network came from the finding that overproduction of the chaperone SecB (Ullers et al, 2004) or the Hsp60 system GroEL/ES can restore growth of ΔtigΔdnaK cells at 30°C (Vorderwulbecke et al, 2005).

While TF binds to ribosomes as a monomer in a 1:1 ratio (Lill et al, 1988) it can also form homodimers when free in the cytosol (Patzelt et al, 2002). In cells, nearly all ribosomes are supposed to carry a TF molecule. TF is present in a 2-3 fold molar excess relative to ribosomes and has an intrinsic affinity for ribosomes (KD ≈ 1.1 µM) (Maier et al, 2003; Patzelt et al, 2002), which is further increased in the presence of nascent polypeptides (Raine et al, 2006; Rutkowska et al, 2008). Moreover, ribosome binding seems to be essential for the in vivo function of TF, as the genetic combination of TF-binding deficient L23 mutants with a deletion mutant of dnaK (ΔdnaK) resulted in conditional lethality (Kramer et al, 2002).

The co-crystallization of a fragment of the N-terminal ribosome-binding domain of TF with the 50S ribosomal subunit of Haloarcula marismortui provided information on the orientation of full-length TF on the ribosome (Figure 9C) (Ferbitz et al, 2004). Based on these structural data it was speculated that TF forms a “molecular cradle” for nascent polypeptides at the tunnel exit site. This may promote cotranslational folding of nascent chains by providing a shielded environment. Indeed, TF was shown to protect nascent proteins from proteolytic digestion in vitro (Hoffmann et al, 2006). Furthermore, hydrophobic interactions of emerging polypeptides with the inner surface of TF may delay folding of some nascent chains (Agashe et al, 2004). The dissociation of TF is most likely driven by compact folding of the growing polypeptide, whereby the hydrophobic residues become buried (Hartl & Hayer-Hartl, 2009;

Kaiser et al, 2006).

Introduction

Figure 9: Structure of TF from E. coli. A) Domain organization of TF. B) Crystal structure of TF (PDB 1W26).

The N-domain (red) contains the TF signature motif (40-GFRxGxxP-49) in a loop region between two α-helices and is connected to the PPIase domain (green) via an extended linker. The C-domain (blue) is located in the center of the molecule and forms two arm-like protrusions. The N-domain and both arms of the C-domain together form a cavity for nascent polypeptide chains. C) Model of TF bound to the 50S ribosomal subunit of H.

marismortui. TF (colors are the same as in A and B) binds to the large ribosomal subunit via the signature motif in its N-domain. Ribosomal RNA is shown in gray. The two ribosomal proteins L23 (orange) and L29 (yellow) are depicted. A nascent polypeptide (pink) was modeled into the exit tunnel. The orientation of TF on the ribosome positions its cavity over the tunnel exit. The model was built using PDB 1W2B.

So far, the precise mechanism of how TF supports initial folding is not fully uncovered.

Recent studies, however, provided new insights in the dynamic reaction cycle of TF on the ribosome. Accordingly, TF cycles on and off the ribosome with a mean residence time of ∼10 seconds (Kaiser et al, 2006). Ribosome binding causes a conformational opening of TF that may activate it for nascent chain interaction (Baram et al, 2005; Kaiser et al, 2006). TF can stay bound to certain nascent polypeptides even after its dissociation from the ribosome, allowing another TF molecule to access the nascent chain via the ribosome. Thus, multiple

N-domainC-domainPPIase

A

90^o linker

signature motif

arm 1 arm 2

B

C

step, nascent chains, which require further folding assistance, may be transferred to DnaK upon release from TF.

Recently, a second, ribosome-independent function of TF was proposed (Martinez-Hackert &

Hendrickson, 2009). Martinez-Hackert and Hendickson found that TF co-purified with a defined set of full-length proteins, many of which were also aggregation prone in ∆tig∆dnaK cells. Interestingly, most of these TF substrates were components of larger molecular assemblies, such as ribosomes. The crystal structure of TF in complex with one of these substrates, the ribosomal protein S7, revealed that S7 adopted a native-like conformation and the interaction surfaces critical for ribosome incorporation were shielded by TF.

Accordingly, it is speculated that TF has a function in the biogenesis of macromolecular assemblies by stabilizing newly synthesized proteins until they become incorporated into complexes.